WO1992001714A1

WO1992001714A1 - Non-a non-b hepatitis virus antigen

Info

Publication number: WO1992001714A1
Application number: PCT/JP1991/000964
Authority: WO
Inventors: Yasuaki Shimizu; Takeshi Imai; Junko Ishida; Emiko Yano; Syuhei Yasuda; Tetsuo Morinaga; Terukatsu Arima
Original assignee: Yamanouchi Pharmaceutical Co., Ltd.
Priority date: 1990-07-24
Filing date: 1991-07-19
Publication date: 1992-02-06

Abstract

Non-A non-B hepatitis virus antigen polypeptide groups Y19 and Y22 or fragments thereof; complex antigen polypeptides prepared by fusing two or more antigens selected from the above polypeptides; and antigen polypeptides having an epitope contained in a group Y19 or Y22 polypeptide. A method of detecting a non-A non-B hepatitis virus antigen by determining an immunological conjugate formed by the reaction of a non-A non-B hepatitis virus antibody with one or a mixture of these antigens.

Description

Specification

Non-A non-B hepatitis virus antigen

The present invention relates to a novel non-A non-B hepatitis virus antigen. Specifically, an antigen polypeptide that specifically shows an antigen-antibody reaction with serum of a non-A non-B hepatitis patient, a polynucleotide encoding the antigen polypeptide, and an antigen polypeptide using the polynucleotide And a method for detecting and diagnosing a non-A non-B hepatitis virus antibody using the antigen polypeptide. Background art

Non-A, non-B hepatitis is the hepatitis A virus (HAV), hepatitis B virus (HBV), Hepatitis caused by hepatitis hepatitis virus (HDV) or cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, etc. Are excluded. Epidemiological evidence suggests that there are three types of non-A, non-B hepatitis: Water-borne epidemics prevalent in developing countries, post-transfusion-type transmission via blood, and sporadic (population-acquisition) types found in developed countries. The waterborne form is caused by a different virus than the other two forms (hepatitis E virus), and its gene fragment has recently been isolated by a group of Genelabs (USA) and the US CDC [ WO 89/12641 Pamphlet (1990), Reyes, GR, et al. Science 247, 1335 (1990)]. In the present specification, unless otherwise specified, the term "non-A non-B hepatitis" means the one caused by the remaining post-ring blood type and sporadic type, excluding this water-borne type.

Ministry of Health estimation (Healthitis Liaison Council of the Ministry of Health and Welfare, 1987 report, p5 1988 It is estimated that more than 90% of posttransfusion acute hepatitis and about 50% of sporadic acute hepatitis are non-A, non-B hepatitis. Approximately half of the resulting hepatitis progresses to chronic hepatitis, 10-20% progress to cirrhosis, and then to liver cancer, but these processes also involve non-A non-B hepatitis virus. Is thought to be involved in persistent infection of Z and associated chronic inflammation. Therefore, establishing a method for preventing and treating non-A non-B hepatitis virus was an extremely important medical issue, but identification of non-A non-B hepatitis virus was difficult despite years of efforts. It was extremely. In 1989, two groups reported the isolation of its non-A, non-B hepatitis-associated antigen gene. One is from the group of Chiron (USA) [Choo, Q-L., Et al. Science, 244, 359 (1989); Kuo, G., et al. Science, 244, 362 (1989). Japanese Patent Application Laid-Open No. 2-500880; European Patent Application Publication No. 318216 (1989); Japanese Patent No. 388232 (1990)]; a virus having this gene is named hepatitis C virus (HCV). did. Another is by Arima et al.'S group at Okayama University (now Kagoshima University) [Arima, T., et al. Gastroenterologia Japonica 24, 685 (1 989); Arima, T., et al. Gastroenterologia Japonica 25,218 (1990); EP-A-363025 (1990)]. Both C100 antigen and clone 14 antigen encoded by these genes specifically react with 60-80% of post-transfusion non-A non-B chronic hepatitis sera and sporadic non-A non-B chronic hepatitis sera. However, it has been shown to be useful for the diagnosis of non-A, non-B chronic hepatitis patients.However, considering the specificity of this disease and the prevention of post-transfusion hepatitis, it is desired to establish a more accurate diagnostic method. Was. For example, although the antibody assay kit for the C100 antigen is already commercially available, it reacts only with about 70% of non-A, non-B hepatitis sera after transfusion [Kuo, G "et al. Science, 244, 362 (1990)], the rate of reaction with non-A non-B hepatitis patients in the chronic phase is high, but the rate of reaction with non-A non-B hepatitis patients in the acute phase is low [Miyamura, T. et al. , et al. Proc. Natl. Acad. Sci. USA, 87, 983 (1990); Hisashiya Nishioka et al., Internal Medicine 64, 1027 (1989)], It takes an average of 4 months for seroconversion [Atler, HJ, et al. N. Engl. J Med. 30, 1494 (1989)] and that the presence of the virus antibody does not always correspond to viremia [Hiroaki Okamoto, Liver, 31 suppl. 40 (1990); Koji Furusawa, Liver, 31 suppl. 42 ( 1990)], etc., and there are still many problems to be solved in diagnostic methods for non-A, non-B hepatitis virus antibodies. It is also believed that the incidence of non-A, non-B chronic hepatitis after blood transfusion decreases only by about 50% when blood donation is screened using the C100 antigen [Toru Katayama, And Organisms, 28, 217 (1990); Esteban. JI et al., N Engl J Med., 323, 1107 (1990)].

