CA2328494A1 - Reporter gene system for use in cell-based assessment of inhibitors of the hepatitis c virus protease - Google Patents

Abstract

A cell-based assay system in which the detection of the reporter gene activity, or secreted alkaline phosphatase (SEAP), is dependent upon the protease activity of the Hepatitis C virus NS3 gene product. This system can be used to assess the activity of candidate protease inhibitors in a mammalian cell-based assay system. The assay system is simpler than previously described assays due to the use of SEAP which allows the reporter gene activity to be quantified by measuring the amount of secreted gene product in the cell media by monitoring the conversion of luminescent or colorimetric alkaline phosphatase substrate.

Description

Reporter Gene System For Use In Cell-Based Assessment Of Inhibitors Of The Hepatitis C Virus Protease Technical and Industrial Applicability of Invention A cell-based assay system in which the detection of reporter gene activity (secreted alkaline phosphatase or SEAP) is dependent upon active Hepatitis C
virus (HCV) NS3 protease. The assay system is useful in the in vitro screening, in a mammalian cell-based assay, of potential protease inhibiting molecules useful in the treatment of HCV. The advantages pf using SEAP over more routinely used reporter genes such as beta-galactosidase or luciferase, is that a cell lysis step is not required since the SEAP protein is secxeted out of the cell. The absence of a cell lysis step decreases infra- and inter-assay variability as well as makes the assay easier to perform then earlier assays.
Background of The Invention HCV is one of the major causes of parenterally transmitted non-A, non-B hepatitis worldwide. HCV is now known as the etiologic agent for Non-A
Non-B hepatitis throughout the world. Mishiro et al., U.S. Patent No.
5,077,193; Mishiro et al., U.S. Patent No. 5,176,994; Takahashi et al, U.S.
Patent No. 5,032,511; Houghton et al., U.S. Patent Nos. 5,714,596 and 5,712,088; as well as (M. Houghton, Hepafitis C Viruses, p.1035-1058 in B.N.
Fields et al.(eds.), Field's Viroloav (3d. ed. 1996). HCV infection is characterized by the high rate (>70%) with which acute infection progresses to chronic infection (Alter, M. J. 1995. Epidemiology of hepatitis C in the west.
Sem~. Liver Dis. 15:5-14.). Chronic HCV infection may lead to progressive liver injury, cirrhosis, and in some cases, hepatocellular carcinoma. Currently, there are no specific antiviral agents available for the treatment of HCV
infection. Although alpha interferon therapy is often used in the treatment of HCV-induced moderate or severe liver disease, only a minority of patients exhibit a sustained response Saracco, G. et al., ,~I. Gastroenterol. He ap tol.
10:668-673 1995. Additionally, a vaccine to prevent HCV infection is not yet available and it remains uncertain whether vaccine development will be complicated by the existence of multiple HCV genotypes as well as viral i _ - WO 00/08469 PCT/US99/17440 variation within infected individuals Martell, M. et al., J.J. Virol. 66:3225-1992; Weiner, et al., Proc. Natl. Acid. Sci. 89:3468-3472 1992. The presence of viral heterogeneity may increase the likelihood that drug resistant virus will emerge in infected individuals unless antiviral therapy effectively suppresses virus replication. Most recently, several of the HCV encoded enrymes, specifically the NS3 protease and NSSB RNA polymerise, have been the focus of intensive research, in vitro screening, andlor rational drug design efforts.
HCV has been classified in the flavivirus family in a genus separate from that of the flaviviruses and the pestiviruses. Rice, C. M., in B. N.
Fields and P. M. Knipe (eds.), Virology, 3rd edn., p. 931-959;1996 Lippincott-Raven, Philadelphia, PA. Although the study of HCV replication is limited by the lack of an efficient cell-based replication system, an understanding of replicative events has been inferred from analogies made to the flaviviruses, pestiviruses, and other positive strand RNA viruses. The HCV virus has a 9.4 kb single positive-strand RNA genome encoding over 3,000 amino acids. The genome expresses over 10 structural and non-structural proteins. Post-translational processing of the viral genome requires cleavage by two proteases. As in the pestiviruses, translation of the large open reading frame occurs by a cap-independent mechanism and results in the production of a polyprotein of 3010-3030 amino acids. Proteolytic processing of the structural proteins (the nucleocapsid protein or core (C)) and two envelope glycoproteins, E1 and E2 is accomplished by the action of host cell signal peptidases. Santolini, E., et al., J.J. Virol. 68:3631-3641, 1994; Ralston, R., et al., J.J. Virol. 67:6753-1993. Cleavage of the nonstructural proteins (NS4A, NS4B, NSSA, and NSSB) is mediated by the action of the NS213 protease or the NS3 protease.
Grakoui, A. et al., J.J. Virol. 67:2832-2843 1993; Hirowatari, Y., et al., ~~[, Biochem. 225:113-120 1995; Bartenschlager, R. et al., J.J. Virol. 68:5045-5055 1994; Eckart, M. R., et al., Biochem. Bio~h_ys. Res. Comm. 192:399-406 1993;
Grakoui, A., et al., J.J. Virol. 67:2832-2843 1993; Tomei, L., et al., J.J.
Virol.
67:4017-40261993; NS4A is a cofactor for NS3 and NSSB is an RNA
dependent RNA polymerise. Bartenschlager, R. et al., (1994}; Failla, C.,et al:, . J.J. Virol. 68:3753-3760 1994; Lin, C. et al., Proc. Natl. Arrad. Sci.
92:7622-7626 1995; Behrens, S.-E., et al., EMBO J.J. 15:12-221996. Functions for the NS4B and NSSA proteins have yet to be defined.
The NS213 is a metalloprotease and has been shown to mediate cleavage at the 213 junction site Grakoui, et al. (1993); Hijikata, M., et al., ~J. Virol.
67:4665-4675 1993. In contrast, the NS3 protease is required for multiple cleavages within the nonstructural segment of the polyprotein, specifically the 314A, 4AI4B, 4BI5A, and 5AI5B junction sites Bartenschlager et al. (1993); Eckart, M. R., et al., Biochem.
Bio~hys. RP,~,,~. Comm. 192:399-4.06 1993; Grakoui et al. (1993); Tomei et al.
(1994).
More recently, it is thought that the NS213 protease might actually be part of the HCV
NS3 protease complex even though they have two functionally distinct activities.
Although NS3 protease is presumed to be essential for HCV viability, definitive proof of its necessity has been hampered by the lack of an infectious molecular clone that can be used in cell-based experiments. However, recently two independent HCV
infectious molecular clones have been developed and have been shown to replicate in chimpanzees. Kofykhalov, A. A., et al., Science 277:570-574 1997; Yanagi, M., et al., Proc. Natl. Acad. Sci. 94:8738-8743 1997. The requirement for NS3 in the HCV
life cycle may be validated in these clones by using oligo nucleotide-mediated site directed mutagenesis to inactivate the NS3 catalytic serine residue and then determining whether infectious virus is produced in chimpanzees. Until these experiments are performed, the necessity of NS3 is inferred from cell-based experiments using the related yellow fever (YFV) and bovine viral diarrhea (BVDV) viruses. Mutagenesis of the YFV and BVDV NS3 protease homologs has shown that NS3 serine protease activity is essential for YFV and BVDV replication.
Chambers, T.
J., et al., Proc. Natl. Acad. Sci. 87:8898-8902 1990; Xu, J., et al., J.J.
Virol. 71:5312-53221997.
In general, when investigators screen potential anti-viral compounds for inhibitory activity, it usually involves initial in vifro testing of putative enzyme inhibitors followed by testing the compounds on actual infected cell lines and animals.
It is obvious that working with live virus in large scale screening activities can be inherently dangerous and problematic. While final testing of putative inhibitors in infected cells and animals is still necessary for preclinical drug development, for initial screening of candidate molecules, such work is cost-prohibitive and unnecessary.
Furthermore, the inability to grow HCV in tissue culture in a reproducible quantitative . - WO 00108469 PCTlUS99/17440 manner prevents the evaluation of potential antiviral agents for HCV in a standard antiviral cytopathic effect assay. In response to this real need in the industry, development of non-infectious, cell-based, screening systems is essential.
For example, Hirowatari, et al. developed a reporter assay system, inter alia, that involves the transfection of mammalian cells with two eukaryotic expression plasmids. Hirowatari, et al., Anal. Biocb~em. 225:113-120 1995. One plasmid has been constructed to express a polyprotein that encompasses the HCV NS2-NS3 domains fused in frame to an NS3 cleavage site followed by the HTLV-1 TAXI
protein. A second plasmid has been constructed to have the expression of the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the HTLV-1 LTR. Thus when COS cells are transfected with both plasmids, NS3-mediated cleavage of the TAX1 protein from the NS2-NS3-TAX1 polyprotein allows the translocation of TAXI to the nucleus and subsequent activation of CAT
transcription from the HTLV-1 LTR. CAT activity can be measured by assaying the acetylation of '4C-chloramphenicol through chromatographic or immunological methods. In the CAT assay generally, cell extracts are incubated in a reaction mix containing '4C- or 3H-labeled chloramphenicol and n-Butyryl Coenzyme A. The CAT
enzyme transfers the n-butyryl moiety of the cofactor to chloramphenicol. For a radiometric scintillation detection (LSC) assay, the reaction products are extracted with a small volume of xylene. The n-butyryl chloramphenicol partitions mainly into the xylene phase, while unmodified chloramphenicol remains predominantly in the aqueous phase. The xylene phase is mixed with a liquid scintillant and counted in a scintillation counter. The assay can be completed in as little as 2-3 hours, is linear for nearly three orders of magnitude, and can detect as little as 3 x 10~' units of CAT
activity. CAT activity also can be analyzed using thin layer chromatography (TLC}.
This method is more time-consuming than the LSC assay, but allows visual confirmation of the data.
Similarly, the other patents of Houghton, et al., U.S. Patent No. 5,371,017, U.S. Patent No. 5,585,258, U.S. Patent No. 5,679,342 and U.S. Patent No.
5,597,691 or Jang et al. WO 98/00548 all disclose a cloned NS3 protease or portion fused to a second gene encoding for a protein which a surrogate expression product can be detected for example, in the '017 patent of Houg hi ton, b-galactosidase, superoxide dismutase, ubiquitin or in Jana. the expression is measured by the proliferation of poliovinrs in cell culture) and its use for candidate screening. It is unclear in the Houghton, et al. patents, however, whether the protease described in the specification is the NS2/3 metalloprotease or NS3 serine protease. Although the serine protease is claimed, the experimental data show putative cleavage of the N-terminal SOD
fusion partner at the NS213 junction, a function which recently has been deemed to be the domain of the NS2/3 metalloprotease (Rice, C.M., et al., Proc. Nat. Acad. Sci.
90:10583-10587 (1993)). Furthermore, an active soluble NS3 serine protease is not disclosed in the Houghton. et al. patents, but a insoluble protein derived from E. coli inclusion bodies and which was N-terminally sequenced. For purposes of the present invention the term "NS2 protease" will refer to the enzymatic activity associated with the NS2/3 metalloprotease as defined by $ice et al., and the term "NS3 protease" will refer to the serine protease located within the NS3 region of the HCV genome.
De Francesco et al., U.S. Patent No. 5,739,002, also describes a cell free in vitro system for testing candidates which activate or inhibit NS3 protease by measuring the amount of cleaved substrate. Hirowatari et al. (1995) discloses another HCV NS3 protease assay, however, it differs from the present invention in several aspects, including the reporter gene, the expression plasmid constructs, and the method of detection. Recently, Cho et al. describe a similar SEAP reporter system for assaying HCV NS3 protease which also differs in its structure and function from the present invention. Cho et al., J. Virol. Meth. 72:109-115 1998. Also of interest is a NS3 protease assay system developed by Chen et al. in WO
98/37180.
In the Chen et al. application, a fusion protein is described which uses NS3 protease polypeptide or various truncation analogs fused to the NS4A polypeptide or various truncation analogs and is not autocleavable. The fusion protein is then incubated with known substrates with or without inhibitors to screen for inhibitory effect.
There are a number of problems inherent in all the abovementioned assay systems. For example, the reporter gene product or analyte is many steps removed from the initial NS3 protease cleavage step, the cells used in the assay system are prokaryotic or Yeast based and must be lysed before the reporter gene product can be measured, and the surrogate marker is proliferation of live virus. All of these problems are overcame in the present invention as summarized below.

Summary of Invention The present invention describes a reporter gene system for~use in the cell based assessment of inhibitors of the HCV protease. Applicants point out that throughout the description of this invention, the reference to specific non-structural (NS) regions or domains of the HCV genome are functional definitions and correspond approximately to the defined sequence locations described by C.M.
Rice and others. The present invention discloses the co-transfection of a target cell line with a viral vector which has been engineered to express from the T7 RNA
polymerase promoter and a recombinant plasmid or viral vector which has been engineered to express a polyprotein that includes NS3 HCV serine protease and the secreted human placental alkaline phosphatase (SEAP) gene (Berger et al. 1988) under control of the T7 promoter. The present invention was designed to have a linkage between the detection of reporter gene activity and NS3 serine protease activity through construction of a segment of the HCV gene encoding the NS2-NS4A-NS4B'-sequence linked to the SEAP reporter.
Detection of NS3 protease activity is accomplished by having the release and hence, the subsequent detection, of the SEAP reporter gene to be dependent upon NS3 serine protease activity. In a preferred embodiment, the target cell line is first infected with a viral vector that expresses the T7 RNA polymerase followed by either co-infection with a second viral vector that encodes the NS3 HCV protease/SEAP
polyprotein, or transfection with a plasmid that contains the same NS3ISEAP
gene elements.
The SEAP enzyme is a truncated form of human placental alkaline phosphatase, in which the cleavage of the transmembrane domain of the protein allows it to be secreted from the cells into the surrounding media. SEAP
activity can be detected by a variety of methods including, but not limited to, measurement of catalysis of a fluorescent substrate, immunoprecipitation, HPLC, and radiometric detection. The luminescent method is preferred due to its increased sensitivity over colorimetric detection methods, and such an assay kit is available from Tropix~. The advantages of using SEAP over more routinely used reporter genes such as beta-galactosidase or luciferase, is that a cell lysis step is not required since the SEAP
protein is secreted out of the cell. The absence of a cell lysis step decreases intra--- WO 00/08469 PCf/US99/17440 and inter-assay variability as well as makes the assay easier to perform then earlier assays in the prior art. When both the T7 promoter and NS31SEAP constructs are present, SEAP can be detected in the cell medium within the usual viral assay timeframe of 24-48 hours, however, the timeframe should not be read as a limitation because it is theoretically possible to detect the SEAP in the media only a few hours after transfection. The medium can then be collected and analyzed . Various examples illustrating the use of this composition and method will be detailed below.
Brief Description of the Drawings Figure 1 illustrates schematically the Vaccinia Virus NS3/SEAP System gene construct.
Figure 1 B illustrates schematically the PlasmidlVaccinia Virus NS3lSEAP
assay.
Figure 2 illustrates schematically how the assay operates.
Figure 3 illustrates schematically the DIIDR Assay.
Figure 4A and 4B shows the SEAP activity dose response curve for a representative plasmidlvirus assay.
Figure 5 shows an experimental 96 well plate diagram for the SEAP protocol on Day 1 in Example 3.
Figure 6 shows an experimental 96 well plate diagram for the SEAP protocol on Day 2 in Example 3.
Figure 7 shows SEAP activity and Cytotoxicity data for Example 4.
Figure 8 shows a summary of DIIDR assay data.
Figure 9 illustrates the experimental plate set-up for Example 2.
Detailed Description of a Preferred Embodiment of the Invention The practice of this invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA
manipulation and production, virology and immunology, which are within the skill of the art. Such techniques are explained fully in the literature: Sambrook, Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I
and II
(D. N. Glover, Ed. 1985); Oligonucleofide Synthesis (M. J. Gait, Ed. 1984);
Nucleic Acid Hybridization (B. D. Hames and S. I. Higgins, Eds. 1984); Transcription and Translation (B. D. Hames and S. 1. Higgins, Eds. 1984); Animal Cell Culture (R. I.
Freshney, Ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B.
Perbal, A
Practical Guide to Molecular Cloning (1984); Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos, Eds. 1987, Cold Spring Harbor Laboratory);
Methods in Enzymology, Volumes 154 and 155 (Wu and Grossman, and Wu, Eds., respectively), (Mayer and Walker, Eds.) (1987); Immunochemical Methods in Cel!
and Molecular Biology (Academic Press, London), Scopes, (1987), Expression of Proteins in Mammalian Cells Using Vaccinia Viral Vectors in Current Profocols in Molecular Biology, Volume 2 (Frederick M. Ausubel, et al., Eds.)(1991 ). All patents, patent applications and publications mentioned herein, both supra and infra, are hereby incorporated by reference.
Both prokaryotic and eukaryotic host cells are useful for expressing desired coding sequences when appropriate control sequences compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used.
Expression control sequences far prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers.
These plasmids are commercially available. The markers may be used to obtain successful transformants by selection. Commonly used prokaryotic control sequences include the (i-lactamase (penicillinase) and lactose promoter systems (Chang et al, Nature (1977) 198:1056), the tryptophan (trp) promoter system (Goeddel et al, Nuc Acids Res (1980) 8:4057) and the lambda-derived P~ promoter and N gene ribosome binding site (Shimatake et al, Nature {1981) 292:128) and the hybrid tac promoter (De Boer et al, Proc NatAcad Sci USA (1983) 292:128) derived from sequences of the trp and lac UV5 promoters. The foregoing systems are particularly compatible with E.
coli; if desired, other prokaryotic hosts such as strains of Bacillus or Pseudomonas may be used, with corresponding control sequences.
Eukaryotic hosts include without limitation yeast and mammalian cells in culture systems. Yeast expression hosts include Saccharomyces, Klebsiella, Picia, and the like. Saccharomyces cerevisiae and Saccharomyces carlsbergensis and K.
lactis are the most commonly used yeast hosts, and are convenient fungal hosts.

Yeast-compatible vectors carry markers which permit selection of successful transformants by conferring prototrophy to auxotrophic mutants or resistance to heavy metals on wild-type strains. Yeast compatible vectors may employ the 2 p origin of replication (Broach et al, Meth Enzymol (1983) 101:307), the combination of and ARS1 or other means for assuring replication, such as sequences which will result in incorporation of an appropriate fragment into the host cell genome.
Control sequences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic enzymes (Hess et al, J Adv Enzyme Reg (1968) 7:149;
Holland et al, Biochem (1978), 17:4900), including the promoter for 3-phosphoglycerate kinase (R. Hitzeman et al, J Blol Chem (1980) 255:2073). Terminators may also be included, such as those derived fram the enolase gene (Holland, J Biol Chem (1981 ) 256:1385).
Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, BSC 1 cells, CV1 cells, and a number of other cell lines.
Suitable promoters for mammalian cells are also known in the art and include vital promoters such as that from Simian Virus 40 (SV40) (Fiers et al, Nature (1978) 273:113}, Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammalian cells may also require terminator sequences and poly-A
addition sequences. Enhancer sequences which increase expression may also be included, and sequences which promote amplification of the gene may also be desirable (for example methotrexate resistance genes). These sequences are known in the art.
Vectors suitable for replication in mammalian cells are known in the art, and may include vital replicons, or sequences which insure integration of the appropriate sequences encoding HCV epitopes into the host genome. For example, another vector used to express foreign DNA is Vaccinia virus. In this case the heterologous DNA is inserted into the Vaccinia genome and transcription can be directed by either endogenous vaccinia promoters or exogenous non-vaccinia promoters (e.g. T7 retroviral promoter) known to those skilled in the art, depending on the characteristics of the constructed vector. Techniques for the insertion of foreign DNA into the vaccinia virus genome are known in the art, and may utilize, for example, homologous recombination. The heterologous DNA is generally inserted into a gene which is non-essential to the virus, for example, the thymidine kinase gene (tk), which also provides a selectable marker. Plasmid vectors that greatly facilitate the construction of recombinant viruses have been described (see, for example, Mackett et al, J
Virol (1984) 49:857; Chakrabarti et al, Mol Celt 8iol (1985) 5:3403; Moss, in GENE
TRANSFER VECTORS FOR MAMMALIAN CELLS (Miller and Calos, eds., Cold Spring Harbor Laboratory, N.Y., 1987), p. 10). Expression of the HCV
polypeptide then occurs in cells or animals which are infected with the live recombinant vaccinia virus.
In order to detect whether or.not the HCV polypeptide is expressed from the vaccinia vector, BSC 1 cells may be infected with the recombinant vector and grown on microscope slides under conditions which allow expression. The cells may then be acetone-fixed, and immunofluorescence assays performed using serum which is known to contain anti-HCV antibodies to a polypeptide(s) encoded in the region of the HCV genome from which the HCV segment in the recombinant expression vector was derived.
Other systems for expression of eukaryotic or vital genomes include insect cells and vectors suitable for use in these cells. These systems are known in the art, and include, for example, insect expression transfer vectors derived from the baculovirus Autographa califomica nuclear polyhedrosis virus (AcNPV), which is a helper-independent, viral expression vector. Expression vectors derived from this system usually use the strong viral polyhedron gene promoter to drive expression of heterologous genes. Currently the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373 (see PCT W089J046699 and U.S. Ser. No.
7/456,637). Many other vectors known to those of skill in the an have also been designed for improved expression. These include, for example, pVL985 (which alters the polyhedron start codon from ATG to ATT, and introduces a BamHl cloning site 32 by downstream from the ATT; See Luckow and Summers, Virol (1989) 17:31 ).
AcNPV
transfer vectors for high level expression of non-fused foreign proteins are described in co-pending applications PCT W089/046699 and U.S. Ser. No. 71456,637. A
unique BamHl site is located following position -8 with respect to the translation initiation codon ATG of the polyhedron gene. There are no cleavage sites for Smal, Pstl, Bglll, Xbal or Sstl. Good expression of non-fused foreign proteins usually requires foreign genes that ideally have a short leader sequence containing suitable translation io WO 00/08469 PC"T/US99I17440 initiation signals preceding an ATG start signal. The plasmid also contains the polyhedron polyadenylation signal and the ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.
Methods for the introduction of heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summer and Smith, Texas Agricultural Experiment Station Bulletin No. 1555; Smith et al, Mol. Cell Biol. (1983) 3:2156-2165;
and Luckow and Summers, Virol. (1989) 17:31 ). For example, the heterologous DNA
can be inserted into a gene such as the polyhedron gene by homologous recombination, or into a restriction enzyme site engineered into the desired baculovirus gene. The inserted sequences may be those which encode all or varying segments of the polyprotein, or other orfs which encode viral polypeptides.
For example, the insert could encode the following numbers of amino acid segments from the polyprotein: amino acids 1-1078; amino acids 332-662; amino acids 406-X62;
amino acids 156-328, and amino acids 199-328.
The signals for post-transiational modifications, such as signal peptide cleavage, proteolytic cleavage, and phosphorylation, appear to be recognized by insect cells. The signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells. Examples of the signal sequences from vertebrate cells which are effective in invertebrate cells are known in the art, for example, the human interleukin-2 signal (IL2S) which signals for secretion from the cell, is recognized and properly removed in insect cells.
Transformation may be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus and transducing a host cell with the virus, and by direct uptake of the polynucleotide. The transformation procedure used depends upon the host to be transformed.
Bacterial transformation by direct uptake generally employs treatment with calcium or rubidium chloride (Cohen, Proc. Nat. Acad. Sci. USA {1972) 69:2110; T. Maniatis et at, "Molecular Cloning; A Laboratory Manual" (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1982). Yeast transformation by direct uptake may be carried out using the method of Hinnen et al, Proc. Naf. Acad. Sci. USA (1978) 75:1929.
Mammalian transformations by direct uptake may be conducted using the calcium phosphate precipitation method of Graham and Van der Eb, Virol. (1978) 52:546, or the various known modifications thereof. Other methods for introducing recombinant polynucleotides into cells, particularly into mammalian cells, include dextran-mediated transfection, calcium phosphate mediated transfection, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the polynucfeotides into nuclei.
Vector construction employs techniques which are known in the art. Site-specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions which generally are specified by the manufacturer of these commercially available enzymes. In general, about 1 mg of plasmid or DNA
sequence is cleaved by 1 unit of enzyme in about 20 mL buffer solution by incubation for 1-2 hr at 37° C. After incubation with the restriction enzyme, protein is removed by phenollchloroform extraction and the DNA recovered by precipitation with ethanol.
The cleaved fragments may be separated using polyacrylamide or agarose gel electrophoresis techniques, according to the general procedures described in'Mefh.
Enzymol. (1980) 65:499-560.
Sticky-ended cleavage fragments may be blunt ended using E. coli DNA
polymerase I (Klenow fragment) with the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with S1 nuclease may also be used, resulting in the hydrolysis of any single stranded DNA portions.
Legations are carried out under standard buffer and temperature conditions using T4 DNA ligase and ATP; sticky end legations require less ATP and less ligase than blunt end legations. When vector fragments are used as part of a legation mixture, the vector fragment is often treated with bacterial alkaline phosphatase (BAP}
or calf intestinal alkaline phosphatase to remove the 5'-phosphate, thus preventing re-legation of the vector. Alternatively, restriction enzyme digestion of unwanted fragments can be used to prevent legation. Legation mixtures are transformed into suitable cloning hosts, such as E. coli, and successful transformants selected using the markers incorporated (e.g., antibiotic resistance), and screened for the correct construction.
Synthetic oligonucleotides may be prepared using an automated oligonucleotide synthesizer as described by Warner, DNA (1984) 3:401. It desired, the synthetic strands may be labeled with 32P by treatment with polynucleotide kinase in the presence of 32P-ATP under standard reaction conditions.
DNA sequences, including those isolated from cDNA libraries, may be modified by known techniques, for example by site directed mutagenesis (see e.g., Zoller, Nuc. Acids Res. (1982} 10:6487). Briefly, the DNA to be modified is packaged into phage as a single stranded sequence, and converted to a double stranded DNA
with DNA polymerase, using as a primer a synthetic oligonucleotide complementary to the portion of the DNA to be modified, where the desired modification is included in the primer sequence. The resulting double stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria which contain copies of each strand .of the phage are plated in agar to obtain plaques.
Theoretically, 50% of the new plaques contain phage having the mutated sequence, and the remaining 50%
have the original sequence. Replicates of the plaques are hybridized to labeled synthetic probe at temperatures and conditions which permit hybridization with the correct strand, but not with the unmodified sequence. The sequences which have been identified by hybridization are recovered and cloned.
DNA libraries may be probed using the procedure of Grunstein and Hogness Proc. Nat. Acad. Sci. USA (1975) 73:3961. Briefly, in this procedure the DNA
to be probed is immobilized on nitrocellulose filters, denatured, and pre-hybridized with a buffer containing 0-50% formamide, 0.75M NaCI, 75 mM Na citrate, 0.02% (wt/v}
each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll~, 50 mM NaH2P04 (pH 6.5), 0.1 % SDS, and 100 m g/mL carrier denatured DNA. The percentage of formamide in the buffer, as well as the time and temperature conditions of the pre-hybridization and subsequent hybridization steps depend on the stringency required.
Oligomeric probes which require lower stringency conditions are generally used with low percentages of formamide, lower temperatures, and longer hybridization times.
Probes containing more than 30 or 40 nucleotides, such as those derived from cDNA
or genomic sequences generally employ higher temperatures, e.g., about 40°-42° C., and a high percentage formamide, e.g., 50%. Following pre-hybridization, 5'-labeled oligonucleotide probe is added to the buffer, and the filters are incubated in this mixture under hybridization conditions. After washing, the treated filters are subjected to autoradiography to show the location of the hybridized probe; DNA
in corresponding locations on the original agar plates is used as the source of the desired DNA.
For routine vector constructions, ligation mixtures are transformed into E.
coli strain HB101 or other suitable hosts, and successful transformants selected by antibiotic resistance or other markers. Plasmids from the transformants are then prepared according to the method of Clewell et al, Proc. Nat. Acad. Sci. USA
(1969) 62:1159, usually following chloramphenicol amplification (Clewell, J.
Bacteriol. {1972) 110:667). The DNA is isolated and analyzed, usually by restriction enzyme analysis andlor sequencing. Sequencing may be performed by the dideoxy method of Sanger et at, Proc. Nat. Acad Sci. USA (1977) 74:5463, as further described by Messing et at, Nuc. Acids Res. (1981) 9:309, or by the method of Maxam et at, Mefh.
Enzymol.
(1980) 65:499. Problems with band compression, which are sometimes observed in GC-rich regions, were overcome by use of T-deazoguanosine according to Barr et al, Biotechniques (1986) 4:428.
Target plasmid sequences are replicated by a polymerizing means which utilizes a primer oligonucleotide to initiate the synthesis of the replicate chain. The primers are selected so that they are complementary to sequences of the plasmid.
Oligomeric primers which are complementary to regions of the sense and antisense strands of the plasmids can be designed from the plasmid sequences already known in the literature.
The primers are selected so that their relative positions along a duplex sequence are such that an extension product synthesized from one primer, when it is separated from its template (complement), serves as a template for the extension of the other primer to yield a replicate chain of defined length.
The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and source of the primer and use of the method.
For WO 00/08469 PC'f/US99/17440 example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains about 15-45 nucleotides, although it may contain more or fewer nucleotides. Short prfmer molecules generally require cooler-temperatures to form sufficiently stable hybrid complexes with the template.
The primers used herein are selected to be "substantially" complementary to the different strands of each specific sequence to be amplified. Therefore, the primers need not reflect the exact sequence of the template, but must be sufficiently complementary to selectively hybridize with their respective strands. For example, a non-complementary nucleotide fragment may be attached to the 5'-end of the primer, with the remainder of the primer sequence being complementary to the strand.
Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer has sufficient complementarity with the sequence of one of the strands to be amplified to hybridize therewith, and to thereby form a duplex structure which can be extended by the polymerizing means. The non-complementary nucleotide sequences of the primers may include restriction enzyme sites. Appending a restriction enzyme site to the ends) of the target sequence would be particularly helpful for cloning of the target sequence.
It will be understood that "primer", as used herein, may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequences) of the target region to be amplified. Hence, a "primer" includes a collection of primer oligonucleotides containing sequences representing the possible variations in the sequence or includes nucleotides which allow a typical basepairing.
The oligonucleotide primers may be prepared by any suitable method.
Methods for preparing oligonucleotides of specific sequence are known in the art, and include, for example, cloning and restriction of appropriate sequences, and direct chemical synthesis. Chemical synthesis methods may include, for example, the phosphotriester method described by Narang et al. (1979), the phosphodiester method disclosed by Brown et al. (1979), the diethylphosphoramidate method disclosed in Beaucage et al. (1981), and the solid support method in U.S. Pat.
No.