After that, HCVcore antigens [Kunitada Shimotono et al., The 38th Annual Meeting of the Japanese Society for Virology, abstract 112; Izumi Saito, et al., Abstract 104; Shizuko Harada et al., 105, 1990 (1990)], including its epitope Peptide fragments CP9, CP10 [Okamoto, H., et al., Japan J. Exp. Med., 60, 223 (1990)] and non-A non-B hepatitis-related antigen GOR derived from cellular genes [K. Akabane et al. And clinical studies, 24, 7021 (1990)], etc., but even when these are used, the response rate in the serum of non-A non-B chronic hepatitis patients is around 80%, It is known that non-A, non-B hepatitis cannot be detected. The existence of non-A, non-B hepatitis that cannot be detected with these existing antigens is attributed to the following factors: 1) HCV diversity, (HCV is known to be a highly mutated virus, [Enomoto. N., et al., BBRC, 170, 1021 (1990); Shuichi Kaneko et al., Liver, 31 suppl. 213 (1990); Nobuyuki Kato et al., Liver, 31 suppl. 214 (1990)] It is unclear whether the viruses share a common epitope), 2) whether there is another virus that causes the same pathology, 3) because it takes time for seroconversion, or 4) testing Whether it is due to inaccuracies in the law is not fully understood. Disclosure of the invention

Thus, studies on the identification of antigens useful for the development of reliable diagnostic methods and therapeutic means for non-A, non-B hepatitis have not yet been sufficiently completed. The present inventors aimed at obtaining a more useful antigen than conventional antigens and establishing an antibody measurement method using them, aiming at a 100% reliable diagnosis and prevention of non-A non-B hepatitis. That is, the present inventors have 1) an antigen having a high reaction rate to a serum of a non-A non-B hepatitis patient after blood transfusion, 2) an antigen having a high reaction rate to a serum of a patient with sporadic non-A non-B hepatitis, 3) acute Antigens that can also react with the serum of patients with non-A and non-B hepatitis in the early stage (antigens that can detect viral antibodies that appear earlier in infection), and 4) antigens that can detect viral antibodies that are highly correlated with viremia. We did intensive research to discover it. As a result, a novel non-A, non-B hepatitis virus antigen different from the existing antigen was obtained from Japanese human plasma. When the nucleotide sequence of the gene was determined, it was found that the nucleotide sequence at positions 4138 to 4521 and 6757 to 6909 of the previously reported nucleotide sequence of HCV (the nucleotide sequence shown in FIG. 17 of European Patent Application Publication No. 388232). It was shown to be a novel non-A, non-B hepatitis virus antigen with 10% and 22% homology to the corresponding amino acid sequence, respectively, but containing different amino acid sequences. By using these antigens alone or in combination, or in combination with other antigens as appropriate, non-A, non-B hepatitis virus antibodies can be detected with a much higher detection rate than existing HCV antibody measurement kits. Knowing that it is possible, they completed the present invention.

That is, the present invention relates to an antigen polypeptide Y19 group, an antigen polypeptide Y22 group or a fragment group thereof, and a composite antigen polypeptide group in which two or more antigens selected from these polypeptide groups are fused. And antigen polypeptides A single antigen selected from the group of antigenic polypeptides having an epitope that can be regarded as immunologically identical to the epitope contained in the Y19 group or the Y22 group, or a mixed antigen obtained by mixing two or more antigens Reacts with non-A, non-B hepatitis virus antibodies present in biological samples. The present invention relates to a method for detecting a non-A non-B hepatitis virus antibody, wherein the immunological conjugate formed is measured to confirm the presence of the non-A non-B hepatitis virus antibody in a biological sample. Also, the present invention relates to a non-A non-B hepatitis virus antibody detection kit for performing this detection method. Further, the present invention provides an antigen polypeptide group used for the method and kit for detecting the non-A non-B hepatitis virus antibody, a polynucleotide encoding the polypeptide of the polypeptide group, and The present invention relates to a method for producing a polypeptide contained in the polypeptide group.

The non-A non-B hepatitis virus antigen polypeptide Y19 group of the present invention is represented by the following general formula (I).

(I)

N-X, lie Pro lie Glu Al a lie Lys G ly Gly

Arg His Leu lie Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser

Ser Leu Gly Val Asn Ala Val A la Tyr Tyr Arg Gly Leu Asp Val Ser lie lie Pro Thr Ser Gly Asp Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val lie Asp Cys Asn Thr Cys Val

Thr Gin Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thrlie Glu Thr Thr Thr Val Pro Gin Asp Ala Val Ser Arg Ser Gin Arg Arg Gly Arg Thr Gly Arg Gly Arg Gly Gly lie Tyr Arg Tyr Val Thr Pro Gly Glu Arg

(In the above formula, X represents a hydrogen atom at the N-terminus or any 1 to 20 amino acids.) Further, the non-A non-B hepatitis virus antigen polypeptide Y22 group of the present invention is represented by the following general formula (次).

(II)

N-X ₂ Leu Asp Ser Phe Asp Pro Leu Arg Ala

Glu Glu Asp Glu Arg Glu Val Ser Val Ala

Ala Glu lie Leu Arg Arg Ser Arg Lys Phe

Pro Ala Ala Leu Pro lie Trp Ala Arg Pro

Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp

(In the above formula, X ₂ represents a hydrogen atom or any 1 to 20 amino acids at the N-terminus.) Fragments of the polypeptide Y22 group include groups represented by the following formulas (III) to (VII). A non-A non-Hepatitis B virus antigen polypeptide selected from the group consisting of:

(III)

Leu Asp Ser Phe Asp Pro Leu Arg Ala Glu Glu Asp Glu Arg Glu Val Ser Val Ala A la Glu lie Leu Arg Arg Ser Arg Lys

(Y22-3 antigen) (IV)

Ser Val Ala Ala Glu lie Leu Arg Arg Ser

Arg Lys Phe Pro Ala Ala Leu Pro lie Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp (Y22-7 antigen) (V)

Glu lie Leu Arg Arg Ser Arg Lys Phe Pro

Ala Ala Leu Pro lie Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp (Y22-6 antigen)

(VI)

Arg Arg Ser Arg Lys Phe Pro Ala Ala Leu Pro lie Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp

(Y22-15 antigen)

(VII)

Ser Arg Lys Phe Pro Ala Ala Leu Pro He

Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp

(Y22-14 antigen) The composite antigen polypeptide group is a composite antigen obtained by fusing two or more types of antigens selected from the above-mentioned group of Y19, Y22, and Y22 groups using genetic recombination technology. It is a polypeptide. _N which may be mentioned as its typical, for example, under Symbol of Poribe petit de is fused included in Y19 group and Y22 group of (VIII) of the polypeptide (Y22- 19) (VIII)