4,458,Ofifi. The primers may be~labeled, if desired, by incorporating means detectable by spectroscopic, photochemical, biochemical, immunochemical, or is WO 00/084b9 PCT/US99/17440 chemical means.
Template-dependent extension of the oligonucleotide primers) is catalyzed by a polymerizing agent in the presence of adequate amounts of the four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP and dTTP) or analogs, in a reaction medium which is comprised of the appropriate salts, metal cations, and pH
buffering system. Suitable polymerizing agents are enzymes known to catalyze primer- and template-dependent DNA synthesis. Known DNA polymerases include, for example, E. coil DNA polymerase I or its Klenow fragment, T4 DNA
polymerase, and Taq DNA polymerase. The reacfion conditions for catalyzing DNA synthesis with these DNA polymerases are known in the art.
The products of the synthesis are duplex molecules consisting of the template strands and the primer extension strands, which inGude the target sequence.
These products, in turn, serve as template for another round of replication. In the second round of replication, the primer extension strand of the first cycle is annealed with its complementary primer, synthesis yields a "short" product which is bounded on both the 5'- and the 3'-ends by primer sequences or their complements. Repeated cycles of denaturation, primer annealing, and extension result in the exponential accumulation of the target region defined by the primers. Sufficient cycles are run to achieve the desired amount of polynucleotide containing the target region of nucleic acid. The desired amount may vary, and is determined by the function which the product polynucleotide is to serve.
The PCR method can be performed in a number of temporal sequences. For example, it can be performed step-wise, where after each step new reagents are added, or in a fashion where all of the reagents are added simultaneously, or in a partial step-wise fashion, where fresh reagents are added after a given number of steps.
In a preferred method, the PCR reaction is carried out as an automated process which utilizes a thermostable enzyme. In this process the reaction mixture is cyGed through a denaturing region, a primer annealing region, and a reaction region.
A machine may be employed which is specifically adapted for use with a thermostabie enzyme, which utilizes temperature cycling without a liquid handling system, since the enzyme need not be added at every cycle. This type of machine is commercially available from Perkin Elmer Cetus Corp.
After amplification by PCR, the target polynucleotides are detected by hybridization with a probe polynucleotide which forms a stable hybrid with that of the target sequence under stringent to moderately stringent hybridization and wash conditions. if it is expected that the probes will be completely complementary (i.e., about 99% or greater) to the target sequence, stringent conditions will be used. If some mismatching is expected, for example if variant strains are expected with the result that the probe will not be completely complementary, the stringency of hybridization may be lessened. However, conditions are chosen which rule out nonspecific/adventitious binding. Conditions which affect hybridization, and which select against nonspecific binding are known in the art, and are described in, for example, Maniatis et al. (1982). Generally, tower salt concentration and higher temperature increase the stringency of binding. For example, it is usually considered that stringent conditions are incubation in solutions which contain approximately 0.1 xSSC, 0.1% SDS, at about 65° C. incubationlwash temperature, and moderately stringent conditions are incubation in solutions which contain approximately 1-2XSSC, 0.1 % SDS and about 50°~5° C. incubationlwash temperature. Low stringency conditions are 2XSSC and about 30°-50°C.
Probes for plasmid target sequences may be derived from well known restriction sites. The plasmid probes may be of any suitable length which span the target region, but which exclude the primers, and which allow specific hybridization to the target region. If there is to be complete complementarity, i.e., if the strain contains a sequence identical to that of the probe, since the duplex will be relatively stable under even stringent conditions, the probes may be short, i.e., in the range of about 10-30 base pairs. If some degree of mismatch is expected with the probe, i.e., if it is suspected that the probe will hybridize to a variant region, the probe may be of greater length, since length seems to counterbalance some of the effect of the mismatch(es).
The probe nucleic acid having a sequence complementary to the target sequence may be synthesized using similar techniques described supra. for the synthesis of primer sequences. If desired, the probe may be labeled.
Appropriate labels are described supra.
In some cases, it may be desirable to determine the length of the PCR product detected by the probe. This may be particularly true if it is suspected that variant plasmid products may contain deletions within the target region, or if one wishes to confirm the length of the PCR product. In such cases it is preferable to subject the products to size analysis as well as hybridization with the probe. Methods for determining the size of nucleic acids are known in the art, and include, for example, gel electrophoresis, sedimentation in gradients, and gel exclusion chromatography.
The presence of the target sequence in a biological sample is detected by determining whether a hybrid has been formed between the polynucieotide probe and the nucleic acid subjected to the PCR amplification technique. Methods to detect hybrids formed between a probe and a nucleic acid sequence are known in the art.
For example, for convenience, an unlabeled sample may be transferred to a solid matrix to which it binds, and the bound sample subjected to conditions which allow specific hybridization with a labeled probe; the solid matrix is than examined for the presence of the labeled probe. Alternatively, if the sample is labeled, the unlabeled probe is bound to the matrix, and after the exposure to the appropriate hybridization conditions, the matrix is examined for the presence of label. Other suitable hybridization assays are described supra. Analysis of the nucleotide sequence of the target regions) may be by direct analysis of the PCR amplified products. A
process for direct sequence analysis of PCR amplified products is described in Saiki et al.
(1988).
Alternatively, the ampl~ed target sequences) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments has been described by Scharf (1986).
In the method, the primers used in the PCR technique are modified near their 5'-ends to produce convenient restriction sites for cloning directly into, for example, an M13 sequencing vector. After amplification, the PCR products are cleaved with~the appropriate restriction enzymes. The restriction fragments are ligated into the M13 vector, and transformed into, for example, a JM 103 host, plated out, and the resulting plaques are screened by hybridization with a labeled oiigonucleotide probe.
Other methods for cloning and sequence analysis are known in the art.
General Method In the first embodiment, the Tropix4 pCMVISEAP expression vector is used as a starting point for construction of the HCV NS3 protease plasmid construct pHCAP1 (Seq. ID. NOS. 1-7). pHCAP1 is constructed from the pTM3 vector (Moss et al., Nature, 348:91-92 {1990)) in which the nucleotide sequence encoding the portion of the HCV-BK polyprotein domains NS2-NS3-NS4A-NS4B was cloned from the pBKCMV/NS2-NS3-NS4A-NS4B-SEAP (the pBKIHCAP) construct. pBKIHCAP is the eukaryotic expression piasmid in which all the original subcloning and ligation of all the HCV NS gene fragments and SEAP gene was created in. pCMVISEAP is a mammalian expression vector designed for studies of promoterlenhancer elements with SEAP as a reporter (Berger et al.. {1988)). The vector contains a polyiinker for promoter/enhancer insertion, as well as an intron and polyadenylation signals from SV40. The vector can be propagated in E.coli due to the pUC19 derived origin of replication and ampicillin resistance gene. Modification of the commercially available plasmids is accomplished by use of PCR techniques including mutational PCR.
Although this particular plasmid is described in the examples that follow, it is not the only plasmid or vector which may be used. The T7 RNA polymerase promoter is part of the pTM3 plasmid which was preferred in construction of the pHCAP vector.
In an alternate embodiment, the pTKgptF2s plasmid (Falkner and Moss, J.
Virol. 62:1849-1854 (1988)) can be used instead of the pTM3 plasmid, which places the HCVISEAP gene construct under transcriptional control of the native vaccinia virus promoter. The only requirement is that the promoter operate when placed in a plasmid having vaccinia virus regions flanking the subcloning region. This requirement allows the plasmid homologous recombination with the wild type vaccinia virus.
Other vaccinia virus intermediate plasmids would be operable here as well.
Example 1 The Tropix~ pCMVISEAP expression vector is first modified so that both Sac1 restriction sites are inactivated. This is done by cleaving the plasmid with BamH1 which results in a 5' cleavage product that contains the plasmid 5' ATG site and about 250 by ending at the Bam H1 site, and a 3' cleavage product having BamH1 sites at its 5' end and at its 3' end. The 5' cleavage fragment was then amplified from the pCMV/SEAP plasmid using primers that were designed to delete the 5' ATG colon and to create a Sac 1 site on the 5' end. The downstream 3' primer spanned the Bam H1 site that is present within the SEAP coding sequence. Thus after PCR, the amplified 5' fragment has a 5' Sac 1 site and a Bam H1 site. The 5' primer introduced an extra colon (a glutamic acid residue) in front of the first leucine residue of the SEAP secretion signal. Furthermore, the first leucine colon was changed from a CTG to a CTC colon {a silent change). The colon change was made to create the second half of the Sac 1 site:

5'-GAGCTC-X-GGATCC-3' (Seq. ID N0:22) Sac 1 site 5' end of SEAP Bam H1 The modified sequence is then cloned into pGEM3Zf(+) (Promega). The Bam H1-Bam H1 SEAP fragment was subcloned into pAlter 1 (Promega) which is a plasmid that has an f1 origin of replication so it produces a single strand DNA for use in oligo mediated site directed mutagenesis. The Sac 1 sites within the SEAP
fragment were mutated by oligo mediated site directed mutagenesis (GAGCT~ to GAGCT~ - a silent change) and the same change at the second Sac 1 site (GAGCT~ to GAGCT~ - an amino acid change from Serine to Cysteine) The complete SEAP pGEM3Zf(+) plasmid is then made by subcloning the PCR modified 5' SEAP fragment into the Sac I- Bam H1 sites of pGEM3Zf(+). The resulting plasmid was then linearized with Bam H1 to allow the subcloning of the 3' SEAP Bam H1-Bam H1 from the pAlter 1 plasmid which was used for the oligo mediated site directed mutagenesis to disrupt the two internal Sac I sites. A clone with the correct orientation of the Bam H1- Bam H1 fragment distal to the 5' SEAP fragment was selected after of purified plasmid DNA by restriction enzyme digest. This clone was used in the subsequent subcloning steps for the construction of the HCV/SEAP
construct.
The coding sequences for the HCV proteins and NS3 cleavage sites that comprise the final HCVISEAP polyprotein were generated in two separate PCRs from cDNA of the HCV-BK strain (Accession No. M58335). Takamizawa, A., et al., J-Viol.
65:1105-1113 1991. The first amplified fragment starts with the amino acid coding sequence of the HCV polyprotein corresponding to the C-terminal 81 amino acids of the putative E2 region, which are upstream of the beginning of the putative region or amino acid 729 (ARVCACLWMMLLIAQAEAALENLWLNSASVAGAHGILSFLVFFCAAWYIKGRLVPG
ATYALYGVWPLLLLLLALPPRAYAMDREMAA) (Seq. ID N0:23) or nucleotide 2187 (GCACGTGTCTGTGCCTGCTTGTGGATGATGCTGCTGATAGCCCAGGCCGAGGC
CGCCTTGGAGAACCTGGTGGTCCTCAATGCGGCGTCTGTGGCCGGCGCACATG
GCATCCTCTCCTTCCTTGTGTTCTTCTGTGCCGCCTGGTACATCAAAGGCAGGCT
GGTCCCTGGGGCGGCATATGCTCTTTATGGCGTGTGGCCGCTGCTCCTGCTCTT
GCTGGCATTACCACCGCGAGCTTACGCCATGGACCGGGAGATGGC) (Seq. ID
N0:24) and contains the DNA encoding the HCV polyprotein domains NS2-NS3-NS4A
through the first 176 amino acids of the NS4B gene (CASHLPYIEQ GMQLAEQFKQ KALGLLQTAT KQAEAAAPW ESKWRALETF
WAKHMWNFIS GIQYLAGLST LPGNPAIASL MAFTASITSPLTTQSTLLFN
ILGGWVAAQL APPSAASAFV GAGIAGAAVG SIGLGKVLVD
ILAGYGAGVAGALVAFKVMS GEMPSTEDLV NLLPAIL) {Seq. ID N0:25) or amino acid 1886 or nucleotide 5658 (TGCGCCTCGCACCTCCCTTACATCGAGCAGGGAATGCAGCTCGCCGAGCAATT
CAAGCAGAAAGCGCTCGGGTTACTGCAAACAGCCACCAAACAAGCGGAGGCTG
CTGCTCCCGTGGTGGAGTCCAAGTGGCGAGCCCTTGAGACATTCTGGGCGAAG
CACATGTGGAATTTCATCAGCGGGATACAGTACTTAGCAGGCTTATCCACTCTGC
CTGGGAACCCCGCAATAGCATCATTGATGGCATTCACAGCCTCTATCACCAGCC
CGCTCACCACCCAAAGTACCCTCCTGTTTAACATCTTGGGGGGGTGGGTGGCTG

CCCAACTCGCCCCCCCCAGCGCCGCTTCGGCTTTCGTGGGCGCCGGCATCGCC
GGTGCGGCTGTTGGCAGCATAGGCCTTGGGAAGGTGCTTGTGGACATTCTGGC
GGGTTATGGAGCAGGAGTGGCCGGCGCGCTCGTGGCCTTTAAGGTCATGAGCG
GCGAGATGCCCTCCACCGAGGACCTGGTCAATCTACTTCCTGCCATC) (Seq. ID
N0:26) The primers used to ampiify the fragment were designed to contain an Eco RI
site and an ATG codon in the 5' primer (Seq. ID N0:27) and an Xho I site in the 3' primer (Seq. ID N0:28). The amplified fragment was accordingly subcloned as an Eco RI
-Xho I fragment into pET24a(+) plasmid (Novagen). The second fragment amplified from the HCV strain BK cDNA encompasses the putative NS5A/5B cleavage site (EEASEDWCCSMSY'(11VTGAL)(Seq. ID N0:29). The 5' primer that was used to amplify the cleavage site was designed to have an Xho I site (Seq. ID N0:30) whereas the 3' primer was designed to have a Sac I site (Seq. ID N0:31 ). The resulting PCR product was subcloned as an Xho I - Sac I fragment into pET24a(+), which had been digested with Xho I- Hind Ill, as part of a three way ligation (Seq. ID
N0:32). The third fragment in the three way ligation was the Sac I - Hind III
fragment from the SEAP pGEM3Zf(+) plasmid. The Sac I - Hind III fragment encompassed the modified SEAP gene and also 30 base pairs of the pGEM3Zf(+) polylinker which included the multiple cloning sites (MCS) between the Bam H1 and Hindlll sites. The final HCVISEAP construct was assembled using pBKCMV as the vector. pBKCMV
was digested with Eco RI and Hind III and then used in a three way ligation with the NS5A/5B - SEAP Xho I -Hind III fragment and the Eco RI-Xho I NS2-NS4B
fragment.
The control plasmids for the assay (pHCAP3, pHCAP4) were constructed in a similar manner to the HCVISEAP construct. The control plasmids have either an inactive form of NS3 protease or inactive forms of both NS2 protease and NS3 protease. Inactivation of NS2 and NS3 proteases was accomplished by oligo mediated site directed mutagenesis performed on the PCR amplified NS2 - NS4B
fragment that had been subcloned into PALTER-1 as an Eco R1 - Xho 1 fragment together with the NS5AI5B Xho 1 - Sac 1 fragment. In order to inactivate the protease, the catalytic serine residue was substituted with an alanine by replacing thyrnidine (ICG) with guanine (~CG)(base 2754). The NS2 protease was inactivated by substitution of the catalytic cysteine residue with an alanine residue (~T ->
~T)(bases 2238-2239). The resulting inactivated NS3 protease and inactivated NS2-NS3 proteases variants of the NS2-NS4B fragment were each subcloned into pBKCMV as separate Eco R1 - Xho 1 fragments together with the NS5A/5B - SEAP
Xho 1 - Hind III fragment.
The pHCAP1 {NS2'"~NS3 ~)(Seq. ID NOS:1-7}, pHCAP3 (NS2~NS3 ""uT)(Seq. ID NOS:B-14}, and pHCAP4 (NS2""uTNS3 ""uT) (Seq. ID NOS:15-21 ) plasmids were constructed using pTM3 as the vector and the appropriate HCV/SEAP
fragment from the corresponding pBKHCV/SEAP constructs. The pBKHCVISEAP
constructs were first digested with Eco R1 and the Eco R1 site was filled in using Klenow fragment in a standard fill in reaction. The pBKHCV/SEAP constructs were then digested with Xba I and the gel purified HCV/SEAP fragment was subcloned into pTM3 that had been digested with Sma 1 and Spe 1. Subcloning the HCV/SEAP
fragment into the Sma 1 site wiN result in an additional 6 amino acids (MGIPQF) (Seq.
ID N0:33) at the N-terminus (codons 1426-1444} if the preferred translational start codon, which is part of the Nco 1 site in pTM3, is used.
The pHCAP1 (NS2~NS3 '"~), pHCAP3 (NS2~"'TNS3 ""uT), and pHCAP4 (NS2""uTNS3 ""uT) plasmids have been used to generate recombinant vaccinia viruses as described in the next section.
Applicants have generated recombinant vaccinia virus using pHCAP1 and the control plasmids, pHCAP3 and pHCAP4. Recombinant vaccinia viruses were generated using standard procedures in which BSC-1 cells were infected with wild type vaccinia virus (strain WR from ATCC) and then transfected with either pHCAP1, pHCAP3, or pHCAP4. Selection of recombinant virus was performed by growth of infected transfected cells in the presence of mycophenolic acid. The recombinant vaccinia viruses are termed vHCAP1, vHCAP3, and vHCAP4 and correspond directly with the pHCAP1, pHCAP3, and pHCAP4 plasmids. Large scale stocks of the vHCAP1, vHCAP3, and vHCAP4 were grown and titered in CV1 cells.

In the first embodiment HeLa cells are transfected with the Hep C/SEAP
reporter gene plasmid, pHCAP1, and co-infection with a vTF7.3, a recombinant vaccinia virus (Fuerst et al., Proc. Nat. Acid. Sci. USA, 86:8122-8126 (1986)).
vTF7.3 expresses T7 RNA polymerise which is required for transcription of the reporter gene since it is under the control of T7 promoter in the pTM3 plasmid. The pTM3 plasmid is a vaccinia intermediate plasmid which can function as an expression vector in cells when T7 RNA polymerise is provided in traps (Figure 2).
As described previously, the Hep C/SEAP reporter gene encodes for a polyprotein with the following gene order: HCV (strain BK) NS2-NS3-NS4A-NS4B' -NS5AI5B cleavage site - SEAP. Thus the HCV sequences for the amino acid coding sequence of the HCV polyprotein corresponding to the C-terminal 81 amino acids of the putative E2 region, which are upstream of the start of the putative NS2 region (as defined by Grakoui et al. ) or amino acid 729 and continues through the first amino acids of the NS4B gene or amino acid 1886 (Seq. ID NOS:23-26), and is proximal to the SEAP protein (see Figure 1 ). The NS5A/5B cleavage site has been engineered between the end of NS4B' and the second codon of SEAP.
The working theory behind the unique design of the reporter gene construct is that the SEAP polyprotein is tethered, as part of the NS2-NS3-NS4A-NS4B' -NS5A/5B cleavage site - SEAP polyprotein, inside the cell. It has been shown that NS2 is a hydrophobic protein and is associated with the outside of the endoplasmic reticulum (ER). Grakoui, et al. (1993). Thus, in the present invention, SEAP
is tethered to the ER via the action of NS2. Release of SEAP from the pofyprotein tether will occur upon NS3-mediated cleavage at the NS5A/5B cleavage site.
SEAP
is then secreted from the cell and can be monitored by assaying media for alkaline phosphatase activity (Figure 1 B). It is assumed that it is NS3-mediated cleavage at the NS5A/5B site which is the necessary cleavage to release SEAP from the upstream polyprotein sequences. However NS3-mediated cleavage at other sites within the poiyprotein may be responsible for SEAP release and hence its subsequent secretion. Both NS3 and NS3lNS4A, where NS4A is a cofactor for NS3, can mediate cleavage at the NS3/4A and NS4A/4B cleavage sites which are present in polyprotein in addition to the engineered NS5A/5B cleavage site. Thus there may be more than one NS3-mediated cleavage event occurring over the length of the polyprotein before SEAP is available to the ceN secretion apparatus and secreted from the cell.
Further, in an alternative embodiments the tether may be changed depending upon the chosen cleavage site. In addition, NS2 is an autocatalytic protease; it mediates the cleavage event between it's carboxy-terminal end and the NS3 N-terminus. In the Hep C/SEAP polyprotein, NS2-mediated cleavage at the NS2INS3 site would release the NS3-NS4A-NS4B'-SEAP polyprotein from the ER.
The above described system can be used to evaluate potent NS3 inhibitors by monitoring the effect of increasing drug concentration on SEAP activity. NS3 inhibition would be detected as a decrease in SEAP activity. Recognizing that a decrease in SEAP activity could also be due to cell cytotoxicity of a given compound or a non-specific effect on vaccinia virus which would adversely effect SEAP
transcription, appropriate controls are used as discussed below.
In an alternate embodiment, a "cis-only° cleavage assay is contemplated. In this assay the NS2""uTNS3 '~'~ variant of the HCVISEAP (HCAP2) is used so the polyprotein remains tethered to the outside of the endoplasmic reticulum because the NS2 protease cannot catalyze the cleavage between the C-terminus and the NS3 N-terminus. Thus the only way for SEAP to be released from the tether is if the protease clips in cis at the NS5AI5B cleavage site. There should not be any traps NS3 mediated cleavage events occurring since NS2 is not available to release the NS3 N-terminus from its tether. The control plasmid or virus for this assay is the NS2""uTNS3 ""uT variant HCAP4.
A preferred embodiment involves the co-infection of BHK (ATCC No. CCL-10) or CV1 cells (a COS1 derived line ATCC No. CCL-70) cells with both vHCAP1 and vTF7.3 (ATCC No, VR-2153), with CV1 being more preferred. The latter virus is necessary since the Hep C/SEAP gene remains under control of the T7 RNA
polymerase promoter in the vHCAP recombinant viruses. Currently both embodiments which are termed the Hep CISEAP transfectionlinfection assay, and the dual recombinant vaccinia virus assay (DIIDR assay) respectively, are useful for HCV
protease candidate compound evaluation (Figure 3).

Protocol for vTF7.3 infection l HCVlSEAP Plasmid Transfection Experiment Day 1 Flat-bottom 96 well plates were seeded with BHK cells at a density of 1 x 104 cellslwell {equivalent to about 85% confluence) after 24 hours. In general, one 96 well plate was used for investigation of each compound of interest (protease inhibitor), plus an additional plate at the same cell density is used where two rows are designated for each compound of interest at increasing concentrations for investigating the cytotoxicity of the compounds themselves in cells alone.
Cytotoxicity was determined by XTT assay (Sigma 4626).
Day 2 The established monolayer was transfected with either pHCAP1, pHCAP3, pHCAP4, or pTM3 plasmids at a concentration of 0.4 Ng/well as part of a DNA
Lipofectamine (Gibco BRL) transfection mixture. Infections of the established monolayer with vTF7.3 preceded the transfection step. A working stock of vTF7.3 was diluted to a.multiplicity of infection (MOI) of 10 with Optimem. The media was aspirated from the wells (2B-10G) 2 rows at a time. A 50 L aliquot of vTF7.3 inoculum was added per well and gently shaken every 10 minutes. 30 minutes after inoculum addition, the transfection mixes were made by adding 1 mL of Optimem in 3 mL polystyrene tubes. To the media, 48 pg of plasmid DNA was then added to the tubes and mixed, followed by 144 p,L of LipofectamineT"", and then the mixture was incubated (R.T.) for 30 minutes. After incubation, 11 mL of Optimem were added to each of the tubes and gently mixed. The vTF7.3 inoculum was aspirated from the wells and 0.1 mL of transfection mix was added to each well and incubated at 34 °C
for 4 hours. Compounds/drugs of interest for testing protease inhibition were prepared as stock solutions of 40 mM in 100% DMSO. For assay use, the compounds were diluted to 640 pM (2X) in Optimem with 4% FBS. The compound dilutions were set up in an unused 96 well plate by adding 100 pL Optimem with 4%
FBS to wells 4-10 and 150 p.L of compound dilutions to all wells in column 3.
A serial dilution of the compounds was then performed by transferring 46 p.L from well to well across the plate. The transfection mixture was then aspirated from the cells.
Then 75 pL of Optimem with 4% FBS was added to the transfected monolayers. Add 75 wL
of the 2X compound dilutions to the transfected monolayers and incubated at 34 °C for 48 hours. The cells were checked microscopically at 24 hours and media is collected at 48 hours for measurement of SEAP activity.
SEAP Activity Measurement After 48 hours, SEAP activity was measured by first transferring 100 NI of media from each well of the 96 well assay plate to a new sterile 96 well plate. Plates) were sealed and heated in a heating block at 65 C for 30 minutes. After 30 minutes, plates) were removed and cooled to room temperature. For each heat treated plate, we transferred 50 NI of heat treated media to a Dynex (Dynex 7416) 96 well plate. To each well was added 50 NI of Tropix assay buffer and incubated at room temperature for 5 minutes, followed by an addition to each well of 50 NI of Tropix reaction buffer/CSPD substrate (Tropix), each was mixed, and incubated for an additional 90 minutes at room temperature. Chemiluminescence was read in the Victor multilabel counter from Wallac, Inc. (model number 1420) as one second counts and data is reported as luminescent units/second.
For Examples 1 and 2:
XTT Cytotoxicity Assay XTT (Sigma 4626) was dissolved in phosphate buffered saline (PBS) to a final concentration of 1 mg/mL. 5 mL was prepared per plate. To this solution was added 5 mM PMS (n-methyldibenzopyrazine methyl sulfate salt) (Sigma P9625) to a final concentration of 20 NM. 50 NL of the XTT solution was added per well to the plate set up for cytotoxicity. The plates were incubated at 37 C in a 5% C02 incubator for about 3.5 hours and then the color change was quantitated by reading absorbance in a Vmax plate reader (Molecular Devices) at 450nm/650 nm. Values were corrected by subtracting media-only background and presented as %viable with the untreated cell control representing 100%.

Representative experiment and resulting data using Protocol of Example 1.
Compounds X, Y, and Z were evaluated in the Vaccinia Virus Infection/
Plasmid Transfection assay as outlined in Example 1. BHK cells were seeded into 96 well plates at a density of 1 x 104 cells/well and grown overnight to approximately 85% confiuency. The SEAP activity was monitored 48 hours post drug addition in cells transfected with either pHCAP1, pHCAP4, pTM3, or no DNA. Concurrently, Compounds X, Y, and Z were evaluated for cell cytotoxicity in a separate dose response assay using XTT to measure cell viability.
For each compound, cells were infected with vTF7.3 followed by the plasmid transfection step. The arrangement of the cells transfected with one of the three plasmids is illustrated in Figure 9.
Results for Compounds X, Y, and Z are shown in Figures 4 A and 4B and Table 1below. In the three graphs, the amount of SEAP activity detected in cells transfected with the pHCAP1 plasmid ranges from 2 to 7-fold above the amount of SEAP detected in cells transfected with the control plasmids, pHCAP4 and pTM3, or cells only. The EC50 (NM) value represents the concentration of drug at which a 50%
reduction in SEAP activity is observed relative to the amount of SEAP activity detected in the absence of drug. The CC50 (NM} value represents the concentration of drug at which a 50% reduction in cell viability is observed relative to cells in the absence of drug. The ratio of ECSpI CC50 yields the therapeutic index (TI) which, by convention, should be greater or equal to 10 in order for a compound to be considered as demonstrating antiviral activity.
Compound EC50 (uM) CC50 (NM) Solubility TI
(NM) X 45 178 = 100 4 Y >320 112 = 100 - -Z >320 112 = 100 -Within the compound dose range that was examined, only an EC50 value for Compound X was obtained. However, since the Ti value for Compound X was below 10, it was concluded that Compound X does not represent a candidate inhibitor of NS3 protease activity. Compounds Y and Z did not demonstrate any efficacy in this system and, therefore, are not considered potential candidates (Figs. 4A and 4B).
For Examples 3 and 4:
XTT Cytotoxicity Assay XTT (Sigma 4626) was dissolved in phosphate buffered saline (PBS) to a final concentration of 1 mg/mL. 5 mL were prepared per plate. To this solution was added 5 mM PMS (n-methyldibenzopyrazine methyl sulfate salt) (Sigma P9625) to a final concentration of 20 pM. This XTT substrate solution was diluted with an equal volume of MEM media containing 4% FBS(V/V). A 100NLIwell of this final solution was added to the original plate which still contains the cell monolayer and about 50 NL
incubation media. The plates were Incubated at 37 C in a 5% C02 incubator for about 3.5 hours and then the color change was quantitated by reading absorbance in a Vmax plate reader (Molecular Devices) at 450nmI650 nm. Values were corrected by subtracting media-only background and presented as %viable with the untreated cell control representing 100%.
Protocol for Dual lnfecflonlDose Response (DIlDR) Assay Day 1 Flat-bottom 96-well plates were seeded with CV1 cells at a density of 1 x 105 cells per well in MEM media containing 10% FBS with no Phenol Red. The plate was set up as shown in Figure 5. Media only was placed in all the wells on the edge of the plate and only one compound is evaluated per plate (Fig. 5).
Day 2 Cells were infected with recombinant vaccinia viruses as follows. There should be about 1.5 x 105 cells per well after incubation for 24 hours. For every plate needed (a plate for each drug in the experiment) 4 mL of vTF7.3 in MEM with 4%
FBS
(-) phenol red at a concentration of 2 x 108 pfulmL was prepared, and divided into 2 mL aliquots. Either vHCAP1 or vHCAP3 was added to the vTF7.3 aliquots for a final concentration of vHCAP of 1 x 10' pfulmL. At 75 wL per well, this concentration of virus stock delivers vTF7.3 at an MOI of 1 and vHCAP1 or vHCAP3 at an MOI of 5.
The arrangement of the experimental plate is shown in Figure 5.
Drug stock solutions for use ~in the assay, were made at a concentration of 40 mM in DMSO as in the previous protocol. The 40 mM drug stock solution was diluted to 640 p.M in MEM with 4% FBS {-) phenol red to yield a 2X drug working stock solution. Using an empty 96 well plate, the drug dilution series was set up as follows:
100 p.L of MEM with 4% FBS (-) phenol red was added to all wells in columns 4-10. 150 uL of 2X drug working stock solution was added to all wells in column 3. 46 pL of media was transferred from column 3 to wells of column 4 and mixed.
Transferring of 46 p,L from column 4 to column 5 and out to row 10 was repeated.
The remaining 46 pL was discarded. The arrangement of the experimental multiwell plate is shown in Figure 6.
Media was aspirated from the CV1 monolayers. After aspiration, 75 ~L per well of appropriate virus inoculum or MEM with 4% FBS (-) phenol red was added to the CV1 monolayers, then 75 p.L was transferred from each well in the drug dilution series plate to the corresponding wells on the cell monolayer plate. The assay plate was incubated at 37 C in a 5% COz incubator for 48 hours.
At Day 3, the cells was microscopically checked for phenotypic changes around the 24 hour time point. At Day 4, 100 p,L of media was collected from each well of which 50 wL was used in the measurement of SEAP activity. The 100 wL
aliquots were transferred to an unused 96 well plate and after the plate was sealed, it was heated to 65 C for 30 minutes. 50 pL of each heat treated sample was then transferred to its corresponding well in a new 96 well opaque plate (Dynex 7416).
Using the Tropix~ SEAP PhosphalightT"" kit, 50 mL of Tropix assay buffer was added WO 00/08469 PCT/IlS99/I7440 to each well and the plate was incubated at room temperature for 5 minutes.
Next, 50 pL of Tropix reaction bufferICPSD substrate was added and mixed. The plate was incubated for 90 minutes at room temperature. The chemiluminescence was then read using a Victor multi-label counter. The XTT assay for measuring cytotoxicity was also performed on Day 4 as described.
Representative Experiment and Resulting Data Using Protocol of Example 3 Compounds A -I were evaluated in the DII DR assay using the standard protocol given in Example 3. The data shown in Figure 7 and Figure 8 represent assay results obtained at a 48 hour time point post drug addition.
The EC50 (NM) value represents the concentration of drug at which a 50%
reduction in SEAP activity is observed relative to the amount of SEAP activity detected in the absence of drug. However, this latter value, the amount of SEAP
activity that is observed in the absence of drug, is first corrected for assay background prior to the calculation of an EC50 value. The correction is made since in the inactive NS3 protease construct, vHCAP3, a background level of SEAP activity is detected (see SEAP Activity graph). This background SEAP activity represents non-NS3 protease mediated SEAP activity and therefore should not be affected by the addition of an NS3 protease inhibitor. It is assumed that a fraction of the SEAP
activity that is observed in the active NS3 protease construct, vHCAPI, represents non-NS3 protease mediated SEAP activity. Therefore the amount of SEAP activity detected vHCAPI is corrected for the fraction that corresponds to non-NS3 protease mediated SEAP activity. The correction is as follows: luminescent units of SEAP
activity of vHCAP1 - luminescent units of SEAP activity of vHCAP3 = Value N (level of NS3 protease dependent SEAP activity). Accordingly, (vHCAP1/SEAP)-NI2 = EC50 value.
The CC50 (NM) value represents the concentration of drug at which a 50%
reduction in cell viability is observed relative to cells in the absence of drug. The ratio of ECSpI CC50 yields the therapeutic index (TI) which, by convention, should be greater or equal to 10 in order for a compound to be considered as demonstrating antiviral activity.
In Figure 7, increasing concentrations of Compound A were observed to have no affect on SEAP activity. In the cell cytotoxicity component of the assay, it was observed that increasing concentrations of Compound A did not result in a reduction of cell viability of cells alone or cells infected with either vHCAP1/vTF7.3 or vHCAP3/vTF7.3. The results obtained with Compounds B - I (Figure 8) demonstrate a range of observed cytotoxicities from 15 uM to >320 NM which is the upper limit of drug concentrations tested in the DI! DR assay although it is theoretically possible to test drug concentrations above 320 pM. The EC50 values that were observed for Compounds B - I ranged from 18 NM to > 320 NM, however, the TI values were under 10. Thus Compounds A -I do not represent potential inhibitors of NS3 protease activity.

I, SEQUENCE LISTING
<110> Potts, Karen E.
Jackson, Roberta L.
Patick, Amy K.
<120> REPORTER GENE SYSTEM FOR USE IN CELL-BASED ASSESSMENT
OF INHIBITORS OF THE HEPATITIS C VIRUS PROTEASE
<130> 0125-0005A
<140>
<141>
<150> 09/129,611 <151> 1999-08-05 <160> 33 <170> PatentIn Ver. 2.0 <210> 1 <211> 13910 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: plasmid phcap 1 <220>
<221> CDS
<222> (497)..(772) <220>
<221> CDS
<222> (1425)..(6500) <220>
<221> CDS
<222> (8579)..(9039) <220>
<221> CDS
<222> (10191)..(10445) <220>
<221> CDS
<222> ;11877)..(I2734) <220>
<221> misc_feature <222> (1). (774) <223> Vaccinia Virus thymidine Kinase gene recombination site <220>
<221> promoter <222> (794)..(816) <223> T7 promoter <220>

. . WO 00/08469 PG"TIUS99/17440 <221> misc_feature -<222> (896)..(1429) <223> EMC/Internal Ribosome Entry Site (IRES) <220>
<221> misc_feature <222> (1426)..(1937) <223> MCS (Multiple Cloning Site) <220>
<221> misc_feature <222> (1446)..(2318) <223> HCV E2/ NS2 domain <220>
<221> misc_feature <222> (2319)..(4231) <223> HCV NS3 Domain containing the~serine protease and helicase enzymes <220>
<221> misc_feature <222> (9203)..(4260) <223> HCV NS3-NS4A cleavage site <220>
<221> misc_feature <222> (4375)..(4424) <223> HCV NS4A-4B clevage site <220>
<221> misc_feature <222> (4'233) .. (4394) <223> HCV NS4A domain <220>
<221> misc_feature <222> (4395)..(4919) <223> HCV NS4B Domain <220>
<221> misc_feature <222> (4920)..(4991) <223> HCV NSSA-NSSB cleavage site <220>
<221> misc feature <222> (9992)..(6501) <223> SEAP Protein <220>
<221> misc_feature <222> (7915)..(7995) <223> MCS (Multiple Cloning Site) <220>
<221> terminator <222> (7938)..(8078) <223> term T7 <220>