N— X ₃ Leu Asp Ser Phe Asp Pro Leu Arg Ala

Glu Glu Asp Glu Arg Glu Val Ser Val Ala

Ala Glu lie Leu Arg Arg Ser Arg Lys Phe

Pro Ala Ala Leu Pro lie Trp Ala Arg Pro

Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp

Lys Asp Y He Pro lie Glu Ala lie Lys

Gly Gly Arg His Leu lie Phe Cys His Ser

Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys

Leu Ser Ser Leu Gly Val Asn Ala Val Ala

Tyr Tyr Arg Gly Leu Asp Val Ser lie lie

Pro Thr Ser Gly Asp Val Val Val Val Ala

Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly

Asp Phe Asp Ser Val lie Asp Cys Asn Thr

Cys Val Thr Gin Thr Val Asp Phe Ser Leu

Asp Pro Thr Phe Thr lie Glu Thr Thr Thr Thr

Val Pro Gin Asp Ala Val Ser Arg Ser Gin

Arg Arg Gly Arg Thr Gly Arg Gly Arg Gly

Gly lie Tyr Arg Tyr Val Thr Pro Gly Glu Arg

(In the above formula, ¾ means a hydrogen atom at the N-terminus or 1 to 20 arbitrary amino acids, and Y means 1 to 20 arbitrary amino acids.) Further, non-A non-B type Representative examples of the hepatitis virus polynucleotide include, for example, the polynucleotide Y19 of the following formula (IX), the polynucleotide Y22 of the formula (X), and the polynucleotide Y22 of the formula (XI). And those represented by the following nucleotide sequence.

OVO

OVV DDI ODD OVO V1X 3 丄 :) VOO X30 OVV OVX

丄 VO 033 003 030 丄 ODD DID VOO VOO 02

303 0 丄 OVV OOV 03 丄 V3V VOO i 丄 :) DXV OVO

030 ODD 丄丄 3 0 丄 V 丄 3 VVO OOV OVO 丄 Vi OVO

OVO 030 VOO 110 033 OVO Oil X3JL 3V0 0X3

(X)

SI

003 VVO 000 V30 XOV 0 丄 3 1Y 1 OOV

DVX VXV 300 OOD VOV 300 OOV XOO 丄 DV OOV

300 VOD 003 OVO DDI ODD DDI 0X0 000 OVO

VVO 333 0X0 30V VOV ODV OVO XIV OOV Oil

VOV 10D OVO Oil OOV 0 丄丄丄 310 VOV OVO 01

OO V 310 101 VOV OVV 丄 3 丄 OVO 3XV 0X0 v〇丄

OVO 〇丄丄 OVO 000 OOV 丄 V 丄 000 VOV OXV 0X0

丄 :) Ό OVO VOV VOO 0X0 11D 11D DID DYD VOO

OOV VOV OOD V 丄 V 丄 V 301 DID OVO 丄丄 D XOO

003 OVX OV 丄 V03 VXO OO IVV 3 丄 3 VOO D 丄 :)

031 DI DID OVV 000 300 D 丄 0 OVO OVO XOX

OVV OVV OVV 00 丄丄 V3 DDI 丄丄 0 丄 V 0 丄〇 OVO

ODV 000 000 OVV OXV 030 OVO DXV 030 XXV

(XI)

6

W600 / l6df / JOd Μ .Ι0 / Γ6 O '(XI)