<221> promoter <222> (8080) . . (8365) <223> Vacinina virus promoter; early/late promoter <220>
<221> misc_feature <222> (8560)..(11317) <223> E. coli gpt; for selection of recombinants <220>
<221> misc_feature <222> (11318)..(13909) <223> remaining DNA from 3' end of Tropix pCMV/SEAP
plasmid <400> 1 ' aagcttttgc gatcaataaa tggatcacaa ccagtatctc ttaacgatgt tcttcgcaga 60 tgatgattca ttttttaagt atttggctag tcaagatgat gaatcttcat tatctgatat 120 attgcaaatc actcaatatc tagactttct gttattatta ttgatccaat caaaaaataa 180 attagaagcc gtgggtcatt gttatgaatc tctttcagag gaatacagac aattgacaaa 240 attcacagac tttcaagatt ttaaaaaact gtttaacaag gtccctattg ttacagatgg 300 aagggtcaaa cttaataaag gatatttgtt cgactttgtg attagtttga tgcgattcaa 360 aaaagaatcc tctctagcta ccaccgcaat agatcctgtt agatacatag atcctcgtcg 420 caatatcgca ttttctaacg tgatggatat attaaagtcg aataaagtga acaataatta 480 attctttatt gtcatc atg~ aac ggc gga cat att cag ttg ata atc ggc ccc 532 Met Asn Gly Gly His Ile Gln Leu Ile Ile Gly Pro atg ttt tca ggt aaa agt aca gaa tta att aga cga gtt aga cgt tat 580 Met Phe Ser Gly Lys Ser Thr Glu Leu Ile Arg Arg Val Arg Arg Tyr caa ata get caa tat aaa tgc gtg act ata aaa tat tct aac gat aat 628 Gln Ile Ala~Gln Tyr Lys Cys Val Thr Ile Lys Tyr Ser As~n Asp Asn aga tac gga acg gga cta tgg acg cat gat aag aat aat ttt gaa gca 676 Arg Tyr Gly Thr Gly Leu Trp Thr His Asp Lys Asn Asn Phe Glu Ala ttg gaa gca act aaa cta tgt gat gtc ttg gaa tca att aca gat ttc 724 Leu Glu Ala Thr Lys Leu Cys Asp Val Leu Glu Ser Ile Thr Asp Phe tcc gtg ata ggt atc gat gaa gga cag ttc ttt cca gac att gtt gaa 772 Ser Val Ile Gly Ile Asp Glu Gly Gln Phe Phe Pro Asp Ile Val Glu ttgatctcga tcccgcgaaa ttaatacgac tcactatagg gagaccacaa cggtttccct 832 ctagcgggat caattccgcc cctctccctc ccccccccct aacgttactg gccgaagccg 892 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt 952 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct 1012 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct 1072 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc 1132 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc 1192 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc 1252 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc 1312 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaacgtcta 1372 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgataata cc atg gga 1930 Met Gly att ccc caa ttc atg gca cgt gtc tgt gcc tgc ttg tgg atg atg ctg 1978 Ile Pro Gln Phe Met Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu ctg ata gcc cag gcc gag gcc gcc ttg gag aac ctg gtg gtc ctc aat 1526 Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn gcg gcg tct gtg gcc ggc gca cat ggc atc ctc tcc ttc ctt gtg ttc 1574 Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe Leu Val Phe ttc tgt gcc gcc tgg tac atc aaa ggc agg ctg gtc cct ggg gcg gca 1622 Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala 195 ~ w 150 155 tat get ctt tat ggc gtg tgg ccg ctg ctc ctg ctc ttg ctg gca tta 1670 Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu cca ccg cga get tac gcc atg gac cgg gag atg get gca tcg tgc gga 1718 Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly ggc gcg gtt ttt gtg ggt ctg gta ctc ctg act ttg tca cca tac tac 1766 Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser Pro Tyr Tyr aag gtg ttc ctc get agg ctc ata tgg tgg tta caa tat ttt acc acc 1814 Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Thr Thr aga gcc gag gcg cac tta cat gtg tgg atc ccc ccc ctc aac get cgg 1862 Arg Ala Glu Ala His Leu His Val Trp Ile Pro Pro Leu Asn Ala Arg gga ggc cgc gat gcc atc atc ctc ctc atc tgc gca gtc cat cca gag 1910 Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu cta atc ttt gac atc acc aaa ctt cta att gcc ata ctc ggt ccg ctc 1958 Leu Ile Phe Asp Ile Thr Lys Leu Leu Ile Ala Ile Leu Gly Pro Leu -. WO 00/08469 PCT/US99/17440 atg gtg ctc caa get ggc ata acc aga gtg ccg tac ttc gtg cgc get 2006 Met Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala caa ggg ctc att cat gca tgc atg tta gtg cgg aag gtc get ggg ggt 2054 Gln Gly Leu Ile His Ala Cys Met Leu Val Arg Lys Val Ala Gly Gly cat tat gtc caa atg gcc ttc atg aag ctg ggc gcg ctg aca ggc acg 2102 His Tyr Val Gln Met Ala Phe Met Lys Leu Gly Ala Leu Thr Gly Thr tac att tac aac cat ctt acc ccg cta cgg gat tgg gcc cac gcg ggc 2150 Tyr Ile Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala His Ala Gly cta cga gac ctt gcg gtg gca gtg gag ccc gtc gtc ttc tcc gac atg 2198 Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Asp Met gag acc aag atc atc acc tgg gga gca gac acc gcg gcg tgt ggg gac 2246 Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp atc atc ttg ggt ctg ccc gtc tcc gcc cga agg gga aag gag ata ctc 2294 Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu ctg ggc ccg gcc gat agt ctt gaa ggg cgg ggg tgg cga ctc ctc gcg 2342 Leu Gly Pro Ala Asp Ser Leu Glu Gly Arg Gly Trp Arg Leu Leu Ala ccc atc acg gcc tac tcc caa cag acg cgg ggc cta ctt'ggt tgc atc 2390 Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile atc act agc ctt aca ggc cgg gac aag aac cag gtc gag gga gag gtt 2438 Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Giy Glu Val cag gtg gtt tcc acc gca aca caa tcc ttc ctg gcg acc tgc gtc aac 2486 Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn ggc gtg tgt tgg acc gtt tac cat ggt get ggc tca aag acc tta gcc 2539 Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu Ala ggc cca aag ggg cca.atc acc cag atg tac act aat gtg gac cag gac 2582 Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp ctc gtc ggc tgg cag gcg ccc ccc ggg gcg cgt tcc ttg aca cca tgc 2630 Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys acc tgt ggc agc tca gac ctt tac ttg gtc acg aga cat get gac gtc 2678 Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val att ccg gtg cgc cgg cgg ggc gac agt agg ggg agc ctg ctc tcc ccc 2726 Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro agg cct gtc tcc tac ttg aag ggc tct tcg ggt ggt cca ctg ctc tgc 2774 Arg Pro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys cct tcg ggg cac get gtg ggc atc ttc cgg get gcc gta tgc acc cgg 2822 Pro Ser Giy His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg ggg gtt gcg aag gcg gtg gac ttt gtg ccc gta gag tcc a.tg gaa act 2870 Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr act atg cgg tct ccg gtc ttc acg gac aac tca tcc ccc ccg gcc gta 2918 Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val ccg cag tca ttt caa gtg gcc cac cta cac get ccc act ggc agc ggc 2966 Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly aag agt act aaa gtg ccg get gca tat gca gcc caa ggg tac.aag gtg 3014 Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val ctc gtc ctc aat ccg tcc gtt gcc get acc tta ggg ttt ggg gcg tat 3062 Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr atg tct aag gca cac ggt att gac ccc aac atc aga act ggg gta agg 3110 Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg accattaccaca ggcgcccccgtcaca tactctacctatggcaag ttt 3158 ThrIleThrThr GlyAlaProValThr TyrSerThrTyrGlyLys Phe cttgccgatggt ggttgctctgggggc gettatgacatcataata tgt 3206 LeuAlaAspGly GlyCysSerGlyGly AlaTyrAspIleIl..eIle Cys gatgagtgccat tcaactgactcgact acaatcttgggcatcggc aca 3254 AspGluCysHis SerThrAspSerThr ThrIleLeuGlyIleGly Thr gtcctggaccaa gcggagacggetgga gcgcggcttgtcgtgctc gcc 3302 ValLeuAspGln AlaGluThrAlaGly AlaArgLeuValValLeu Ala accgetacgcct ccgggatcggtcacc gtgccacacccaaacatc gag 3350 ThrAlaThrPro ProGlySerValThr ValProHisPrnAsnIle Glu gaggtggccctg tctaatactggagag atccccttctatggcaaa gcc 3398 GluValAlaLeu SerAsnThrGlyGlu IleProPheTyrGlyLys Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile at - - J
atc ccc att gaa gcc atc agg ggg gga agg cat ctc att ttc tgt cat 3446 -Ile Pro Ile Glu Ala Ile Arg Gly Gly Arg His Leu Ile Phe Cys His tcc aag aag aag tgc gac gag ctc gcc gca aag ctg tca ggc ctc gga 3994 Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly atc aac get gtg gcg tat tac cgg ggg ctc gat gtg tcc gtc ata cca 3542 Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro act atc gga gac gtc gtt gtc gtg gca aca gac get ctg atg acg ggc 3590 Thr Ile Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly tat acg ggc gac ttt gac tca gtg atc gac tgt aac aca tgt gtc acc 3638 Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr cag aca gtc gac ttc agc ttg gat ccc acc ttc acc att gag acg acg 3686 Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr acc gtg cct caa gac gca gtg tcg cgc tcg cag cgg cgg ggt agg act 3739 Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr ggc agg ggt agg aga ggc atc tac agg ttt gtg act ccg gga gaa cgg 3782 Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg ccc tcg ggc atg ttc gat tcc tcg gtc ctg tgt gag tgc tat gac gcg 3830 Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala ggc tgt get tgg tac gag ctc acc ccc gcc gag acc tcg gtt agg ttg 3878 Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu cgg gcc tac ctg aac aca cca ggg ttg ccc gtt tgc cag gac cac ctg 3926 Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu gag ttc tgg gag agt gtc ttc aca ggc ctc acc cat ata gat gca cac 3974 Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala His ttc ttg tcc cag acc aag cag gca gga gac aac ttc ccc tac ctg gta 4022 Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val gca tac caa gcc acg gtg tgc gcc agg get cag gcc cca cct cca tca 4070 Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser tgg gat caa atg tgg aag tgt ctc ata cgg ctg aaa cct acg ctg cac 4118 Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His ggg cca aca ccc ttg ctg tac agg ctg gga gcc gtc caa aat gag gtc 4166 Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val acc ctc acc cac ccc ata acc aaa tac atc atg gca tgc atg tcg get 4214 Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala gac ctg gag gtc gtc act agc acc tgg gtg ctg gtg ggc gga gtc ctt 4262 Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu gca get ctg gcc gcg tat tgc ctg aca aca ggc agt gtg gtc att gtg 9310 Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val ggt agg att atc ttg tcc ggg agg ccg,gcc att gtt ccc gac agg gag 4358 Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Ile Val Pro Asp Arg Glu ctt ctc tac cag gag ttc gat gaa atg gaa gag tgc gcc tcg cac ctc 4406 Leu Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His Leu cct tac atc gag cag gga atg cag ctc gcc gag caa ttc aag cag aaa 4454 Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu G.ln Phe Lys Gln Lys gcg ctc ggg tta ctg caa aca gcc acc aaa caa gcg gag get get get 9502 Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala ccc gtg gtg gag tcc aag tgg cga gcc~ctt gag aca ttc tgg gcg aag 4550 Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys cac atg tgg aat ttc atc agc ggg ata cag tac tta gca ggc tta tcc 4598 His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser act ctg cct ggg aac ccc gca ata gca tca ttg atg gca ttc aca gcc 9646 Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Ptie Thr Ala tct atc acc agc ccg ctc acc acc caa agt acc ctc ctg ttt aac atc 4694 Ser Ile Thr Ser Pro Leu Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile ttg ggg ggg tgg gtg get gcc caa ctc gcc ccc ccc agc gcc get tcg 4742 Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala Ser get ttc gtg ggc gcc qgc atc gcc ggt gcg get gtt ggc agc ata ggc 4790 Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly ctt ggg aag gtg ctt gtg gac att ctg gcg ggt tat gga gca gga gtg 4838 Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly,Tyr Gly Ala Gly Val gcc ggc gcg ctc gtg gcc ttt aag gtc aLg agc ggc gag atg ccc tcc 4886 Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser acc gag gac ctg gtc aat cta ctt cct gcc atc ctc gag gaa get agt 4934 Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Glu Glu Ala Ser gag gat gtc gtc tgc tgc tca atg tcc tac aca tgg aca ggc gcc ttg 4982 Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu gag ctg ctg ctg ctg ctg ctg ctg ggc ctg agg cta cag ctc tcc ctg 5030 Glu Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu ggc atc atc cca gtt gag gag gag aac.ccg gac ttc tgg aac cgc gag 5078 Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu gca gcc gag gcc ctg ggt gcc gcc aag aag ctg cag cct gca cag aca 5126 Ala Ala Glu Ala Leu Gly Ala Ala- Lys Lys Leu Gln Pro Ala Gln Thr gcc gcc aag aac ctc atc atc ttc ctg ggc gat ggg atg ggg gtg tct 5174 Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser acg gtg aca get gcc agg atc cta aaa ggg cag aag aag gac aaa ctg 5222 Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu ggg cct gag ata ccc ctg gcc atg gac cgc ttc cca tat gtg get ctg 5270 Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu tcc aag aca tac aat gta gac aaa cat gtg cca gac agt gga gcc aca 5318 Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr gcc acg gcc tac ctg tgc ggg gtc aag ggc aac ttc cag acc att ggc 5366 Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly ttg agt gca gcc gcc cgc ttt aac cag tgc aac acg aca cgc gg.c aac 5419 Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn gag gtc atc tcc gtg atg aat cgg gcc aag aaa gca ggg aag tca gtg 5962 Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val gga gtg gta acc acc aca cga gtg cag cac gcc tcg cca gcc ggc acc 5510 Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr tac gcc cac acg gtg aac~cgc aac tgg tac tcg gac gcc gac gtg cct 5558 Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro gcc tcg gcc cgc cag gag ggg tgc cag gac atc get acg cag ctc atc 5606 Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile tcc aac atg gac att gac gtg atc cta ggt gga ggc cga aag tac atg 5654 Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met ttt ccc atg gga acc cca gac cct gag tac cca gat gac tac agc caa 5702 Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln ggt ggg acc agg ctg gac ggg aag aat ctg gtg cag gaa tgg ctg gcg 5750 Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala aag cgc cag ggt gcc cgg tat gtg tgg aac cgc act gag ctg atg cag 5798 Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln get tcc ctg gac ccg tct gtg acc cat ctc atg ggt ctc ttt gag cct 5846 Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro gga gac atg aaa tac gag atc cac cga gac tcc aca ctg gac ccc tcc 5894 Gly Asp Met Lys Tyr Glu Ile His Arg Asp 5er Thr Leu Asp Pro Ser ctg atg gag atg aca gag get gcc ctg cgc ctg ctg agc agg aac ccc 5942 Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro 1585 1590 ~ 1595 cgc ggc ttc ttc ctc ttc gtg gag ggt ggt cgc ett gac cat ggt cat 5990 Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His cat gaa agc agg.gct tac cgg gca ctg act gag acg atc atg ttc gac 6038 His Glu Ser Arg Ala Tyr Arg Ala Leu Thr.Glu Thr Ile Met Phe Asp gac gcc att gag agg gcg ggc cag ctc acc agc gag gag gac acg ctg 6086 Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu G1u Asp Thr Leu agc ctc gtc act gcc gac cac tcc cac gtc ttc tcc ttc gga ggc tac 6134 Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr ccc ctg cga ggg agc tgc atc ttc ggg ctg gcc cct ggc aag gcc cgg 6182 Pro Leu Arg Gly Ser Cys Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg gac agg aag gcc tac acg gtc ctc cta tac gga aac ggt cca ggc tat 6230 Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr gtg ctc aag gac ggc gcc cgg ccg gat gtt acc gag agc gag agc ggg 6278 Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly io agc ccc gag tat cgg cag cag tca gca gtg ccc ctg gac gaa gag acc 6326 Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr cac gca ggc gag gac gtg gcg gtg ttc gcg cgc ggc ccg cag gcg cac 6379 His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His ctg gtt cac ggc gtg cag gag cag acc ttc ata gcg cac gtc atg gcc 6922 Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala ttc gcc gcc tgc ctg gag ccc tac acc gcc tgc gac ctg gcg ccc ccc 6470 Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro gcc ggc acc acc gac gcc gcg cac ccg ggt taacccgtgg tccccgcgtt 6520 Ala Gly Thr Thr Asp Ala Ala His Pro Gly gcttcctctg ctggccggga catcaggtgg cccccgctga attggaatcg atattgttac 6580 aacaccccaa catcttcgac gcgggcgtgg caggtcttcc cgacgatgac gccggtgaac 6690 ttcccgccgc cgttgttgtt ttggagcacg gaaagacgat gacggaaaaa gagatcgtgg 6700 attacgtcgc cagtcaagta acaaccgcga aaaagttgcg cggaggagtt gtgtttgtgg 6760 acgaagtacc gaaaggtctt accggaaaac tcgacgcaag aaaaatcaga gagatcctca 6820 taaaggccaa gaagggcgga aagtccaaat tgtaaaatgt aactgtattc agcgatgacg 6880 aaattcttag ctattgtaat actgcgatga gtggcagggc ggggcgtaat ttttttaagg 6940 cagttattgg tgcccttaaa cgcctggtgc tacgcctgaa taagtgataa taagcggatg 7000 aatggcagaa attcgccgga tctttgtgaa ggaaccttac ttctgtggtg tgacataatt 7060 ggacaaacta cctacagaga tttaaagctc taaggtaaat ataaaatttt taagtgtata 7120 atgtgttaaa ctactgattc taattgtttg tgtattttag attccaacct atggaactga 7180 tgaatgggag cagtggtgga atgcctttaa tgaggaaaac ctgttttgct cagaagaaat 7240 gccatctagt gatgatgagg ctactgctga ctctcaacat tctactcctc caaaaaagaa 7300 gagaaaggta gaagacccca aggactttcc ttcagaattg ctaagttttt tgagtcatgc 7360 tgtgtttagt aatagaactc ttgcttgctt tgctatttac accacaaagg aaaaagctgc 7420 actgctatac aagaaaatta tggaaaaata ttctgtaacc tttataagta ggcataacag 7480 ttataatcat aacatactgt tttttcttac tccacacaqg catagagtgt ctgctattaa 7590 taactatgct caaaaattgt gtacctttag ctttttaatt tgtaaagggg ttaataagga 7600 atatttgatg tatagtgcct tgactagaga tcataatcag ccataccaca tttgtagagg 7660 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 7720 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 7780 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 7840 tcatcaatgt atcttatcat gtctggatcc tctagagtcg acctgcaggc atgcaagctt 7900 ctcgagagta cttctagtgg atccctgcag ctcgagaggc ctaattaatt aagtcgacga 7960 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 8020 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 8080 aactatatcc ggagttaact cgacatatac tatatagtaa taccaatact caagactacg 8140 aaactgatac aatctcttat catgtgggta atgttctcga tgtcgaatag ccatatgccg 8200 gtagttgcga tatacataaa ctgatcacta attccaaacc cacccgcttt ttatagtaag 8260 tttttcaccc ataaataata aatacaataa ttaatttctc gtaaaagtag aaaatatatt 8320 ctaatttatt gcaeggtaag gaagtagaat cataaagaac agtgacggat cgatccccca 8380 agcttggaca caagacaggc ttgcgagata tgtttgagaa taccacttta tcccgcgtca 8440 gggagaggca gtgcgtaaaa agacgcggac tcatgtgaaa tactggtttt tagtgcgcca 8500 gatctctata atctcgcgca acctattttc ccctcgaaca ctttttaagc cgtagataaa 8560 caggctggga cacttcac atg agc gaa aaa tac atc gtc acc tgg gac atg 8611 Met Ser Glu Lys Tyr Ile Val Thr Trp Asp Met ttg cag atc cat gca cgt aaa ctc gca agc cga ctg atg cct tct gaa 8659 Leu Gln Ile His Ala Arg Lys Leu Ala Ser Arg Leu Met Pro ~Se'r GIu caa tgg aaa ggc att att gcc gta agc cgt ggc ggt ctg gta ccg ggt 8707 Gln Trp Lys G1y Ile Ile Ala Val Ser Arg Gly Gly Leu Val Pro Gly gcg tta ctg gcg cgt gaa ctg ggt att cgt cat gtc gat acc gtt tgt 8755 Ala Leu Leu Ala Arg Glu Leu Gly Ile Arg His Val Asp Thr Val Cys att tcc agc tac gat cac gac aac cag cgc gag ctt aaa gtg ctg aaa 8803 Ile Ser Ser Tyr Asp His Asp Asn Gln Arg Glu Leu Lys Val Leu Lys cgc gca gaa ggc gat ggc gaa ggc ttc atc gtt att gat gac ctg gtg 8851 Arg Ala Glu Gly Asp Gly Glu Gly Phe Ile Val Ile Asp Asp Leu Val gat acc ggt ggt act gcg gtt gcg att cgt gaa atg tat cca aaa gcg 8899 Asp Thr Gly Gly Thr Ala Val Ala Ile Arg Glu Met Tyr Pro Lys Ala cac ttt gtc acc atc ttc gca aaa ccg get ggt cgt ccg ctg gtt gat 8947 His Phe Val Thr Ile Phe Ala Lys Pro Ala Gly Arg Pro Leu Val Asp gac tat gtt gtt gat atc ccg caa gat acc tgg att gaa cag ccg tgg 8995 Asp Tyr Val Val Asp Ile Pro Gln Asp Thr Trp Ile Glu Gln Pro Trp gat atg ggc gtc gta ttc gtc ccg cca atc tcc ggt cgc taatcttttc 9049 Asp Met Gly Val Val Phe Val Pro Pro Ile Ser Gly Arg aacgcctggc actgccgggc gttgttcttt ttaacttcag gcgggttaca atagtttcca 9109 gtaagtattc tggaggctgc atccatgaca caggcaaacc tgagcgaaac cctgttcaaa 9169 ccccgcttta aacatcctga aacctcgacg ctagtccgcc gctttaatca cggcgcacaa 9224 ccgcctgtgc agtcggccct tgatggtaaa accatccctc actggtatcg catgattaac 9284 cgtctgatgt ggatctggcg cggcattgac ccacgcgaaa tcctcgacgt ccaggcacgt 9344 attgtgatga gcgatgccga acgtaccgac gatgatttat acgatacggt gattggctac 9404 cgtggcggca actggattta tgagtgggcc ccggatcttt gtgaaggaac cttacttctg 9464 tggtgtgaca taattggaca aactacctac agagatttaa agctctaagg taaatataaa 9524 atttttaagt gtataatgtg ttaaactact gattctaatt gtttgtgtat tttagattcc 9584 aacctatgga actgatgaat gggagcagtg gtggaatgcc tttaatgagg aaaacctgtt 9644 ttgctcagaa gaaatgccat ctagtgatga tgaggctact gctgactctc aacattctac 9704 tcctccaaaa aagaagagaa aggtagaaga ccccaaggac tttccttcag aattgctaag 9764 ttttttgagt catgctgtgt ttagtaatag aactcttgct tgctttgcta tttacaccac 9829 aaaggaaaaa gctgcactgc tatacaagaa aattatggaa aaatattctg taacctttat 9884 aagtaggcat aacagttata atcataacat actgtttttt cttactccac acaggcatag 9944 agtgtctgct attaataact atgctcaaaa attgtgtacc tttagctttt taatttgtaa 10004 aggggttaat aaggaatatt tgatgtatag tgccttgact agagatcata atcagccata 10069 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 10124 aacataaaat gaatgcaatt gttgttgtta agcttggggg aattgcatgc tccggatcga 10184 gatcaa ttc tgt gag cgt atg gca aac gaa gga aaa ata gtt ata gta 10232 Phe Cys Glu Arg Met Ala Asn Glu Gly Lys Ile Val Ile Val gcc gca ctc gat ggg aca ttt caa cgt aaa ccg ttt aat aat att ttg 10280 Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Asn Asn Ile Leu aat ctt att cca tta tct gaa atg gtg gta aaa cta act get gtg tgt 10328 Asn Leu Ile Pro Leu Ser Glu Met Val Val Lys Leu Thr Ala Val Cys atg aaa tgc ttt aag gag get tcc ttt tct aaa cga ttg ggt gag gaa 10376 Met Lys Cys Phe Lys-Glu Ala Ser Phe Ser.Lys Arg Leu Gly Glu Glu 1'3 acc gag ata gaa ata ata gga ggt aat gat atg tat caa tcg gtg tgt 10424 Thr Glu Ile Glu Ile Ile Gly Gly Asn Asp Met Tyr Gln Ser Val Cys aga aag tgt tac atc gac tca taatattata ttttttatct aaaaaactaa 10475 Arg Lys Cys Tyr Ile Asp Ser aaataaacat tgattaaatt ttaatataat acttaaaaat ggatgttgtg tcgttagata 10535 aaccgtttat gtattttgag gaaattgata atgagttaga ttacgaacca gaaagtgcaa 10595 atgaggtcgc aaaaaaactg ccgtatcaag gacagttaaa actattacta ggagaattat 10655 tttttcttag taagttacag cgacacggta tattagatgg tgccaccgta gtgtatatag 10715 gatctgctcc cggtacacat atacgttatt tgagagatca tttctataat ttaggagtga 10775 tcatcaaatg gatgctaatt gacggccgcc atcatgatcc tattttaaat ggattgcgtg 10835 atgtgactct agtgactcgg ttcgttgatg aggaatatct acgatccatc aaaaaacaac 10895 tgcatcctte taagattatt ttaatttctg atgtgagatc caaacgagga ggaaatgaac 10955 ctagtacggc ggatttacta agtaattacg ctctacaaaa tgtcatgatt agtattttaa 11015 accccgtggc gtctagtctt aaatggagat gcccgtttcc agatcaatgg atcaaggact 11075 tttatatccc acacggtaat aaaatgttac aaccttttgc tccttcatat tcagggccgt 11135 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 11195 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 11255 acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg cattaagcgc 11315 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 11375 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 11435 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg,accccaaaaa 11495 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 11555 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 11615 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 11675 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 11735 tacaatttcc caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 11795 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 11855 taatattgaa aaaggaagag t atg agt att caa cat ttc cgt gtc gcc ctt 11906 Met Ser Ile Gln His Phe Arg Val Ala Leu WO pp/pg469 PCT/US99/17440 att ccc ttt ttt .gcg gca ttt tgc ctt cct gtt ttt get cac cca gaa 11954 Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu acg ctg gtg aaa gta aaa gat get gaa gat cag ttg ggt gca cga gtg 12002 Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val ggt tac atc gaa ctg gat ctc aac agc ggt aag atc ctt gag agt ttt 12050 Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe cgc ccc gaa gaa cgt ttt cca atg atg agc act ttt aaa gtt ctg cta 12098 Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu tgt ggc gcg gta tta tcc cgt att gac.gcc ggg caa gag caa ctc ggt 12146 Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly cgc cgc ata cac tat tct cag aat gac ttg gtt gag tac tca cca gtc 12194 Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa tta tgc agt 12292 Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser get gcc ata acc atg agt gat aac act gcg gcc aac tta ctt ctg aca 12290 Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr acg atc'gga gga ccg aag gag cta acc get ttt ttg cac aac atg ggg 12338 Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc 12386 Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala ata cca aac gac gag cgt gac acc acg atg cct gta gca atg gca aca 12439 Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr acg ttg cgc aaa cta tta act ggc gaa cta ctt act cta get tcc cgg 12982 Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg caa caa tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt 12530 Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu ctg cgc tcg gcc ctt ccg get ggc tgg ttt att get gat aaa tct gga 12578 Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Aia Asp Lys Ser Gly gcc ggt gag cgt ggg tct cgc ggt atc att gca gca ctg ggg cca gat 12626 Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp - - WO 00/08469 PCT/tJS99117440 ggt aag ccc tcc cgt atc gta gtt atc tac acg acg ggg agt cag gca 12674 Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly Ser Gln Ala act atg gat gaa cga aat aga cag atc get gag ata ggt gcc tca ctg 12722 Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu att aag cat tgg taactgtcag accaagttta ctcatatata ctttagattg 12774 Ile Lys His Trp atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 12834 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 12894 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 12954 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 13014 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 13074 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 13139 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 13194 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 13259 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 13314 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 13374 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 13434 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 13494 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 13559 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 13614 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 13674 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 13734 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 13794 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 13854 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgcc 13910 <210> 2 <211> 2307 <212> PRT
<213> Artificial Sequence <400> 2 Met Asn Gly Gly His Ile Gln Leu Ile Ile Gly Pro Met Phe Ser Gly Lys Ser Thr Glu Leu Ile Arg Arg Val Arg Arg Tyr Gln Ile Ala Gln Tyr Lys Cys Val Thr Ile Lys Tyr Ser Asn Asp Asn Arg Tyr Gly Thr Gly Leu Trp Thr His Asp Lys Asn Asn Phe Glu Ala Leu Glu Ala Thr Lys Leu Cys Asp Val Leu Glu Ser Ile Thr Asp Phe Ser Val Ile Gly Ile Asp Giu Gly Gln Phe Phe Pro Asp Ile Val Glu Met Gly Ile Pro Gln Phe Met Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala i15 120 125 Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Thr Thr Arg Ala Glu Ala His Leu His Val Trp Ile Pro Pro Leu Asn Ala Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Ile Ala Ile Leu Gly Pro Leu Met Val . 260 265 270 Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile His Ala Cys Met Leu Val Arg Lys Val Ala Gly Gly His Tyr Val Gln Met Ala Phe Met Lys Leu Gly Ala Leu Thr Gly Thr Tyr Ile Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Leu Glu G1y Arg Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val 920 925 . 430 Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn.Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile _. _ WO Op/08469 PCT/US99/17440 _ ' Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Ala Ile Arg Gly Giy Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ile Gly Asp Val V~1 Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro~Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala IIe Val Pro Asp Arg Glu Leu Leu 1060 1065 ~ 1070 Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Glu Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr Ala Thr 1380 1385 . 1390 Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly Leu Ser Ala Aia Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro Leu Arg Gly Ser Cys Ile Phe Gly Leu Ala Pro Gly Lys Aia Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Va1 Thr Glu Ser Glu Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro Ala Gly Thr Thr Asp Ala Ala His Pro Gly Met Ser Glu Lys Tyr Ile Val Thr Trp Asp Met Leu Gln Ile His Ala Arg Lys Leu Ala Ser Arg Leu Met Pro Ser G1u Gln Trp Lys Gly Ile Ile Ala Val Ser Arg Gly Gly Leu Val Pro Gly Ala Leu Leu Ala Arg Glu Leu Gly Ile Arg His Val Asp Thr Val Cys Ile Ser Ser Tyr Asp His Asp Asn Gln Arg Glu Leu Lys Val Leu Lys Arg Ala Glu Gly Asp Gly Glu Gly Phe Ile Val Ile Asp Asp Leu Val Asp Thr Gly Gly Thr Ala Val Ala Ile Arg Glu Met Tyr Pro Lys Ala His Phe Val Thr Ile Phe Ala Lys Pro Ala Gly Arg Pro Leu Val Asp Asp Tyr Val Val Asp Ile Pro Gln Asp Thr Trp Ile Glu Gln Pro Trp Asp Met Gly Val Val Phe Val Pro Pro Ile Ser Gly Arg Phe Cys Glu Arg Met Ala Asn Glu Gly Lys Ile Val Ile Val Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Asn Asn Ile Leu Asn Leu Ile Pro Leu Ser Glu Met Val Val Lys Leu Thr Ala Val Cys Met Lys Cys Phe Lys Glu Ala Ser Phe Ser Lys Arg Leu Gly Glu Glu Thr Glu Ile Glu Ile Ile Gly Gly Asn Asp Met Tyr Gln Ser Val Cys Arg Lys Cys Tyr Ile Asp Ser Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile G1u Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu.Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly Ser Gln Ala.Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp <210> 3 <211> 92 <212> PRT
<213> Artificial Sequence .
<400> 3 Met Asn Gly Gly His Ile Gln Leu Ile Ile Gly Pro Met Phe Ser Gly Lys Ser Thr Glu Leu Ile Arg Arg Val Arg Arg Tyr Gln Ile Ala Gln Tyr Lys Cys Val Thr Ile Lys Tyr Ser Asn Asp Asn Arg Tyr Gly Thr Gly Leu Trp Thr His Asp Lys Asn Asn Phe Glu Ala Leu Glu Ala Thr Lys Leu Cys Asp Val Leu Glu Ser Ile Thr Asp Phe Ser Val Ile Gly Ile Asp Glu Gly Gln Phe Phe Pro Asp Ile Val Glu <210> 4 <211> 1692 <212> PRT
<213> Artificial Sequence <400> 9 Met Gly Ile Pro Gln Phe Met Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe' Thr Thr Arg Ala Glu Ala His Leu His Val Trp Ile Pro Pro Leu Asn Ala Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Ile Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile His Ala Cys Met Leu Val Arg Lys Val Ala Gly Gly His Tyr Val Gln Met Ala Phe Met Lys Leu Gly Ala Leu Thr Gly Thr Tyr Ile Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser ' 245 250 255 Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Leu Glu Gly Arg Gly Trp Arg Leu 290 ~ 295 300 Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro 485 490 ' 495 Ala Vai Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly 500 505 . 510 Ser Gly Lys Sex Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn I1e Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Ala Ile Arg Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ile Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly 770 775 780 ' Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu Glu Val.Va1 Thr Ser Thr T~rp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Ile Val Pro Asp Arg Glu Leu Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala 1090 logs lloo Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met 1140 1145 . 1150 Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Glu Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp ' 28 Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe 1460 1465 . 1470 Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro Leu Arg Gly Ser Cys Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro Ala Gly Thr Thr Asp Ala Ala His Pro Gly _ _ WO pp/08469 PCTIITS99/17440 <210> 5 <211> 152 <212> PRT
<213> Artificial Sequence <400> 5 Met Ser Glu Lys Tyr Ile Val Thr Trp Asp Met Leu Gln Ile His Ala Arg Lys Leu Ala Ser Arg Leu Met Pro Ser Glu Gln Trp Lys Gly Ile 20 25 30' Ile Ala Val Ser Arg Gly Gly Leu Val Pro Gly Ala Leu Leu Ala Arg Glu Leu Gly Ile Arg His Val Asp Thr Val Cys Ile Ser Ser Tyr Asp 50 55 , 60 His Asp Asn Gln Arg Glu Leu Lys Val Leu Lys Arg Ala Glu Gly Asp Gly Glu Gly Phe Ile Val Ile Asp Asp Leu Val Asp Thr Gly Gly Thr Ala Val Ala Ile Arg Glu Met Tyr Pro Lys Ala His Phe Val Thr Ile Phe Ala Lys Pro Ala Gly Arg Pro Leu Val Asp Asp Tyr Val Val Asp Ile Pro Gln Asp Thr Trp Ile Glu Gln Pro Trp Asp Met Gly Val Val Phe Val Pro Pro Ile Ser Gly Arg <210> 6 <211> 85 <212> PRT
<213> Artificial Seauence <900> 6 Phe Cys Glu Arg Met Ala Asn Glu Gly Lys Ile Val Ile Val Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Asn Asn Ile Leu Asn Leu Ile Pro Leu Ser Glu Met Val Val Lys Leu Thr Ala Val Cys Met Lys Cys Phe Lys Glu Ala Ser Phe Ser Lys Arg Leu Gly Glu Glu Thr Glu Ile Glu Ile Ile Gly Gly Asn Asp Met Tyr Gln Ser Val Cys Arg Lys Cys Tyr Ile Asp Ser <210> 7 <211> 286 <2I2> PRT
<213> Artificial Sequence <400> 7 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 i5 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys Asp Ala G1u Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Giu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Aia Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu Asg Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp <210> 8 <211> 13910 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: plasmid phcap 3 <220>
<221> CDS
<222> (997)..(772) <220>
<221> CDS
<222> (1925)..(6500) <220>
<221> CDS
<222> (8579)..(9039) <220>
<221> CDS
<222> (10191)..(10445) <220>
<221> CDS
<222> (11877)..(12734) <220>
<221> misc_feature <222> (1). (779) <223> Vaccinia Virus thymidine Kinase gene recombination site <220>
<221> promoter <222> (794)..(816) <223> T7 promoter <220>
<221> misc_feature <222> (896)..(1424) <223> EMC/Internal Ribosome Entry Site (IRES) <220>
<221> misc_feature <222> (1426)..(1437) <223> MCS (Multiple Cloning Site) <220>
<221> misc_feature <222> (1446)..(2318) <223> IiCV E2/ NS2 domain <220>
<221> misc_feature <222> (2319)..(4231) <223> HCV NS3 Domain containing the serine protease and helicase enzymes <220>
<221> misc_feature <222> (4203)..(4260) <223> HCV NS3-NS4A cleavage site <220>
<221> misc_feature <222> (4375)..(4424) <223> HCV NS4A-4B clevage site .
<220>
<221> misc_feature <222> (4233)..(4399) <223> HCV NS4A domain <220>
<221> misc_feature <222> (4395)..(4919) <223> HCV NS4B Domain <220>
<221> misc_feature <222> (4920)..(4991) <223> HCV NSSA-NSSH cleavage site <220>
<221> misc_feature <222> (9992)..(6501) <223> SEAP Protein <220>
<221> misc_feature <222> (7915)..(7945) <223> MCS (Multiple Cloning Site) <220>
<221> terminator <222> (7938)..;8078) <223> term T7 <220>
<221> promoter <222> (8080)..(8365) <223> Vacinina virus promoter; early/late promoter <220>
<221> misc_feature <222> (8560)..(11317) <223> E. coli gpt; for selection of recombinants <220>
<221> misc_feature <222> (11318)..(13909) <223> remaining DNA from 3' end of Tropix pCMV/SEAP
plasmid <400> B
aagcttttgc gatcaataaa tggatcacaa ccagtatctc ttaacgatgt tcttcgcaga 60 tgatgattca ttttttaagt atttggctag tcaagatgat gaatcttcat tatctgatat 120 attgcaaatc actcaatatc tagactttct gttattatta ttgatccaat caaaaaataa 180 attagaagcc gtgggtcatt gttatgaatc tctttcagag gaatacagac aattgacaaa 240 attcacagac tttcaagatt ttaaaaaact gtttaacaag gtccctattg ttacagatgg 300 aagggtcaaa cttaataaag gatatttgtt cgactttgtg attagtttga tgcgattcaa 360 aaaagaatcc tctctagcta ccaccgcaat agatcctgtt agatacatag atcctcgtcg 420 caatatcgca ttttctaacg tgatggatat attaaagtcg aataaagtga acaataatta 980 attctttatt gtcatc atg aac ggc gga cat att cag ttg ata atc ggc ccc 532 Met Asn Gly Gly His Ile Gln Leu Ile Ile Gly Pro atg ttt tca ggt aaa agt aca gaa tta att aga cga gtt aga cgt tat 580 Met Phe Ser Gly Lys Ser Thr Glu Leu Ile Arg Arg Val Arg Arg Tyr caa ata get caa tat aaa tgc gtg act ata aaa tat tct aac gat aat 628 Gln Ile Ala Gln Tyr Lys Cys Val Thr Ile Lys Tyr Ser Asn Asp Asn aga tac gga acg gga cta tgg acg cat gat aag aat aat ttt gaa gca 676 Arg Tyr Gly Thr Gly Leu Trp Thr His Asp Lys Asn Asn Phe Glu Ala ttg gaa gca act aaa cta tgt gat gtc ttg gaa tca att aca gat ttc 724 Leu Glu Ala Thr Lys Leu Cys Asp Val Leu Glu Ser Ile Thr Asp Phe tcc gtg ata ggt atc gat gaa gga cag ttc ttt cca gac att gtt gaa 772 Ser Val Ile Gly Ile Asp Glu Gly Gln Phe Phe Pro Asp Ile Val Glu ttgatctcga tcccgcgaaa ttaatacgac tcactatagg gagaccacaa cggtttccct 832 ctagcgggat caattccgcc cctctccctc ccccccccct aacgttactg gccgaagccg 892 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt 952 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct 1012 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct 1072 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc 1132 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc 1192 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc 1252 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc 1312 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaacgtcta 1372 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgataata cc atg gga 1430 Met Gly -- WO 00/08469 PCf/US99/17440 att ccc caa ttc atg gca cgt gtc tgt gcc tgc ttg tgg atg atg ctg 1478 Ile Pro Gln Phe Met Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu ctg ata gcc cag gcc gag gcc gcc ttg gag aac ctg gtg gtc ctc aat- 1526 Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn gcg gcg tct gtg gcc ggc gca cat ggc atc ctc tcc ttc ctt gtg ttc 1574 Ala Ala Ser Val Ala G1y Ala His Gly Ile Leu Ser Phe Leu Val Phe ttc tgt gcc gcc tgg tac atc aaa ggc agg cLg gtc cct ggg gcg gca 1622 Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala tat get ctt tat ggc gtg tgg ccg ctg ctc ctg ctc ttg ctg gca tta 1670 Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu cca ccg cga get tac gcc atg gac cgg gag atg get gca tcg tgc gga 1718 Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly ggc gcg gtt ttt gtg ggt ctg gta ctc ctg act ttg tca cca tac tac 1766 Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser Pro Tyr Tyr aag gtg ttc ctc get agg ctc ata tgg tgg tta caa tat ttt acc acc 1814 Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Thr Thr aga gcc gag gcg cac tta cat gtg tgg atc ccc ccc ctc aac get cgg 1862 Arg Ala Glu Ala His Leu His Val Trp Ile Pro Pro Leu Asn Ala Arg gga ggc cgc gat gcc atc atc ctc ctc atg tgc gca gtc cat cca gag 1910 Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu cta atc ttt gac atc acc aaa ctt cta att gcc ata ctc ggt ccg ctc 1958 Leu Ile Phe Asp Ile Thr Lys Leu Leu Ile Ala Ile Leu G1"y Pro Leu atg gtg c~c caa get ggc ata acc aga gtg ccg tac ttc gtg cgc get 2006 Met Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala caa ggg ctc att cat gca tgc atg tta gtg cgg aag gtc get ggg ggt 2054 Gln Gly Leu Ile His Ala Cys Met Leu Val Arg Lys Val Ala Gly Gly cat tat gtc caa atg gcc ttc atg aag ctg ggc gcg ctg aca ggc acg 2102 His Tyr Val Gln Met Ala Phe Met Lys Leu Gly Ala Leu Thr Gly Thr tac att tac aac cat ctt acc ccg cta cgg gat tgg gcc cac gcg ggc 2150 Tyr Ile Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala His Ala Gly cta cga gac ctt gcg gtg gca gtg gag ccc gtc gtc ttc tcc gac atg 2198 Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Asp Met gag acc aag atc atc acc tgg gga gca gac acc gcg gcg tgt ggg gac 2246 Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp atc atc ttg ggt ctg ccc gtc tcc gcc cga agg gga aag gag ata ctc 2294 Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu ctg ggc ccg gcc gat agt ctt gaa ggg cgg ggg tgg cga ctc ctc gcg 2342 Leu Gly Pro Ala Asp Ser Leu Glu Gly Arg Gly Trp Arg Leu Leu Ala ccc atc acg gcc tac tcc caa cag acg cgg ggc cta ctt ggt tgc atc 2390 Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile atc act agc ctt aca ggc cgg gac aag aac cag gtc gag gga gag gtt 2438 Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val cag gtg gtt tcc acc gca aca caa tcc ttc ctg gcg acc tgc gtc aac 2486 Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn ggc gtg tgt tgg acc gtt tac cat ggt get ggc tca aag acc tta gcc 2539 Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu Ala ggc cca aag ggg cca atc acc cag atg tac act aat gtg'gac cag gac 2582 Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp ctc gtc ggc tgg cag gcg ccc ccc ggg gcg cgt tcc ttg aca cca tgc 2630 Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys acc tgt ggc agc tca gac ctt tac ttg gtc acg aga cat get gac gtc 2678 Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His A1a Asp Val att ccg gtg cgc cgg cgg ggc gac agt agg ggg agc ctg ctc tcc ccc 2726 Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro agg cct gtc tcc tac ttg aag ggc tct gcg ggt ggt cca ctg ctc tgc 2779 Arg Pro Val Ser Tyr Leu Lys Gly Ser Ala Gly Gly Pro Leu Leu Cys cct tcg ggg cac get gtg ggc atc ttc cgg get gcc gta tgc acc cgg 2822 Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg ggg gtt gcg aag gcg gtg gac ttt gtg ccc gta gag tcc atg gaa act 2870 Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr . _. _ WO 00/08469 PGT/US99117440 act atg cgg tct ccg gtc ttc acg gac aac tca tcc ccc ccg gcc gta 2918 Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val ccg cag tca ttt caa gtg gcc cac cta cac get ccc act ggc agc ggc 2966 Pro GIn Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly aag agt act aaa gtg ccg get gca tat gca gcc caa ggg tac aag gtg 3019 Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val ctc gtc ctc aat ccg tcc gtt gcc get acc tta ggg ttt ggg gcg tat 3062 Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr atg tct aag gca cac ggt att gac ccc aac atc aga act ggg gta agg 3110 Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg acc att acc aca ggc gcc ccc gtc aca tac tct acc tat ggc aag ttt 3158 Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr Gly Lys Phe 655 660 ~ 665 670 ctt gcc gat ggt ggt tgc tct ggg ggc get tat gac atc ata ata tgt 3206 Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys gat gag tgc cat tca act gac tcg act aca atc ttg ggc atc ggc aca 3254 Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr gtc ctg gac caa gcg gag acg get gga gcg cgg ctt gtc gtg ctc gcc 3302 Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala acc get acg cct ccg gga tcg gtc acc gtg cca cac cca aac atc gag 3350 Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu gag gtg gcc ctg tct aat act gga gag atc ccc ttc tat ggc aaa gcc 3398 Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr G7.y Lys Ala atc ccc att gaa gcc atc agg ggg gga agg cat ctc att ttc tgt cat 3446 Ile Pro Ile Glu Ala Ile Arg Gly Gly Arg His Leu Ile Phe Cys His tcc aag aag aag tgc gac gag ctc gcc gca aag ctg tca ggc ctc gga 3494 Ser Lys Lys Lys Cys Asp GIu Leu Ala Ala Lys Leu Ser Gly Leu Gly atc aac get gtg gcg tat tac cgg ggg ctc gat gtg tcc gtc ata cca 3542 Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro act atc gga gac gtc gtt gtc gtg gca aca gac get ctg atg acg ggc 3590 Thr Ile Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly tat acg ggc gac ttt gac tca gtg atc gac tgt aac aca tgt gtc acc 3638 Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr cag aca gtc gac ttc agc ttg gat ccc acc ttc acc att gag acg acg 3686 Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr acc gtg cct caa gac gca gtg tcg cgc tcg cag cgg cgg ggt agg act 3734 Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr ggc agg ggt agg aga ggc atc tac agg ttt gtg act ccg gga gaa cgg 3782 Gly Arg GIy Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg ccc tcg ggc atg ttc gat tcc tcg gtc ctg tgt gag tgc tat gac gcg 3830 Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala ggc tgt get tgg tac gag ctc acc ccc gcc gag acc tcg gtt agg ttg 3878 Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu cgg gcc tac ctg aac aca cca ggg ttg ccc gtt tgc cag gac cac ctg 3926 Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu gag ttc tgg gag agt gtc ttc~aca ggc ctc acc cat ata gat gca cac 3974 Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala His ttc ttg tcc cag acc aag cag gca gga gac aac ttc ccc tac ctg~gta 4022 Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val gca tac caa gcc acg gtg tgc gcc agg get cag gcc cca cct cca tca 4070 Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser tgg gat caa atg tgg aag tgt ctc ata cgg ctg aaa cct acg ctg cac 4118 Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His ggg cca aca ccc ttg ctg tac agg ctg gga gcc gtc caa aat gag gtc 4166 Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val acc ctc acc cac ccc ata acc aaa tac atc atg gca tgc atg tcg get 4214 Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala gac ctg gag gtc gtc act agc acc tgg gtg ctg gtg ggc gga gtc ctt 4262 Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu gca get ctg gcc gcg tat tgc ctg aca aca ggc agt gtg gtc att gtg 4310 Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val 1040 1045 lOSO

WO ~~~9 PCT/US99/I74W0 ggt agg att atc ttg tcc ggg agg ccg gcc att gtt ccc gac agg gag 4358 Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Ile Val Pro Asp Arg Giu ctt ctc tac cag gag ttc gat gaa atg gaa gag tgc gcc tcg cac ctc 9906 Leu Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His Leu cct tac atc gag cag gga atg cag ctc gcc gag caa ttc aag cag aaa 9459 Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln Lys gcg ctc ggg tta ctg caa aca gcc acc aaa caa gcg gag get get get 4502 Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala ccc gtg gtg gag tcc aag tgg cga gcc ~ctt gag aca ttc tgg gcg aag 4550 Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys cac atg tgg aat ttc atc agc ggg ata cag tac tta gca ggc tta tcc 4598 His Met Trp Asn Phe Ile Sex Gly Ile Gln Tyr Leu Ala Gly Leu Ser act ctg cct ggg aac ccc gca ata gca tca ttg atg gca ttc aca gcc 9646 Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala tct atc acc agc ccg ctc acc acc caa agt acc ctc ctg ttt aac atc 9694 Ser Ile Thr Ser Pro Leu Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile ttg ggg ggg tgg gtg get gcc caa ctc gcc ccc ccc agc gcc get tcg 4742 Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala Ser get ttc gtg ggc gcc ggc atc gcc ggt gcg get gtt ggc agc ata ggc 4790 Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly ctt ggg aag gtg ctt gtg gac att ctg gcg ggt tat gga gca gga gtg 4838 Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ana Gly Val gcc ggc gcg ctc gtg gcc ttt aag gtc atg agc ggc gag atg ccc tcc 9886 Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser acc gag gac ctg gtc aat cta ctt cct gcc atc ctc gag gaa get agt 4934 Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu G1u Glu Ala Ser gag gat gtc gtc tgc tgc tca atg tcc tac aca tgg aca ggc gcc ttg 4982 Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu gag ctg ctg ctg ctg ctg ctg ctg ggc ctg agg cta cag ctc tcc ctg 5030 Glu Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu ggc atc atc cca gtt gag gag gag aac ccg gac ttc tgg aac cgc gag 5078 ' Gly Iie Ile Pro Val Glu Glu Glu Asr. Pro Asp Phe Trp Asn Arg GIu gca gcc gag gcc ctg ggt gcc gcc aag aag ctg cag cct gca cag aca 5126 Ala Ala Glu Ala Leu GIy Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr gcc gcc aag aac ctc atc atc ttc ctg ggc gat ggg atg ggg gtg tct 5174 Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser acg gtg aca get gcc agg atc cta aaa ggg cag aag aag gac aaa ctg 5222 Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu ggg cct gag ata ccc ctg gcc atg gac cgc ttc cca tat gtg get ctg 5270 Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu tcc aag aca tac aat gta gac aaa cat gtg cca gac agt gga gcc aca 5318 Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr gcc acg gcc tac ctg tgc ggg gtc aag ggc aac ttc cag acc att ggc 5366 Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly ttg agt gca gcc gcc cgc ttt aac cag tgc aac acg aca cgc ggc aac 5414 Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg GIy Asn gag gtc atc tcc gtg atg aat cgg gcc aag aaa gca~ggg aag tca gtg 5962 Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val gga gtg gta acc acc aca cga gtg cag cac gcc tcg cca gcc ggc acc 5510 Gly Val Val Thr Thr Thr Arg VaI Gln His Ala Ser Pro Ala Gly Thr tac gcc cac acg gtg aac cgc aac tgg tac tcg gac gcc gac gtg cct 5558 Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro gcc tcg gcc cgc cag gag ggg tgc cag gac atc get acg cag ctc atc 5606 Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile tcc aac atg gac att gac gtg atc cta ggt gga ggc cga aag tac atg 5659 Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met 1490. 1995 1500 ttt ccc atg gga acc cca gac cct gag tac cca gat gac tac agc caa 5702 Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln ggt ggg acc agg ctg gac ggg aag aat ctg gtg cag gaa tgg ctg gcg 5750 Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala aag cgc cag ggt gcc cgg tat gtg tgg aac cgc act gag ctg atg cag 5798 Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln get tcc ctg gac ccg tct gtg acc cat ctc atg ggt ctc ttt gag cct 5846 Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro gga gac atg aaa tac gag atc cac cga gac tcc aca ctg gac ccc tcc 5894 Gly Asp Met Lys Tyr Giu Ile His Arg Asp Ser Thr Leu Asp Pro Ser '_570 1575 1580 ctg atg gag atg aca gag get gcc ctg cgc ctg ctg agc agg aac ccc 5942 Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro cgc ggc ttc ttc ctc ttc gtg gag ggt ggt cgc atc gac cat ggt cat 5990 A~g Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His cat gaa agc agg get tac cgg gca ctg act gag acg atc atg ttc gac 6038 His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp gac gcc att gag agg gcg ggc cag ctc acc agc gag gag gac acg ctg 6086 Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu agc ctc gtc act gcc gac cac tcc cac gtc ttc tcc ttc gga ggc tac 6139 Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr ccc ctg cga ggg agc tgc atc ttc ggg ctg gcc cct ggc aag gcc cgg 6182 Pro Leu Arg Gly Ser Cys Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg gac agg aag gcc tac acg gtc ctc cta tac gga aac ggt cca ggc tat 6230 Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr gtg ctc aag gac ggc gcc cgg ccg gat gtt acc gag agc gag agc ggg 6278 Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly agc ccc gag tat cgg cag cag tca gca gtg ccc ctg gac gaa gag acc 6326 Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr cac gca ggc gag gac gtg gcg gtg ttc gcg cgc ggc ccg cag gcg cac 6374 His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His ctg gtt cac ggc gtg cag gag cag acc ttc ata gcg cac gtc atg gcc 6422 Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala ttc gcc gcc tgc ctg gag. ccc tac acc gcc tgc gac ctg gcg ccc ccc 6970 Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro . . I
gcc ggc acc acc gac gcc gcg cac ccg ggt taacccgtgg tccccgcgtt 6520 Ala Gly Thr Thr Asp Ala Ala His Pro Gly gcttcctctg ctggccggga catcaggtgg cccccgctga attggaatcg atattgttac 6580 aacaccccaa catcttcgac gcgggcgtgg caggtcttcc cgacgatgac gccggtgaac 6640 ttcccgccgc cgttgttgtt ttggagcacg gaaagacgat gacggaaaaa gagatcgtgg 6700 attacgtcgc cagtcaagta acaaccgcga aaaagttgcg cggaggagtt gtgtttgtgg 6760 acgaagtacc gaaaggtctt accggaaaac tcgacgcaag aaaaatcaga gagatcctca 6820 taaaggccaa gaagggcgga aagtccaaat tgtaaaatgt aactgtat~c agcgatgacg 6880 aaattcttag ctattgtaat actgcgatga gtggcagggc ggggcgtaat ttttttaagg 6940 cagttattgg tgcccttaaa cgcctggtgc tacgcctgaa taagtgataa taagcggatg 7000 aatggcagaa attcgccgga tctttgtgaa ggaaccttac ttctgtggzg tgacataatt 7060 ggacaaacta cctacagaga tttaaagctc taaggtaaat ataaaattt~_ taagtgtata 7120 atgtgttaaa ctactgattc taattgtttg tgtattttag attccaacct atggaactga 7180 tgaatgggag cagtggtgga atgcctttaa tgaggaaaac ctgttttgct cagaagaaat 7240 gccatctagt gatgatgagg ctactgctga ctctcaacat tctactcctc caaaaaagaa 7300 gagaaaggta gaagacccca aggactttcc ttcagaattg ctaagttttt tgagtcatgc 7360 tgtgtttagt aatagaactc ttgcttgctt tgctatttac accacaaagg aaaaagctgc 7420 actgctatac aagaaaatta tggaaaaata ttctgtaacc tttataagta ggcataacag 7480 ttataatcat aacatactgt tttttcttac tccacacagg catagagtgt ctgctattaa 7540 taactatgct caaaaattgt gtacctttag ctttttaatt tgtaaagggg ttaataagga 7600 atatttgatg tatagtgcct tgactagaga tcataatcag ccataccaca tttgtagagg 7660 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 7720 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 7780 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 7840 tcatcaatgt atcttatcat gtctggatcc tctagagtcg acctgcaggc atgcaagctt 7900 ctcgagagta cttctagtgg atccctgcag ctcgagaggc ctaattaatt aagtcgacga 7960 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 8020 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg~8080 aactatatcc ggagttaact cgacatatac tatatagtaa taccaatact caagactacg 8140 aaactgatac aatctcttat catgtgggta atgttctcga tgtcgaatag ccatatgccg 8200 gtaqttgcga tatacataaa~ctgatcacta attccaaacc cacccgcttt ttatagtaag 8260 tttttcaccc ataaataata aatacaataa ttaatttctc gtaaaagtag aaaatatatt 8320 ctaatttatt gcacggtaag gaagtagaat cataaagaac agtgacggat cgatccccca 8380 agcttggaca caagacaggc ttgcgagata tgtttgagaa taccacttta tcccgcgtca 8440 gggagaggca gtgcgtaaaa agacgcggac tcatgtgaaa tactggtttt tagtgcgcca 8500 gatctctata atctcgcgca acctattttc ccctcgaaca ctttttaagc cgtagataaa 8560 caggctggga cacttcac atg agc gaa aaa tac atc gtc acc tgg gac atg 8611 Met Ser Glu Lys Tyr Ile Val Thr Trp Asp Met ttg cag atc cat gca cgt aaa ctc gca agc cga ctg atg cct tct gaa 8659 Leu Gln Ile His Ala Arg Lys Leu Ala Ser Arg Leu Met Pro Ser Glu caa tgg aaa ggc att att gcc gta agc cgt ggc ggt ctg gta ccg ggt 8707 Gln Trp Lys Gly Ile Ile Ala Val Ser Arg Gly Gly Leu Val Pro Gly gcg tta ctg gcg cgt gaa ctg ggt att cgt cat gtc gat acc gtt tgt 8755 Ala Leu Leu Ala Arg Glu Leu Gly Ile Arg His Val Asp Thr Val Cys att tcc agc tac gat cac gac aac cag cgc gag ctt aaa gtg ctg aaa 8803 Ile Ser Ser Tyr Asp His Asp Asn Gln Arg Glu Leu Lys Val Leu Lys cgc gca gaa ggc gat ggc gaa ggc ttc atc gtt att gat gac ctg gtg 8851 Arg Ala Glu Gly Asp Gly Glu Gly'Phe Ile Val Ile Asp Asp Leu Val gat acc ggt ggt act gcg gtt gcg att cgt gaa atg tat cca aaa gcg 8899 Asp Thr Gly Gly Thr Ala Val Ala Ile Arg Glu Met Tyr Pro Lys Ala cac ttt gtc acc atc ttc gca aaa ccg get ggt cgt ccg ctg gtt gat 8947 His Phe Val Thr Ile Phe Ala Lys Pro Ala Gly Arg Pro Leu Val Asp 1895 1900 190,5 gac tat gtt gtt gat atc ccg caa gat acc tgg att gaa cag ccg tgg 8995 Asp Tyr Val Val Asp Ile Pro Gln Asp Thr Trp Ile Glu Gln Pro Trp gat atg ggc gtc gta ttc gtc ccg cca atc tcc ggt cgc taatcttttc 9044 Asp Met Gly Val Val Phe Val Pro Pro Ile Ser Gly Arg aacgcctggc actgccgggc gttgttcttt ttaacttcag gcgggttaca atagtttcca 9104 gtaagtattc tggaggctgc atccatgaca caggcaaacc tgagcgaaac cctgttcaaa 9164 ccccgcttta aacatcctga aacctcgacg ctagtccgcc gctttaatca cggcgcacaa 9224 ccgcctgtgc agtcggccct tgatggtaaa accatccctc actggtatcg catgattaac 9289 cgtctgatgt ggatctggcg cggcattgac ccacgcgaaa tcctcgacgt ccaggcacgt 9349 attgtgatga gcgatgccga acgtaccgac gatgatttat acgatacggt gattggctac 9404 cgtggcggca actggattta tgagtgggcc ccggatcttt gtgaaggaac cttacttctg 9464 tggtgtgaca taattggaca aactacctac agagatttaa agctctaagg taaatataaa 9524 atttttaagt gtataatgtg ttaaactact gattctaatt gtttgtgtat tttagattcc 9584 aacctatgga actgatgaat gggagcagtg gtggaatgcc tttaatgagg aaaacctgtt 9694 ttgctcagaa gaaatgccat ctagtgatga tgaggctact gctgactctc aacattctac 9744 tcctccaaaa aagaagagaa aggtagaaga ccccaaggac tttccttcag aattgctaag 9769 ttttttgagt catgctgtgt ttagtaatag aactcttgct tgctttgcta tttacaccac 9824 aaaggaaaaa gctgcactgc tatacaagaa aattatggaa aaatattccg taacctttat 9889 aagtaggcat aacagttata atcataacat actgtttttt cttactccac acaggcatag 9944 agtgtctgct attaataact atgctcaaaa attgtgtacc tttagctttt taatttgtaa 10009 aggggttaat aaggaatatt tgatgtatag tgccttgact agagatcata atcagccata 10069 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 10124 aacataaaat gaatgcaatt gttgttgtta agcttggggg aattgcatgc tccggatcga 10189 gatcaa ttc tgt gag cgt atg gca aac gaa gga aaa ata gtt ata gta 10232 Phe Cys Glu Arg Met Ala Asn Glu Gly Lys Ile Val Ile Val gcc gca ctc gat ggg aca ttt caa cgt aaa ccg ttt aat aat att ttg 10280 Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Asn Asn Ile Leu aat ctt att cca tta tct gaa atg gtg gta aaa cta act get gtg tgt 10328 Asn Leu Ile Pro Leu Ser Glu Met Val Val Lys Leu Thr Ala Val Cys atg aaa tgc ttt aag gag get tcc ttt tct aaa cga ttg ggt gag gaa 10376 Met Lys Cys Phe Lys Glu Ala Ser Phe Ser Lys Arg Leu Gly Glu Glu acc gag ata gaa ata ata gga ggt aat gat atg tat caa tcg gtg tgt 10424 Thr Glu Ile Glu Ile Ile Gly Gly Asn Asp Met Tyr Gln Ser Val Cys aga aag tgt tac atc gac tca taatattata ttttttatct aaaaaactaa 10475 Arg Lys Cys Tyr Ile Asp Ser aaataaacat tgattaaatt ttaatataat acttaaaaat ggatgttgtg tcgttagata 10535 aaccgtttat gtattttgag gaaattgata atgagttaga ttacgaacca gaaagtgcaa 10595 atgaggtcgc aaaaaaactg ccgtatcaag gacagttaaa actattacta ggagaattat 10655 tttttcttag taagttacag cgacacggta tattagatgg tgccaccgta gtgtatatag 10715 gatctgctcc cggtacacat atacgttatt tgagagatca tttctataat ttaggagtga 10775 tcatcaaatg gatgctaatt gacggccgcc atcatgatcc tattttaaat ggattgcgtg 10835 atgtgactct agtgactcgg ttcgttgatg aggaatatct acgatccatc aaaaaacaac 10895 tgcatccttc taagattatt ttaatttctg atgtgagatc caaacgagga ggaaatgaac 10955 ctagtacggc ggatttacta agtaattacg ctctacaaaa tgtcatgatt agtattttaa 11015 accccgtggc gtctagtctt aaatggagat gcccgtttcc agatcaatgg atcaaggact 11075 tttatatccc acacggtaat aaaatgttac aaccttttgc tccttcatat tcagggccgt 11135 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 11195 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 11255 acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg cattaagcgc 11315 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 11375 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 11435 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 11495 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 11555 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 11615 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 11675 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 11735 tacaatttcc caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 11795 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 11855 taatattgaa aaaggaagag t atg agt att caa cat ttc cgt gtc gcc ctt 11906 Met Ser Ile Gln His Phe Arg Val Ala Leu att ccc ttt ttt gcg gca ttt tgc ctt cct gtt ttt get cac cca gaa 11954 Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu acg ctg gtg aaa gta aaa gat get gaa gat cag ttg ggt gca cga gtg 12002 Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val ggt tac atc gaa ctg gat ctc aac agc ggt aag atc ctt gag agt ttt 12050 Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe cgc ccc gaa gaa cgt ttt cca atg atg agc act ttt aaa gtt ctg cta 12098 Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu tgt ggc gcg gta tta tcc cgt att gac gcc ggg caa gag caa ctc ggt 12146 Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly WO 00/08469 PCT/US99/1~440 cgc cgc ata cac tat tct cag aat gac ttg gtt gag tac tca cca gtc 12194 Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa tta tgc agt 12242 Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser get gcc ata acc atg agt gat aac act gcg gcc aac tta ctt ctg aca 12290 Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr acg atc gga gga ccg aag gag cta acc get ttt ttg cac aac atg ggg 12338 Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly gat cat gta act cgc ctt gat cgt tgg ,gaa ccg gag ctg aat gaa gcc 12386 Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala ata cca aac gac gag cgt gac acc acg atg cct gta gca atg gca aca 12434 Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr acg ttg cgc aaa cta tta act ggc gaa cta ctt act cta get tcc cgg 12982 Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg caa caa tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt 12530 Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu ctg cgc tcg gcc ctt ccg get ggc tgg ttt att get gat aaa tct gga 12578 Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly gcc ggt gag cgt ggg tct cgc ggt atc att gca gca ctg ggg cca gat 12626 Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp ggt aag ccc tcc cgt atc gta gtt atc tac acg acg ggg agt cag gca 12674 Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly Sar Gln Ala act atg gat gaa cga aat aga cag atc get gag ata ggt gcc tca ctg 12722 Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu att aag cat tgg taactgtcag accaagttta ctcatatata ctttagattg 12774 Ile Lys His Trp atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 12834 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 12894 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 12954 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 13014 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 13074 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 13139 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 13194 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 13254 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 13314 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 13374 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 13434 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 13494 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 13554 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 13614 ctgataccqc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 13674 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 13734 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 13799 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 13854 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgcc 13910 <210> 9 <211> 230.7 <212> PRT
<213> Artificial Sequence <400> 9 Met Asn Gly Gly His Ile Gln Leu Ile Ile Gly Pro Met Phe Ser Gly Lys Ser Thr Glu Leu Ile Arg Arg Val Arg Arg Tyr Gln Ile Ala Gln Tyr Lys Cys Val Thr Ile Lys Tyr Ser Asn Asp Asn Arg Tyr Gly Thr Gly Leu Trp Thr His Asp Lys Asn Asn Phe Glu Ala Leu Glu Ala Thr Lys Leu Cys Asp Val Leu Glu Ser Ile Thr Asp Phe Ser Val Ile Gly 65 70 75 ~ 80 Ile Asp Glu Gly Gln Phe Phe Pro Asp Ile Val Glu Met Gly Ile Pro Gln Phe Met Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met AIa Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser Pro Tyr 'Tyr Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Thr Thr Arg Ala Glu Ala His Leu His Val Trp Ile Pro Pro Leu Asn Ala Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Ile Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile His Ala Cys Met Leu Val Arg Lys Val Ala Gly Gly His Tyr Val Gln Met Ala Phe Met Lys Leu Gly Ala Leu Thr Gly Thr Tyr Ile Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Leu Glu Gly Arg Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ala Gly Gly Pro Leu Leu Cys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Ala Ile Arg Gly Gly Arg His Leu Ile Phe Cys His Ser Lys WO ~ro~9 PGTNS99/17440 Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ile Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro.Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met 5er Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Ile Val Pro Asp Arg Glu Leu Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Glu Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly Leu Ser _ _ Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly Val 425 1430 1935 1440.
Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro Ala Ser Ala Arg Gin Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro Leu Arg Gly Ser Cys Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His Ala - WO 00/08469 PC"T/US99/17440 Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu Val _ His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro Ala Gly Thr Thr Asp Ala Ala His Pro Gly Met Ser Glu Lys Tyr Ile Val Thr 178b 785 1790 Trp Asp Met Leu Gln Ile His Ala Arg Lys Leu Ala Ser Arg Leu Met Pro Ser Glu Gln Trp Lys Gly Ile ile Ala Val Ser Arg Gly Gly Leu Val Pro Gly Ala Leu Leu Ala Arg Glu Leu Gly Ile Arg His Val Asp Thr Val Cys Ile Ser Ser Tyr Asp His Asp Asn Gln Arg Glu Leu Lys Val Leu Lys Arg Ala Glu Gly Asp Gly Glu Gly Phe Ile Val Ile Asp Asp Leu Val Asp Thr Gly Gly Thr Ala Val Ala Ile Arg Glu Met Tyr Pro Lys Ala His Phe Val Thr Ile Phe Ala Lys Pro Ala Gly Arg Pro Leu Val Asp Asp Tyr Val Val Asp Ile Pro Gln Asp Thr Trp Ile Glu Gln Pro Trp Asp Met Gly Val Val Phe Val Pro Pro Ile Ser Gly Arg Phe Cys Glu Arg Met Ala Asn Glu Gly Lys Ile Val Ile Val Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Asn Asn Ile Leu Asn Leu Ile Pro Leu Ser Glu Met Val Val Lys Leu Thr Ala Val Cys Met Lys Cys Phe Lys Glu A1a Ser Phe Ser Lys Arg Leu Gly Glu Glu Thr Glu Ile Glu Ile Ile Gly Gly Asn Asp Met Tyr Gln Ser Val Cys Arg Lys Cys Tyr Ile Asp Ser Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys 2085 2090 . 2095 Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn_Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly w Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp <210> 10 <211> 92 <212> PRT
<213> Artificial Sequence <400> 10 Met Asn Gly Gly His Ile Gln Leu Ile Ile Gly Pro Met Phe Ser Gly Lys Ser Thr Glu Leu Ile Arg Arg Val Arg Arg Tyr Gln Ile Ala Gln Tyr Lys Cys Val Thr Ile Lys Tyr Ser Asn Asp Asn Arg Tyr Gly Thr Gly Leu Trp Thr His Asp Lys Asn Asn Phe Glu Ala Leu Glu Ala Thr Lys Leu Cys Asp Val Leu Glu Ser Ile Thr Asp Phe Ser Val Ile Gly Ile Asp Glu Gly Gln Phe Phe Pro Asp Ile Val Glu <210> 11 <211> 1692 <212> PRT
<213> Artificial Sequence <400> 11 Met Gly Ile Pro Gln Phe Met Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Thr Thr Arg Ala Glu Ala His Leu His Val Trp Ile Pro Pro Leu Asn Ala Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Ile Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile His Ala Cys Met Leu Val Arg Lys Val Ala _ _ WO pp/084169 PGT/US99/17440 Gly Gly His Tyr Val Gln Met Ala Phe Met Lys Leu Gly Ala Leu Thr Gly Thr Tyr Ile Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Leu Glu Gly Arg Gly Trp Arg Leu 290 295 . 300 Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu w Ser Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ala Gly Gly Pro Leu Leu Cys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met 465 9?0 475 480 Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Ala Ile Arg Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu 5er Gly Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ile Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr.Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp - - WO 00/08469 PGT/US99/1?440 Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met 915 , 920 925 Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala I1e Val Pro Asp Arg Glu Leu Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp 025 1030 1035 1040 ' Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala G1y Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala I1e Leu Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Glu Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp 1250 1255 . 1260 Lys Leu Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val 265 . 1270 1275 1280 Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser VaI Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp 13?5 Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp - -Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro Leu Arg Gly Ser Cys Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala Phe Ala Ala .Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro Ala Gly Thr Thr Asp Ala Ala His Pro Gly <210> 12 <211> 152 <212> PRT
<213> Artificial Sequence <400> 12 Met Ser Glu Lys Tyr Ile Val Thr Trp Asp Met Leu Gln Ile His Ala Arg Lys Leu Ala Ser Arg Leu Met Pro Ser Glu Gln Trp Lys Gly Ile Ile Ala Val Ser Arg Gly Gly Leu Val Pro Gly Ala Leu Leu Ala Arg Glu Leu Gly Ile Arg His Val Asp Thr Val Cys Ile Ser Ser Tyr Asp His Asp Asn Gln Arg Glu Leu Lys Val Leu Lys Arg Ala Glu Gly Asp -- WO 00/0$469 PCT/US99/17440 Gly Glu Gly Phe Ile Val Ile Asp Asp Leu Val Asp Thr Gly Gly Thr -Ala Val Ala Ile Arg Glu Met Tyr Pro Lys Ala His Phe Val Thr Ile Phe Ala Lys Pro Ala Gly Arg Pro Leu Val Asp Asp Tyr Val Val Asp Ile Pro Gln Asp Thr Trp Ile Glu Gln Pro Trp Asp Met Gly Val Val Phe Val Pro Pro Ile Ser Gly Arg <210> 13 <211> 85 <212> PRT
<213> Artificial Sequence <900> 13 Phe Cys Glu Arg Met Ala Asn Glu Gly Lys Ile Val Ile Val Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Asn Asn Ile Leu Asn Leu Ile Pro Leu Ser Glu Met Val Val Lys Leu Thr Ala Val Cys Met Lys Cys Phe Lys Glu Ala Ser Phe Ser Lys Arg Leu Gly Glu Glu Thr Glu Ile Glu Ile Ile Gly Gly Asn Asp Met Tyr Gln Ser Val Cys Arg Lys Cys Tyr Ile Asp Ser <210> 14 <211> 286 <212> PRT
<213> Artificial Sequence <400> 14 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser . _ WO 00/08469 PC1'/US99/17440 Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val~Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg 165 ~ 170 175 Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu '":~r Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 210 215 ~ 220 Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile 245 . . . 250 255 Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu _Tle Gly Ala Ser Leu Ile Lys His Trp <210> 15 <211> 139.0 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: plasmid phcap 4 <220>
<221> CDS
<222> (497)..(772) <220>
<221> CDS
<222> (1425)..(6500) <220>
<221> CDS
<222> (8579)..(9034) i, s <220>
<221> CDS
<222> (10191)..(10445) <220>
<221> CDS .
<222> (11877)..(I2739) <220>
<221> misc_feature <222> (1). (774) <223> Vaccinia Virus thymidine Kinase gene recombination site <220>
<221> promoter <222> (799)..(816) <223> T7 promoter <220>
<221> misc_feature <222> (846)..(1424) <223> EMC/Internal Ribosome Entry Site (IRES) <220>
<221> misc_feature <222> (1426)..(1937) <223> MCS (Multiple Cloning Site) <220>
<221> misc_feature <222> (1496)..(2318) <223> HCV E2/ NS2 domain <220>
<221> misc_feature <222> (2319)..(9231) <223> HCV NS3 Domain containing the serine protease and helicase enzymes <220>
<221> misc_feature <222> (9203)..(4260) <223> HCV NS3-NS4A cleavage site <220>
<221> misc_feature <222> (9375)..(4429) <223> HCV NS4A-4B clevage site <220>
<221> misc_feature <222> (4233)..(9394) <223> HCV NS4A domain <220>
<221> misc_feature <222> (4395)..(4919) <223> HCV NS4B Domain <220>

<221> misc_feature <222> (4920)..(4991) <223> HCV NSSA-NSSB cleavage site <220>
<221> rnisc_feature <222> (4992)..(6501) <223> SEAP Protein <220>
<221> misc_feature <222> (7915)..(7945) <223> MCS (Multiple Cloning Site) <220>
<221> terminator <222> (7938)..(8078) <223> term T7 <220>
<221> promoter <222> (8080)..(8365) <223> Vacinina virus promoter; early/late promoter <220>
<221> misc_feature <222> (8560)..(11317) <223> E. coli gpt; for selection of recombinants <220>
<221> misc_feature <222> (11318)..(13909) .
<223> remaining DNA from 3' end of Tropix~pGMV/SEAP
plasmid <900> 15 aagcttttgc gatcaataaa tggatcacaa ccagtatctc ttaacgatgt tcttcgcaga 60 tgatgattca ttttttaagt atttggctag tcaagatgat gaatcttcat tatctgatat 120 attgcaaatc actcaatatc tagactttct gttattatta ttgatccaat caaaaaataa 180 attagaagcc gtgggtcatt gttatgaatc tctttcagag gaatacagac aattgacaaa 240 attcacagac tttcaagatt ttaaaaaact gtttaacaag gtccctattg ttacagatgg 300 aagggtcaaa cttaataaag gatatttgtt cgactttgtg attagtttga tgcgattcaa 360 aaaagaatcc tctctagcta ccaccgcaat agatcctgtt agatacatag atcctcgtcg 420 caatatcgca ttttctaacg tgatggatat attaaagtcg aataaagtga acaataatta 980 attctttatt gtcatc atg aac ggc gga cat att cag ttg ata atc ggc ccc 532 Met Asn Gly Gly His Ile Gln Leu Iie Ile Gly Pro atg ttt tca ggt aaa agt aca gaa tta att aga cga gtt aga cgt tat 580 Met Phe Ser Gly~Lys Ser Thr Glu Leu Ile Arg Arg Val Arg Arg Tyr 1.5 20 25 ' . i caa ata get caa tat aaa tgc gtg act ata aaa tat tct aac gat aat 628 Gln Ile Ala Gln Tyr Lys Cys Val Thr Ile Lys Tyr 5er Asn Asp Asn aga tac gga acg gga cta tgg acg cat gat aag aat aat ttt gaa gca 676 Arg Tyr Gly Thr Gly Leu Trp Thr His Asp Lys Asn Asn Phe Glu Ala ttg gaa gca act aaa cta tgt gat gtc ttg gaa tca att aca gat ttc 729 Leu Glu Ala Thr Lys Leu Cys Asp Val Leu Glu Ser Ile Thr Asp Phe tcc gtg ata ggt atc gat gaa gga cag ttc ttt cca gac att gtt gaa 772 Ser Val Ile Gly Ile Asp Glu Gly Gln Phe Phe Pro Asp Ile Val Glu ttgatctcga tcccgcgaaa ttaatacgac tcactatagg gagaccacaa cggtttccct 832 ctagcgggat caattccgcc cctctccctc ccccccccct aacgttactg gccgaagccg 892 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt 952 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct 1012 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct 1072 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc 1132 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc 1192 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc 1252 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc 1312 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaacgtcta 1372 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgataata cc atg gga 1430 Met Gly att ccc caa ttc atg gca cgt gtc tgt gcc tgc ttg tgg atg atg ctg 1978 Ile Pro Gln Phe Met Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu ctg ata gcc cag gcc gag gcc gcc ttg gag aac ctg gtg gtc ctc aat 1526 Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn gcg gcg tct gtg gcc ggc gca cat ggc atc ctc tcc ttc ctt gtg ttc 1574 Ala Ala Sez Val Ala Gly Ala His Gly Ile Leu Ser Phe Leu Val Phe ttc tgt gcc gcc tgg tac atc aaa ggc agg ctg gtc cct ggg gcg gca 1622 Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala tat get ctt tat ggc gtg tgg ccg ctg ctc ctg ctc ttg ctg gca tta 1670 Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu . . WO 00108469 PCT/US99/17440 cca ccg cga get tac gcc atg gac cgg gag atg get gca tcg tgc gga 1718 Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly ggc gcg gtt ttt gtg ggt ctg gta ctc ctg act ttg tca cca tac ta-c 1766 Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser Pro Tyr Tyr aag gtg ttc ctc get agg ctc ata tgg tgg tta caa tat ttt acc acc 1814 Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Thr Thr aga gcc gag gcg cac tta cat gtg tgg atc ccc ccc ctc~aac get cgg 1862 Arg Ala Glu Ala His Leu His Val Trp Ile Pro Pro Leu Asn Ala Arg gga ggc cgc gat gcc atc atc ctc ctc atg tgc gca gtc cat cca gag 1910 Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu cta atc ttt gac atc acc aaa ctt cta att gcc ata ctc ggt ccg ctc 1958 Leu Ile Phe Asp Ile Thr Lys Leu Leu Ile Ala Ile Leu Gly Pro Leu atg gtg ctc caa get ggc ata acc aga gtg ccg tac ttc gtg cgc get 2006 Met Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala caa ggg ctc att cat gca tgc atg tta gtg cgg aag gtc get ggg ggt 2054 Gln Gly Leu Ile His Ala Cys Met Leu Val Arg Lys Val Ala Gly Gly cat tat gtc caa atg gcc ttc atg aag ctg ggc gcg ctg aca ggc acg 2102 His Tyr Val Gln Met Ala Phe Met Lys Leu Gly Ala Leu Thr Gly Thr tac att tac aac cat ctt acc ccg cta cgg gat tgg gcc cac gcg ggc 2150 Tyr Ile Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala His Ala Gly cta cga gac ctt gcg gtg gca gtg gag ccc gtc gtc ttc tcc gac atg 2198 Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Asp Met gag acc aag atc atc acc tgg gga gca gac acc gcg gcg get ggg gac 2246 Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr Ala Ala Ala Gly Asp atc atc ttg ggt ctg ccc gtc tcc gcc cga agg gga aag gag ata ctc 2294 Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu ctg ggc ccg gcc gat agt ctt gaa ggg cgg ggg tgg cga ctc ctc gcg 2342 Leu Gly Pro Ala Asp Ser Leu Glu Gly Arg Gly Trp Arg Leu Leu Ala ccc atc acg gcc tac tcc caa cag acg cgg ggc cta ctt ggt tgc atc 2390 Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile _ _ WO 00/08469 PCT/US99/17440 atc act agc ctt aca ggc cgg gac aag aac cag gtc gag gga gag gtt 2438 Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val cag gtg gtt tcc acc gca aca caa tcc ttc ctg gcg acc tgc gtc aac 2986 Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn ggc gtg tgt tgg acc gtt tac cat ggt get ggc tca aag acc tta gcc 2534 Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu Ala ggc cca aag ggg cca atc acc cag atg tac act aat gtg gac cag gac 2582 Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp ctc gtc ggc tgg cag gcg ccc ccc ggg,gcg cgt tcc ttg aca cca tgc 2630 Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys acc tgt ggc agc tca gac ctt tac ttg gtc acg aga cat get gac gtc 2678 Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val att ccg gtg cgc cgg cgg ggc gac agt agg ggg agc ctg ctc tcc ccc 2726 Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro agg cct gtc tcc tac ttg aag ggc tct gcg ggt ggt cca ctg ctc tgc 2779 Arg Pro Val Ser Tyr Leu Lys Gly Ser Ala Gly Gly Pro Leu Leu Cys cct tcg ggg cac get gtg ggc atc ttc cgg get gcc gta tgc acc cgg 2822 Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg ggg gtt gcg aag gcg gtg gac ttt gtg ccc gta gag tcc atg gaa act 2870 Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr act atg cgg tct ccg gtc ttc acg gac aac tca tcc ccc ccg gcc gta 2918 Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val ccg cag tca ttt caa gtg gcc cac cta cac get ccc act ggc agc ggc 2966 Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly aag agt act aaa gtg ccg get gca tat gca gcc caa ggg tac aag gtg 3014 Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val ctc gtc ctc aat ccg tcc gtt gcc get acc tta ggg ttt ggg gcg tat 3062 Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr atg tct aag gca cac ggt att gac ccc aac atc aga act ggg gta agg ~ 3110 Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg -- WO 00/08469 PCT/US99/1'I440 acc att acc aca ggc gcc ccc gtc aca tac tct acc tat ggc aag ttt 3158 Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr Gly Lys Phe ctt gcc gat ggt ggt tgc tct ggg ggc get tat gac atc ata ata tgt 3206 Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys gat gag tgc cat tca act gac tcg act aca atc ttg ggc atc ggc aca 3254 Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile G1y Thr gtc ctg gac caa gcg gag acg get gga gcg cgg ctt gtc gtg ctc gcc 3302 Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala acc get acg cct ccg gga tcg gtc acc .gtg cca cac cca aac atc gag 3350 Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu gag gtg gcc ctg tct aat act gga gag atc ccc ttc tat ggc aaa gcc 3398 Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala atc ccc att gaa gcc atc agg ggg gga agg cat ctc att ttc tgt cat 3496 Ile Pro Ile Glu Ala Ile Arg Gly Gly Arg His Leu Ile Phe Cys His tcc aag aag aag tgc gac gag ctc gcc gca aag ctg tca ggc ctc gga 3499 Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly atc aac get gtg gcg tat tac cgg ggg ctc gat gtg tcc gtc ata cca ~ 3592 Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro act atc gga gac gtc gtt gtc gtg gca aca gac get ctg atg acg ggc 3590 Thr Ile Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly tat acg ggc gac ttt gac tca gtg atc gac tgt aac aca tgt gtc acc 3638 Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr cag aca gtc gac ttc agc ttg gat ccc acc ttc acc att gag acg acg 3686 Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr acc gtg cct caa gac gca gtg tcg cgc tcg cag cgg cgg ggt agg act 3734 Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr ggc agg ggt agg aga ggc atc tac agg ttt gtg act ccg gga gaa cgg 3782 Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg ccc tcg ggc atg ttc gat tcc tcg gtc ctg tgt gag tgc tat gac gcg 3830 Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Tyr Val Leu Lys As - . WO 00/08469 PCT/ITS99/I7440 ggctgtgettgg tacgagctcaccccc gccgagacctcggttagg ttg 3878 GlyCysAlaTrp TyrGluLeuThrPro AlaGluThrSerValArg Leu cgggcctacctg aacacaccagggttg cccgtttgccaggaccac ctg 3926 ArgAlaTyrLeu AsnThrProGlyLeu ProValCysGlnAspHis Leu gagttctgggag agtgtcttcacaggc ctcacccatatagatgca cac 3974 GluPheTrpGlu SerValPheThrGly LeuThrHisIleAspAla His ttcttgtcccag accaagcaggcagga gacaacttcccc~tacctg gta 9022 PheLeuSerGln ThrLysGlnAlaGly AspAsnPheProTyrLeu Val gcataccaagcc acggtgtgcgccagg getcaggccccacctcca tca 4070 AlaTyrGlnAla ThrValCysAlaArg AlaGlnAlaProProPro Ser tgggatcaaatg tggaagtgtctcata cggctgaaacctacgctg cac 4118 TrpAspGlnMet TrpLysCysLeuIle ArgLeuLysProThrLeu His gggccaacaccc ttgctgtacaggctg ggagccgtccaaaatgag gtc 4166 GlyProThrPro LeuLeuTyrArgLeu GlyAlaValGlnAsnGlu Val accctcacccac cccataaccaaatac atcatggcatgcatgtcg get 4214 ThrLeuThrHis ProIleThrLysTyr IleMetAlaCysMetSer Ala gacc-tg;gaggtc gtcactagcacctgg gtgctggtgggcggagtc ctt 9262 AspLeuGluVal ValThrSerThrTrp ValLeuValGlyGlyVal Leu gcagetctggcc gcgtattgcctgaca acaggcagtgtggtcatt gtg 4310 AlaAlaLeuAla AlaTyrCysLeuThr ThrGlySerValValIle Val ggtaggattatc ttgtccgggaggccg gccattgttcccgacagg gag 4358 GlyArgIleIle LeuSerGlyArgPro AlaIleValProAbpArg Glu cttctctaccag gagttcgatgaaatg gaagagtgcgcctcgcac ctc 4906 LeuLeuTyrGln GluPheAspGluMet GluGluCysAlaSerHis Leu ccttacatcgag cagggaatgcagctc gccgagcaattcaagcag aaa 4454 ProTyrIleGlu GlnGlyMetGlnLeu AlaGluGlnPheLysGln Lys gcgctcgggtta ctgcaaacagccacc aaacaagcggaggetget get 4502 AlaLeuGlyLeu LeuGlnThrAlaThr LysGlnAlaGluAlaAla Ala cccgtggtggag tccaagtggcgagcc cttgagacattctgggcg aag 4550 ProValValGlu SerLysTrpArgAla LeuGluThrPheTrpAla Lys wo oaros469 pcrius99n 7aao cac atg tgg aat ttc atc agc ggg ata cag tac tta gca ggc tta tcc 4598 His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser act ctg cct ggg aac ccc gca ata gca tca ttg atg gca ttc aca gcc 4646 Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala tct atc acc agc ccg ctc acc acc caa agt acc ctc ctg ttt aac atc 4694 Ser Ile Thr Ser Pro Leu Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile ttg ggg ggg tgg gtg get gcc caa ctc gcc ccc ccc agc gcc get tcg 4742 Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala Ser get ttc gtg ggc gcc ggc atc gcc ggt gcg get gtt ggc agc ata ggc 4790 Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly ctt ggg aag gtg ctt gtg gac att ctg gcg ggt tat gga gca gga gtg 9838 Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val gcc ggc gcg ctc gtg gcc ttt aag gtc atg agc ggc gag atg ccc tcc 9886 Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser acc gag gac ctg gtc aat cta ctt cct gcc atc ctc gag gaa get agt 9934 Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Glu Glu Ala Ser gag gat gtc gtc tgc tgc tca atg tcc tac aca tgg aca ggc gcc ttg 9982 Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu gag ctg ctg ctg ctg ctg ctg ctg ggc ctg agg cta cag ctc tcc ctg 5030 Glu Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu ggc atc atc cca gtt gag gag gag aac ccg gac ttc tgg aac cgc gag 5078 Gly Iie Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu gca gcc gag gcc ctg ggt gcc gcc aag aag ctg cag cct gca cag aca 5126 Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr gcc gcc aag aac ctc atc atc ttc ctg ggc gat ggg atg ggg gtg tct 5174 Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser aeg gtg aca get gcc agg atc eta aaa ggg cag aag aag gac aaa ctg 5222 Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu ggg cct gag ata cce ctg gcc atg gac cgc ttc cca tat gtg get ctg 5270 Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu tccaagacatac aatgtagacaaacatgtg ccagacagtgga gccaca 5318 SerLysThrTyr AsnValAspLysHisVal ProAspSerGly AlaThr gccacggcctac ctgtgcggggtcaagggc aacttccagacc attggc 5366 AlaThrAlaTyr LeuCysGlyValLysGly AsnPheGlnThr IleGly ttgagtgcagcc gcccgctttaaccagtgc aacacgacacgc ggcaac 5414 LeuSerAlaAla AlaArgPheAsnGlnCys AsnThrThrArg GlyAsn gaggtcatctcc gtgatgaatcgggccaag aaagcagggaag tcagtg 5462 GluValIleSer ValMetAsnArgAlaLys LysAlaGlyLys SerVal ggagtggtaacc accacacgagtgcagcac gcctcgccagcc ggcacc 5510 GlyValValThr ThrThrArgValGln'HisAlaSerProAla GlyThr tacgcccacacg gtgaaccgcaactggtac tcggacgccgac gtgcct 5558 TyrAlaHisThr ValAsnArgAsnTrpTyr SerAspAlaAsp ValPro gcctcggcccgc caggaggggtgccaggac atcgetacgcag ctcatc 5606 AlaSerAlaArg GlnGluGlyCysGlnAsp IleAiaThrGln LeuIle tccaacatggac attgacgtgatcctaggt ggaggccgaaag tacatg 5654 SerAsnMetAsp IleAspValIleLeuGly GlyGlyArgLys TyrMet tttcccatggga.accccagaccctgagtac ccagatgactac agccaa 5702 PheProMetGly ThrProAspProGluTyr ProAspAspTyr SerGln ggtgggaccagg ctggacgggaagaatctg gtgcaggaatgg ctggcg 5750 GlyGlyThrArg LeuAspGlyLysAsnLeu ValGlnGluTrp LeuAla aagcgccagggt gcccggtatgtgtggaac cgcactgagctg atgcag 5798 LysArgGlnGly AlaArgTyrValTrpAsn ArgThrGluLeu MetGln gettccctggac ccgtctgtgacccatctc atgggtctcttt gagcct 5896 AlaSerLeuAsp ProSerValThrHisLeu MetGlyLeuPhe GluPro ggagacatgaaa tacgagatccaccgagac tccacactggac ccctcc 5894 GlyAspMetLys TyrGluIleHisArgAsp SerThrLeuAsp ProSer ctgatggagatg acagaggetgccctgcgc ctgctgagcagg aacccc 5942 LeuMetGluMet ThrGluAlaAlaLeuArg LeuLeuSerArg AsnPro cgcggcttcttc ctcttcgtggagggtggt cgcatcgaccat ggtcat 5990 ArgGlyPhePhe LeuPheValGluGlyGly ArgIleAspHis GlyHis WO 00/08469 PC"T/US99/17440 cat gaaagcagg gettaccgggcactgact gagacgatcatgttc gac 6038 His GluSerArg AlaTyrArgAlaLeuThr GluThrIleMetPhe Asp gac gccattgag agggcgggccagctcacc agcgaggaggacacg ctg 6086 Asp AlaIleGlu ArgAlaGlyGlnLeuThr SerGluGluAspThr Leu agc ctcgtcact gccgaccactcccacgtc ttctccttcggaggc tac 6134 Ser LeuValThr AlaAspHisSerHisVal PheSerPheGlyGly Tyr ccc ctgcgaggg agctgcatcttcgggctg gcccctggcaaggcc cgg 6182 Pro LeuArgGly SerCysIlePheGlyLeu AlaProGlyLysAla Arg gac aggaaggcc tacacggtcctcctatac ggaaacggtccaggc tat 6230 Asp ArgLysAla TyrThrValLeuLeufiyrGlyAsnGlyProGly Tyr gtg ctcaaggac ggcgcccggccggatgtt accgagagcgagagc ggg 6278 Val LeuLysAsp GlyAlaArgProAspVal ThrGluSerGluSer Gly agc cccgagtat cggcagcagtcagcagtg cccctggacgaagag acc 6326 Ser ProGluTyr ArgGlnGlnSerAlaVal ProLeuAspGluGlu Thr 1715. 1720 1725 cac gca ggc gag gac gtg gcg gtg ttc gcg cgc ggc ccg cag gcg cac 6374 His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His ctg gtt cac ggc gtg cag gag cag acc ttc ata gcg cac gtc atg gcc 6422 Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala ttc gcc gcc tgc ctg gag ccc tac acc gcc tgc gac ctg gcg ccc ccc 6470 Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro gcc ggc acc acc gac gcc gcg cac ccg ggt taacccgtgg tccccgcgtt 6520 Ala Gly Thr Thr Asp Ala Ala His Pro Gly gcttcctctg ctggccggga catcaggtgg cccccgctga attggaatcg atattgttac 6580 aacaccccaa catcttcgac gcgggcgtgg caggtcttcc cgacgatgac gccggtgaac 6640 ttcccgccgc cgttgttgtt ttggagcacg gaaagacgat gacggaaaaa gagatcgtgg 6700 attacgtcgc cagtcaagta acaaccgcga aaaagttgcg cggaggagtt gtgtttgtgg 6760 acgaagtacc gaaaggtctt accggaaaac tcgacgcaag aaaaatcaga gagatcctca 6820 taaaggccaa gaagggcgga aagtccaaat tgtaaaatgt aactgtattc agcgatgacg 6880 aaattcttag ctattgtaat actgcgatga gtggcagggc ggggcgtaat ttttttaagg 6940 cagttattgg tgcccttaaa cgcctggtgc tacgcctgaa taagtgataa taagcggatg 7000 aatggcagaa attcgccgga tctttgtgaa ggaaccttac ttctgtggtg tgacataatt 7060 WO 00/08469 PG"T/US99/17440 ggacaaacta cctacagaga tttaaagctc taaggtaaat ataaaatttt taagtgtata 7120 atgtgttaaa ctactgattc taattgtttg tgtattttag attccaacct atggaactga 7180 tgaatgggag cagtggtgga atgcctttaa tgaggaaaac ctgttttgct cagaagaaat 7240 gccatctagt gatgatgagg ctactgctga ctctcaacat tctactcctc caaaaaagaa 7300 gagaaaggta gaagacccca aggactttcc ttcagaattg ctaagttttt tgagtcatgc 7360 tgtgtttagt aatagaactc ttgcttgctt tgctatttac accacaaagg aaaaagctgc 7420 actgctatac aagaaaatta tggaaaaata ttctgtaacc tttataagta ggcataacag 7480 ttataatcat aacatactgt tttttcttac tccacacagg catagagtgt ctgctattaa ?540 taactatgct caaaaattgt gtacctttag ctttttaatt tgtaaagggg ttaataagga 7600 atatttgatg tatagtgcct tgactagaga tcataatcag ccataccaca tttgtagagg 7660 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 7?20 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 7780 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 7840 tcatcaatgt atcttatcat gtctggatcc tctagagtcg acctgcaggc atgcaagctt 7900 ctcgagagta cttctagtgg atccctgcag ctcgagaggc ctaattaatt aagtcgacga 7960 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 8020 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 8080 aactatatcc ggagttaact cgacatatac tatatagtaa taccaatact caagactacg 8140 aaactgatac aatctcttat catgtgggta atgttctcga tgtcgaatag ccatatgccg 8200 gtagttgcga tatacataaa ctgatcacta attccaaacc cacccgcttt ttatagtaag 8260 tttttcaccc ataaataata aatacaataa ttaatttctc gtaaaagtaq aaaatatatt 8320 ctaatttatt gcacggtaag gaagtagaat cataaagaac agtgacggat cgatccccca 8380 agcttggaca caagacaggc ttgcgagata tgtttgagaa taccacttta tcccgcgtca 8440 gggagaggca gtgcgtaaaa agacgcggac tcatgtgaaa tactggtttt tagtgcgcca 8500 gatctctata atctcgcgca acctattttc ccctcgaaca ctttttaagc cgtagataaa 8560 caggctggga cacttcac atg agc gaa aaa tac atc gtc acc tgg gac atg 8611 Met Ser Glu Lys Tyr Ile Val Thr Trp Asp Met ttg cag atc cat gca cgt aaa ctc gca agc cga ctg atg cct tct gaa 8659 Leu Gln Ile His Ala Axg Lys Leu Ala Ser Arg Leu Met Pro Ser Glu . 1800 1805 1810 caa tgg aaa ggc att att gcc gta agc cgt ggc ggt ctg gta ccg ggt 8707 -Gln Trp Lys Gly Ile Ile Ala Val Ser Arg Gly Gly Leu Val Pro Gly gcg tta ctg gcg cgt gaa ctg ggt att cgt cat gtc gat acc gtt tgt 8755 A1a Leu Leu Ala Arg Glu Leu Gly Ile Arg His Val Asp Thr Val Cys att tcc agc tac gat cac gac aac cag cgc gag ctt aaa gtg ctg aaa 8803 Ile Ser Ser Tyr Asp His Asp Asn Gln Arg Glu Leu Lys Val Leu Lys cgc gca gaa ggc gat ggc gaa ggc ttc atc gtt att gat.gac ctg gtg 8851 Arg Ala Glu Gly Asp Gly Glu Gly Phe Ile Val Ile Asp Asp Leu Val gat acc ggt ggt act gcg gtt gcg att cgt gaa atg tat cca aaa gcg 8899 Asp Thr Gly Gly Thr A1a Val Ala Ile-Arg Glu Met Tyr Pro Lys Ala cac ttt gtc acc atc ttc gca aaa ccg get ggt cgt ccg ctg gtt gat 894?
His Phe Val Thr Ile Phe Ala Lys Pro Ala Gly Arg Pro Leu Val Asp gac tat gtt gtt gat atc ccg caa gat acc tgg att gaa cag ccg tgg 8995 Asp Tyr Val Val Asp Ile Pro Gln Asp Thr Trp Ile Glu Gln Pro Trp gat atg ggc gtc gta ttc gtc ccg cca atc tcc ggt cgc taatcttttc 9044 Asp Met Gly Val Val Phe Val Pro Pro Ile Ser Gly Arg aacgcctggc actgccgggc gttgttcttt ttaacttcag gcgggttaca atagtttcca 9109 gtaagtattc tggaggctgc atccatgaca caggcaaacc tgagcgaaac cctgttcaaa 9164 ccccgcttta aacatcctga aacctcgacg ctagtccgcc gctttaatca cggcgcacaa 9224 ccgcctgtgc agtcggccct tgatggtaaa accatccctc actggtatcg catgattaac 9284 cgtctgatgt ggatctggcg cggcattgac ccacgcgaaa tcctcgacgt ccaggcacgt 9344 attgtgatga gcgatgccga acgtaccgac gatgatttat acgatacggt gattggctac 9404 cgtggcggca actggattta tgagtgggcc ccggatcttt gtgaaggaac cttacttctg 9469 tggtgtgaca taattggaca aactacctac agagatttaa agctctaagg taaatataaa 9524 atttttaagt gtataatgtg ttaaactact gattctaatt gtttgtgtat tttagattcc 9584 aacctatgga actgatgaat gggagcagtg gtggaatgcc tttaatgagg aaaacctgtt 9644 ttgctcagaa gaaatgccat ctagtgatga tgaggctact gctgactctc aacattctac 9704 tcctccaaaa aagaagagaa aggtagaaga ccccaaggac tttccttcag aattgctaag 9769 ttttttgagt catgctgtgt ttagtaatag aactcttgct tgctttgcta tttacaccac 9829 aaaggaaaaa gctgcactgc tatacaagaa aattatggaa aaatattctg taacctttat 9884 aagtaggcat aacagttata atcataacat actgtttttt cttactccac acaggcatag 9949 agtgtctgct attaataact atgctcaaaa attgtgtacc tttagctttt taatttgtaa 10004 aggggttaat aaggaatatt tgatgtatag tgccttgact agagatcata atcagccata 10069 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 10129 aacataaaat gaatgcaatt gttgttgtta agcttggggg aattgcatgc tccggatcga 10184 gatcaa ttc tgt gag cgt atg gca aac gaa gga aaa ata gtt ata gta 10232 Phe Cys Glu Arg Met Ala Asn Glu Gly Lys Ile Val Ile Val gcc gca ctc gat ggg aca ttt caa cgt aaa ccg ttt aat aat att ttg 10280 Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Asn Asn Ile Leu aat ctt att cca tta tct gaa atg gtg gta aaa cta act get gtg tgt 10328 Asn Leu Ile Pro Leu Ser Glu Met Val Val Lys Leu Thr Ala Val Cys atg aaa tgc ttt aag gag get tcc ttt tct aaa cga ttg ggt gag gaa 10376 Met Lys Cys Phe Lys Glu Ala Ser Phe Ser Lys Arg Leu Gly Glu Glu acc gag ata gaa ata ata gga ggt aat gat atg tat caa tcg gtg tgt 10424 Thr Glu Ile Glu Ile Ile Gly Gly Asn Asp Met Tyr Gln Ser Val Cys aga aag tgt tac atc gac tca taatattata ttttttatct aaaaaactaa 10975 Arg Lys Cys Tyr Ile Asp 5er aaataaacat tgattaaatt ttaatataat acttaaaaat ggatgttgtg tcgttagata 10535 aaccgtttat gtattttgag gaaattgata atgagttaga ttacgaacca gaaagtgcaa 10595 atgaggtcgc aaaaaaactg ccgtatcaag gacagttaaa actattacta ggagaattat 10655 tttttcttag taagttacag cgacacggta tattagatgg tgccaccgta gtgtatatag 10715 gatctgctcc cggtacacat atacgttatt tgagagatca tttctataat ttaggagtga 10775 tcatcaaatg gatgctaatt gacggccgcc atcatgatcc tattttaaat ggattgcgtg 10835 atgtgactct agtgactcgg ttcgttgatg aggaatatct acgatccatc aaaaaacaac 10895 tgcatccttc taagattatt ttaatttctg atgtgagatc caaacgagga ggaaatgaac 10955 ctagtacggc ggatttacta agtaattacg ctctacaaaa tgtcatgatt agtattttaa 11015 accccgtggc gtctagtctt aaatggagat gcccgtttcc agatcaatgg atcaaggact 11075 tttatatccc acacggtaat aaaatgttac aaccttttgc tccttcatat tcagggccgt 11135 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 11195 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gccct:.ccca 11255 acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg cattaagcgc 11315 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 11375 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 11935 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 11495 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 11555 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 11615 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 11675 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 11735 tacaatttcc caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 11795 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 11855 taatattgaa aaaggaagag t atg agt att caa cat ttc cgt gtc gcc ctt 11906 Met Ser Ile Gln His Phe Arg Val Ala Leu att ccc ttt ttt gcg gca ttt tgc ctt cct gtt ttt get cac cca gaa 11954 Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu aeg etg gtg aaa gta aaa gat get gaa gat cag ttg ggt gca ega gtg 12002 Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val ggt tac atc gaa ctg gat ctc aac agc ggt aag atc ctt gag agt ttt 12050 Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe cgc ccc gaa gaa cgt ttt cca atg atg agc act ttt aaa gtt ctg cta 12098 Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu tgt ggc gcg gta tta tcc cgt att gac gcc ggg caa gag caa ctc ggt 12146 Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly cgc cgc ata cac tat tct cag aat gac ttg gtt gag tac tca cca gtc 12194 Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa tta tgc agt 12242 Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser get gcc ata acc atg agt gat aae act gcg gcc aac tta ctt ctg aca 12290 Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr acg ate gga gga ccg aag gag cta acc get ttt ttg cac aac atg ggg 12338 Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc 12386 Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala ata cca aac gac gag cgt gac acc acg atg cct gta gca atg gca aca 12434 Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr acg ttg cgc aaa cta tta act ggc gaa cta ctt act cta get tcc cgg 12982 Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg caa caa tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt 12530 Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Va1 Ala Gly Pro Leu ctg cgc tcg gcc ctt ccg get ggc tgg ttt att get gat aaa tct gga 12578 Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser G1y gcc ggt gag cgt ggg tct cgc ggt atc att gca gca ctg ggg cca gat 12626 Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp 2260 2265 22?0 ggt aag ccc tcc cgt atc gta gtt atc tac acg acg ggg agt cag gca 12674 Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly Ser Gln Ala act atg gat gaa cga aat aga cag atc get gag ata ggt gcc tca ctg 12722 Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu att aag cat tgg taactgtcag accaagttta ctcatatata ctttagattg 12774 Ile Lys His Trp atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 12834 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 12894 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 12954 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 13014 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtc~ tagccgtagt 13074 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 13134 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 13194 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 13254 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 13314 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 13374 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 13934 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 13494 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 13554 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 13614 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 13679 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 13734 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 13794 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 13859 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgcc 13910 <210> i6 <211> 2307 <212> PRT
<213> Artificial Sequence <400> 16 Met Asn Gly Gly His Ile Gln Leu Ile'Ile Gly Pro Met Phe Ser Gly Lys Ser Thr Glu Leu Ile Arg Arg Val Arg Arg Tyr Gln Ile Ala Gln Tyr Lys Cys Val Thr Ile Lys Tyr Ser Asn Asp Asn Arg Tyr Gly Thr Gly Leu Trp Thr His Asp Lys Asn Asn Phe Glu Ala Leu Glu Ala Thr Lys Leu Cys Asp Val Leu Glu Ser Ile Thr Asp Phe Ser Val Ile Gly Ile Asp Glu Gly Gln Phe Phe Pro Asp Ile Val Glu Met Gly Ile Pro Gln Phe Met Ala Arg Vai Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala~Arg Leu Ile Trp Trp Leu Gln Tyr Phe Thr Thr Arg Ala Glu Ala His Leu His Val Trp Ile Pro Pro Leu Asn Ala Arg Gly Gly . _ WO 00/08469 PCT/US99/17440 _ Arg Asp Ala Iie Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Ile Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile His Ala Cys Met Leu Val Arg Lys Val Ala Gly Gly His Tyr Val Gln Met Ala Phe Met Lys Leu Gly Ala Leu Thr Gly Thr Tyr Ile Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala His Ala Gly Leu Arg 325 '330 335 Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr Ala Ala Ala Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Leu Glu Gly Arg Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr 905 410 ~ :415 Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ala Gly Gly Pro Leu Leu Cys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val - _ Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala G1n Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala.Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly A1a T_yr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp 5er Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Ala Ile Arg Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pio Thr Ile Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser . .. WO 00/08469 PCT/US99/17440 Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His I1e Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Ile Val Pro Asp Arg Glu Leu Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu Thr'Thr Gln Ser Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala A1a Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Glu Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly Pro G1u Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln Gly Gly WO 00/08469 PG"T/US99l17440 Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn.Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro Leu Arg Gly Ser Cys Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly Ser Pro 1700 1?05 1710 Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe I1~ Ala His Val Met Al.a Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro Ala Gly Thr Thr Asp Ala Ala His Pro Gly Met Ser Glu Lys Tyr Ile Val Thr Trp Asp Met Leu Gln Ile His Ala Arg Lys Leu Ala Ser Arg Leu Met 179s 1800 leo5 Pro Ser Glu Gln Trp Lys Gly Ile Ile Ala Val Ser Arg Gly.Gly Leu Val Pro Gly Ala Leu Leu Ala Arg Glu Leu Gly Ile Arg His Val Asp Thr Val Cys Ile Ser Ser Tyr Asp His Asp Asn Gln Arg Glu Leu Lys Val Leu Lys Arg Ala Glu Gly Asp Gly Glu Gly Phe Ile Val Ile Asp Asp Leu Val Asp Thr Gly Gly Thr Ala Val Ala Ile Arg Glu Met Tyr Pro Lys Ala His Phe Val Thr Ile Phe Ala Lys Pro Ala Gly Arg Pro Leu Val Asp Asp Tyr Val Val Asp Ile Pro Gln Asp Thr Trp Ile Glu Gln Pro Trp Asp Met Gly Val Val Phe Val Pro Pro Ile Ser Gly Arg Phe Cys Glu Arg Met Ala Asn Glu Gly Lys Ile Val Ile Val Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Asn Asn Ile Leu Asn Leu Ile Pro Leu Ser Glu Met Val Val Lys Leu Thr Ala Val Cys Met Lys Cys Phe Lys Glu Ala Ser Phe Ser Lys Arg Leu Gly Glu Glu Thr Glu Ile Glu Ile Ile Gly Gly Asn Asp Met Tyr Gln Ser Val Cys Arg Lys Cys Tyr Ile Asp Ser Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Giy Ala Ser Leu Ile Lys His Trp <210> 17 <211> 92 <212> PRT
<213> Artificial Sequence <900> 17 Met Asn Gly Gly His Ile Gln Leu Ile Ile Gly Pro Met Phe Ser Gly Lys Ser Thr Glu Leu Ile Arg Arg Val Arg Arg Tyr Gln Ile Ala Gln 20 25 ~,0 Tyr Lys Cys Val Thr Ile Lys Tyr Ser Asn Asp Asn Arg Tyr Gly Thr Gly Leu Trp Thr His Asp Lys Asn Asn Phe Glu Ala Leu Glu Ala Thr Lys Leu Cjrs Asp Val Leu Glu Ser Ile Thr Asp Phe Ser Val Ile Gly Ile Asp Glu Gly Gln Phe Phe Pro Asp Ile Val Giu <210> 18 <211> 1692 <212> PRT
<213> Artificial Sequence <400> 18 Met Gly Ile Pro Gln Phe Met Ala Arg Val Cys Ala Cys Leu Trp Met 1 ~ 10 15 Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Val Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr Phe Thr Thr Arg Ala Glu Ala His Leu His Val Trp Ile Pro Pro Leu Asn Ala Arg Gly Gly Arg Asp Ala Ile Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr Lys Leu Leu Ile Ala Ile Leu Gly 165 ~ :170 175 Pro Leu Met Val Leu Gln Ala Gly Ile Thr Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile His Ala Cys Met Leu Val Arg Lys Val Ala Gly Gly His Tyr Val Gln Met Ala Phe Met Lys Leu Gly Ala Leu Thr Gly Thr Tyr Ile Tyr Asn His Leu Thr Pro Leu Arg Asp Trp Ala His Ala Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr Trp Gly Ala Asp Thr Ala Ala Ala Gly Asp Ile Ile Leu Gly Leu Pro Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser Leu Glu Gly Arg Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ala Gly Gly Pro Leu Leu Cys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn _ _ Ile Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Ala Ile Arg Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ile Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met 70s 710 71s 720 Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys ?25 730 735 Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp 5er Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala Ile Val Pro Asp Arg Glu Leu Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr A1a Ser Ile Thr Ser Pro Leu Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Glu Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val . _ WO pp/p8469 PCT/US99/17440 Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly Leu Ser A1a Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp 1365 13?0 1375 Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Giu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro Leu Arg Gly Ser Cys Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val~Thr Glu Ser mu Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His Ala Gly Glu Asp Val Ala Vai Phe Ala Arg Gly Pro Gln Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Iie Ala His Val Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro Ala Gly Thr Thr Asp Ala Ala His Pro Gly <210> 19 <211> 152 <212> PRT
<213> Artificial Sequence <400> 19 Met Ser Glu Lys Tyr Ile Val Thr Trp Asp Met Leu Gln Ile His Ala Arg Lys Leu Ala Ser Arg Leu Met Pro Ser flu Gln Trp Lys Gly Ile Ile Ala Val Ser Arg Gly Gly Leu Val Pro Gly Ala Leu Leu Ala Arg Glu Leu Gly Ile Arg His Val Asp Thr Val Cys Ile Ser Ser Tyr Asp His Asp Asn Gln Arg Glu Leu Lys Val Leu Lys Arg Ala Glu Gly Asp Gly Glu Gly Phe Ile Val Ile Asp Asp Leu Val Asp Thr Gly Gly Thr Ala Val Ala Ile Arg Glu Met Tyr Pro Lys Ala His Phe Val Thr Ile Phe Ala Lys Pro Ala Gly Arg Pro Leu Val Asp Asp Tyr Val Val Asp Ile Pro Gln Asp Thr Trp Ile Glu Gln Pro Trp Asp Met Gly Val Val Phe Val Pro Pro Ile Ser Gly Arg <210> 20 <211> 85 <212> PRT
<213> Artificial Sequence - - _ J
<900> 20 Phe Cys Glu Arg Met Ala Asn Glu Gly Lys Ile Val Ile Val Ala Ala Leu Asp Gly Thr Phe Gln Arg Lys Pro Phe Asn Asn Ile Leu Asn Leu Ile Pro Leu Ser Glu Met Val Val Lys Leu Thr Ala Val Cys Met Lys Cys Phe Lys Glu Ala Ser Phe Ser Lys Arg Leu Gly Glu Glu Thr Glu Ile Glu Ile Ile Gly Gly Asn Asp Met Tyr Gln Ser Val Cys Arg Lys Cys Tyr Ile Asp Ser <210> 21 <211> 286 <212> PRT
<213> Artificial Sequence <400> 21 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp ., Leu Asn Ser Gly Lys Ile Leu G1u Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Glu Thr His Ala Gly Glu Asp Val Ala Vai Phe Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Va1 Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp 275 280 . 285 <210> 22 <211> 220 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Sac 1/SEAP/Bam H1 construct <400> 22 gcgcgcgagc tcctgctgct gctgctgctg ggcctgaggc tacagctctc cctgggcatc 60 atcccagttg aggaggagaa cccggacttc tggaaccgcg aggcagccga ggccctgggt 120 gccgccaaga agctgcagcc tgcacagaca gccgccaaga acctcatcat cttcctgggc 180 gatgggatgg gggtgtctac ggtgacagct gccaggatcc 220 <210> 23 <211> 88 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: amino acid fragment of the HCV polyprotein <400> 23 Ala Arg Val Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn Ser Ala Ser Val Ala Gly Ala His Gly Ile Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Thr Tyr Ala Leu Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala <210> 24 <211> 260 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: DNA fragment coding for an amino acid fragment of the HCV
polyprotein <900> 24 gcacgtgtct gtgcctgctt gtggatgatg ctgctgatag cccaggccga ggccgccttg 60 gagaacctgg tggtcctcaa tgcggcgtct gtggccggcg cacatggcat cctctccttc 120 cttgtgttct tctgtgccgc ctggtacatc aaaggcaggc tggtccctgg ggcggcatat 180 gctctttatg gcgtgtggcc gctgctcctg ctcttgctgg cattaccacc gcgagcttac 240 gccatggacc gggagatggc 260 <210> 25 <211> 177 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: amino acid fragment of the HCV polyprotein <400> 25 Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala P.he Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro - WO 00/08469 p~~s~/~ ~~0 Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Mgt Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu <210> 26 <211> 528 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: DNA fragment coding for an amino acid fragment of the HCV
polyprotein <400> 26 tgcgcctcgc acctccctta catcgagcag ggaatgcagc tcgccgagca attcaagcag 60 aaagcgctcg ggttactgca aacagccacc aaacaagcgg aggctgctgc tcccgtggtg 120 gagtccaagt ggcgagccct tgagacattc tgggcgaagc acatgtggaa tttcatcagc 180 gggatacagt acttagcagg cttatccact ctgcctggga accccgcaat agcatcattg 240 atggcattca cagcctctat caccagcccg ctcaccaccc aaagtaccct cctgtttaac 300 atcttggggg ggtgggtggc tgcccaactc gcccccccca gcgccgcttc ggctttcgtg 360 ggcgccggca tcgccggtgc ggctgttggc agcataggcc ttgggaaggt gcttgtggac 420 attctggcgg gttatggagc aggagtggcc ggcgcgctcg tggcctttaa ggtcatgagc 480 ggcgagatgc cctccaccga ggacctggtc aatctacttc ctgccatc 528 <210> 27 <211> 33 <212> DNA
<213> primer <900> 27 gcgcgcgaat tcatggcacg tgtctgtgcc tgc 33 <210> 28 <211> 33 <212> DNA
<213> primer <900> 28 WO 00/08469 PCT/US99117440 .
cgcgcgctcg aggatggcag gaagtagatt gac 33 <210> 29 <211> 20 <212> PRT
<213> putative NSSA/58 cleavage site <400> 29 Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu <210> 30 <211> 33 <212> DNA
<213> primer <400> 30 gcgcgcctcg aggaagctag tgaggatgtc gtc 33 <210> 31 <211> 36 <212> DNA
<213> primer <400> 3I
cgcgcggagc tccaaggcgc ctgtccatgt gtagga 36 <210> 32 <211> 69 <212> DNA
<213> primer <400> 32 ctcgaggaag ctagtgagga tgtcgtctgc tgctcaatgt cctacacatg gacaggcgcc 60 ttggagctc 69 <210> 33 <211> 6 <212> PRT
<213> HCV/SEAP 6 amino acid fragment <900> 33 Met Gly Ile Pro Gln Phe

Claims (41)

We Claim:

1. A reporter gene system useful in the assessment of compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising:
a) a recombinant viral vector comprising a DNA molecule encoding an RNA polymerise promoter compatible with said viral vector and which is expressed upon infection of a target mammalian cell;
b) a recombinant plasmid comprising a DNA molecule encoding the HCV/SEAP reporter gene polyprotein which is expressed when transfected into a target mammalian cell;
c) said target mammalian cell line being infected first with said recombinant viral vector then transfected with said recombinant plasmid such that the DNA molecule encoding the HCV/SEAP reporter gene is under transcriptional control of said promoter; and d) the target mammalian cell expressing said HCV/SEAP reporter gene polyprotein such that SEAP is secreted from said target mammalian cell.

2. A reporter gene system useful in the assessment of compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising:
a) a first recombinant viral vector comprising a DNA molecule encoding an RNA polymerise promoter compatible with said viral vector and which is expressed upon infection of a target mammalian cell;
b) a second recombinant viral vector comprising a DNA molecule encoding the HCV/SEAP reporter gene polyprotein which is expressed upon infection of a target mammalian cell;
c) said target mammalian cell line being infected first with said first recombinant viral vector then co-infected with said second recombinant plasmid such that the DNA molecule encoding the HCV/SEAP reporter gene is under control of said promoter; and d) the target mammalian cell expresses said HCV/SEAP reporter gene polyprotein such that SEAP is secreted from said target mammalian cell.

3. The reporter gene system of claim 1 wherein said recombinant plasmid is the pTM3 plasmid containing said HepC/SEAP construct.

4. The recombinant plasmid of claim 3 wherein said recombinant plasmid comprises the pHCAP1 plasmid having a DNA molecule encoding the NS2 and NS3 protease polyproteins in a fusion protein fused with the SEAP gene according to the sequence in Seq. ID NO: 1.

5. The recombinant plasmid of claim 3 wherein said recombinant plasmid further comprises the pHCAP3 plasmid containing the active NS2 protease and a mutant NS3 protease in a fusion protein fused with the SEAP gene according to the sequence in Seq. ID NO: 8.

6. The recombinant plasmid of claim 3 wherein said recombinant plasmid further comprises the pHCAP4 plasmid containing the mutant inactive NS2 and mutant inactive NS3 protease in a fusion protein fused with the SEAP gene according to the sequence in Seq. ID NO: 15.

7. The reporter gene system of claim 2 wherein said second recombinant viral vector further comprises the vHCAP1 vector having a DNA molecule encoding the NS2 and NS3 protease polyproteins in a fusion protein fused with the SEAP gene according to the sequence in Seq. 1D NO: 1.

8. The reporter gene system of claim 2 wherein said second recombinant viral vector further comprises the vHCAP3 vector containing the active NS2 protease and a mutant NS3 protease in a fusion protein fused with the SEAP
gene according to the sequence in Seq. ID NO: 9.

9. The reporter gene system of claim 2 wherein said second recombinant viral vector further comprises the vHCAP4 vector containing the active NS2 protease and a mutant NS3 protease in a fusion protein fused with the SEAP
gene according to the sequence in Seq. ID NO: 16.

10. The reporter gene system of claim 1 wherein said recombinant viral vector comprises a virus containing the DNA sequence encoding T7 RNA
polymerase promoter.

11. The recombinant viral vector of claim 7 wherein said vector is the vTF7.3 vector.

12. The reporter gene system of claim 2 wherein said first recombinant viral vector comprises a virus containing the DNA sequence encoding the T7 RNA
polymerase promoter.

13. The recombinant viral vector of claim 9 wherein said vector is the vTF7.3 vector.

14. The reporter gene system of claim 1 wherein said first recombinant viral vector comprises a virus containing the DNA sequence encoding a vaccinia virus compatible promoter.

15. The first recombinant viral vector of claim 11 wherein said vector is a vaccinia virus derived vector.

16. The reporter gene system of claim 2 wherein said first recombinant viral vector comprises a virus containing the DNA sequence encoding a vaccinia virus compatible promoter.

17. The first recombinant viral vector of claim 13 wherein said vector is a vaccinia virus derived vector.

18. A first recombinant viral vector according to claim 2 wherein the vector is pTM3 plasmid, a Listeria vector, an orthopox virus, avipox virus, canarypox virus, suipox virus, vaccinia virus, baculovirus, human adenovirus, SV40, Herpes Virus or bovine papilloma virus.

19. A second recombinant viral vector according to claim 2 wherein the vector is pTM3 plasmid, a Listeria vector, an orthopox virus, avipox virus, canarypox virus, suipox virus, vaccinia virus, baculovirus, human adenovirus, SV40, Herpes Virus or bovine papilloma virus.

20. The reporter gene system of claim 1 wherein said recombinant viral vector comprises a virus containing a the DNA sequence encoding a promoter selected from the group of mammalian viral vectors consisting of:
Simian Virus 40 (SV40), Rous Sarcoma Virus (RSV), Adenovirus (ADV) and Bovine Papilloma Virus (BPV).

21. The reporter gene system of claim 2 wherein said recombinant viral vector comprises a virus containing a the DNA sequence encoding a promoter selected from the group of mammalian viral vectors consisting of:
Simian Virus 40 (SV40), Rous Sarcoma Virus (RSV), Adenovirus (ADV) and Bovine Papilloma Virus (BPV).

22. The reporter gene system of claim 1 wherein said target cell line is selected from the group consisting of:
HeLa cells, Chinese Hamster Ovary cells, CV1 African Green Monkey cells, BSC 1 cells and Baby Hamster Kidney cells.

23. The reporter gene system of claim 2 wherein said target cell line is selected from the group consisting of:

HeLa cells, Chinese Hamster Ovary cells, CV1 African Green Monkey cells, BSC 1 cells and Baby Hamster Kidney cells.

24. An isolated DNA sequence comprising a DNA sequence or variants thereof encoding the HepC/SEAP reporter gene construct according to claim 1.

25. The isolated DNA sequence of claim 24 comprising a DNA sequence or variants thereof in SEQ. ID NO. 1.

26. An isolated DNA sequence comprising a DNA sequence or variants thereof encoding the sequence defined as pHCAP1.

27. An isolated DNA sequence comprising a DNA sequence or variants thereof encoding the sequence defined as pHCAP3.

28. An isolated DNA sequence comprising a DNA sequence or variants thereof encoding the sequence defined as pHCAP4.

29. An isolated DNA sequence comprising a DNA sequence or variants thereof encoding the sequence defined as vHCAP1.

30. An isolated DNA sequence comprising a DNA sequence or variants thereof encoding the sequence defined as vHCAP3.

31. An isolated DNA sequence comprising a DNA sequence or variants thereof encoding the sequence defined as vHCAP4.

32. A method of assessing compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising:
a) a control target mammalian cell;
b) a first target mammalian cell expressing the pHCAP1 polyprotein;
c) a second target mammalian cell expressing the pHCAP4 polyprotein;

d) a third target mammalian cell expressing the viral promoter only;
e) incubating said control, first, second, and third target mammalian cells for about 24 hours in a suitable growth medium in the presence and/or absence of pharmacologically effective concentrations of candidate compounds;
f) measuring the amount of SEAP activity; and g) determining whether said candidate compounds augmented or inhibited hepatitis C NS3 protease by comparing the SEAP activity of said control, first, second, and third target mammalian cells.

33. A method of assessing compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising:
a) a control target mammalian cell;
b) a first target mammalian cell expressing the vHCAP1 polyprotein;
c) a second target mammalian cell expressing the vHCAP4 polyprotein;
d) a third target mammalian cell expressing the viral promoter only;
e) incubating said control, first, second, and third target mammalian cells for about 24 hours in a suitable growth medium in the presence and/or absence of pharmacologically effective concentrations of candidate compounds;
f) measuring the amount of SEAP activity; and g) determining whether said candidate compounds augmented or inhibited hepatitis C NS3 protease by comparing the SEAP activity of said control, first, second, and third target mammalian cells.

34. A method of assessing compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease cis-only cleavage comprising:
a) a control target mammalian cell;
b) a first target mammalian cell expressing the pHCAP3 polyprotein;
c) a second target mammalian cell expressing the pHCAP4 polyprotein;
d) a third target mammalian cell expressing the viral promoter only;
e) incubating said control, first, second, and third target mammalian cells for about 24 hours in a suitable growth medium in the presence and/or absence of pharmacologically effective concentrations of candidate compounds;
f) measuring the amount of SEAP activity; and g) determining whether said candidate compounds augmented or inhibited hepatitis C NS3 protease by comparing the SEAP activity of said control, first, second, and third target mammalian cells.

35. A process for constructing a reporter gene system useful in the assessment of compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising:
a) providing a recombinant viral vector comprising a DNA molecule encoding an RNA polymerase promoter compatible with said viral vector and which is expressed upon infection of a target mammalian cell;
b) providing a recombinant plasmid comprising a DNA molecule encoding the HCV/SEAP reporter gene polyprotein which is expressed when transfected into a target mammalian cell further comprising the steps of cloning into a suitable vector the NS2-NS3-NS4A-NS4B' -NS5A/5B
cleavage site - SEAP polyprotein;
c) said target mammalian cell line being infected first with said recombinant viral vector then transfected with said recombinant plasmid such that the DNA molecule encoding the HCV/SEAP reporter gene is under transcriptional control of said promoter; and d) the target mammalian cell expressing said HCV/SEAP reporter gene polyprotein such that SEAP is secreted from said target mammalian cell.

36. A process for constructing a reporter gene system useful in the assessment of compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising:
a) providing a first recombinant viral vector comprising a DNA molecule encoding an RNA polymerase promoter compatible with said viral vector and which is expressed upon infection of a target mammalian cell;
b) providing a second recombinant viral vector comprising a DNA
molecule encoding the HCV/SEAP reporter gene polyprotein which is expressed when transfected into a target mammalian cell further comprising the steps of cloning into a suitable vector the NS2-NS3-NS4A-NS4B' -NS5A/5B cleavage site - SEAP polyprotein;
c) said target mammalian cell line being infected first with said first recombinant viral vector then co-infected with said second recombinant plasmid such that the DNA molecule encoding the HCV/SEAP reporter gene is under control of said promoter; and d) the target mammalian cell expresses said HCV/SEAP reporter gene polyprotein such that SEAP is secreted from said target mammalian cell.

37. The isolated DNA sequence of claim 27 comprising a DNA sequence or variants thereof in SEQ. ID NO. 8.

38. The isolated DNA sequence of claim 28 comprising a DNA sequence or variants thereof in SEQ. ID NO. 15.

39. A composition comprising the pHCAP1 polyprotein as described in SEQ.ID
NO.2.

40. A composition comprising the pHCAP3 polyprotein as described in SEQ.ID
NO.9.

41. A composition comprising the pHCAP4 polyprotein as described in SEQ.ID
NO.16.