CTG GAC TCT TTC GAC CCG CTT CGA GCG GAG

GAG GAT GAG AGG GAA GTA TCC GTT GCG GCG

GAG ATC CTG CGA AGA TCC AGG AAG TTC CCC

GCA GCA CTG CCC ATA TGG GCG CGG CCG GAT

TAC AAC CCT CCA CTG TTA GAG CCC TGG AAG

GAC GGT GGC GCC ATG GCT ATT CCC ATC GAG

GCC ATC AAG GGG GGG AGG CAC CTC ATC TTC

TGC CAT TCC AAG AAG AAG TGT GAC GAG CTC

GCC GCG AAG CTG TCG TCC CTC GGA GTC AAT

GCT GTA GCA TAC TAC CGG GGT CTT GAC G TG

TCC ATC ATA CCG ACA AGC GGA GAC GTC GTT

GTT GTG GCA ACA GAC GCT CTG ATG ACA GGC

TAT ACC GGC GAC TTC GAC TCA GTG ATC GAC

TGT AAC ACA TGT GTC ACC CAG ACA GTC GAT

TTC AGC TTG GAC CCT ACA TTC ACC ATT GAG

ACG ACA ACC GTG CCC CAA GAC GCG GTG TCG

CGC TCG CAG CGG CGA GGC AGG ACT GGT AGG

GGC AGA GGG GGC ATA TAC AGG TAT GTG ACT

CCA GGG GAA CGG Further, the present invention relates to a polynucleotide probe for detecting a polynucleotide derived from a non-A non-B hepatitis virus, comprising a continuous sequence derived from the sequence of the complementary strand of the polynucleotide. Invention relating to a polynucleotide probe containing a sequence consisting of 8 bases or more, and reverse transcription-PCR method using a polynucleotide derived from a non-A non-B hepatitis virus (polymerase-chain reaction method) A) a pair of polynucleotide primers for detection in the above, comprising a sequence of the polynucleotide and a continuous 8 bases derived from its complementary strand; Or an invention involving a pair of polynucleotide primers containing a sequence having a longer length, and an epitope which can be regarded as immunologically identical to an epitope contained in a polypeptide of the Y19 group or the Y22 group. The invention also includes inventions relating to polypeptides having a teep. Hereinafter, the present invention will be described in detail. As used herein, the term "polynucleotide" refers to a polymer of a nucleotide (ribonucleotide or deoxyribonucleotide) having any length. The term also includes both single and double stranded forms.

The term "epitope" refers to an antigenic determinant of a polypeptide. Epitope can also be formed with three amino acids located within the conformational hormone that is unique to epitope, but generally consists of at least 5 to 10 amino acids.

The term “non-A non-B hepatitis-related antigen” means an antigen other than the antigen of the present invention and capable of diagnosing non-A non-B hepatitis, and includes both antigens derived from viral genomes and cell genes. For example, C100 antigen, Core antigen, Clone 14 antigen, GOR antigen, etc.)

The “non-A non-B hepatitis virus antigen” means a non-A non-B hepatitis-related antigen derived from the virus genome.

The “fusion antigen” refers to a non-A non-hepatitis B virus antigen of the present invention, which contains N-terminal and Z- or C-terminal other proteins (eg, ^ -gal, Protein A, T7 gene 10, maltose-binding protein, Glutathione S-transferase) or a part or all of them. In practicing the present invention, unless otherwise indicated, conventional techniques of molecular biology, microbiology, genetic engineering, and immunology known in the art are employed. Please refer to the following experiment for such a method. Experiment 1: Sambrook 丄, Fritsch. E.F., and Maniatis, T. Molecular Cloning 2nd ed .; Cold Spring Harbor (1989) Experiment 2: DNA cloning 1 roll (Glover, DM) IRL Press. (1985)

Experiment 3: DNA Cloning 3 volume (Glover, D.M.ed.) IRL Press (1987)

Experiment 4: Handbook of experimental immunology, 4th ed. 1-IV (Weir, D.M.ed.), BlackweU (1986)

Experiment 5: Current Protocols in Molecular Biology (Ausubel, F.M. et al., Ed.) John Wiley & Sons (1988)

Test Book 6: Method in Enzymology Volume 185 (Edited by Goeddel.D.V.) Academic Press (1990)

(1) Cloning policy for non-A non-B hepatitis virus antigen gene cloning As described below, cloning of non-A non-B hepatitis virus antigen gene is based on 1) RNA from starting material that is thought to contain the viral genome. 2) Preparation of λ gtll cDNA library, 3) Immunoscreening with serum from patients with non-A and non-B hepatitis. HCV is known to be rich in mutations, but multiple (10 or more) individuals are required to reliably and efficiently clone highly comprehensive antigens (epitopes) that react with any virus antibody. It is desirable to prepare cDNA based on RNA derived from Escherichia coli and perform immunoscreening on the mixed serum of multiple (10 or more) non-A non-B hepatitis patients. This makes it easier to obtain antigens that react with antibodies shared among non-A, non-B hepatitis patients.

(2) Preparation of viral RNA

a) Preparation of RNA from patient blood

HBs antigen-negative, HBV-DNA-negative, human plasma with high ALT (GPT) activity (> 35 IU / L) or non-A non-B hepatitis patient serum is pooled and used as starting material for viral RNA preparation . The virus particles are concentrated and recovered as a precipitate by polyethylene glycol precipitation. Sinking Extraction of RNA from the precipitate is performed by the AGPC method using guanidine * thiosyanate [Chomczynski, P. and Sacchi, N .: Analytical Biochemistry 162, 159 (1987)] and the like.

Alternatively, chimpanzee serum infected with non-A, non-B hepatitis virus can be used as a starting material in place of human plasma or serum, but in general, when the virus is passaged in a heterologous animal, a mutation in the virus genome occurs. It is desirable to use human plasma or serum as a starting material when cloning the antigen, as they may occur or sometimes alter the pathogenicity of the virus. Because HCV is transmitted through the blood or in a collective manner, certain virus strains may be endemic in certain areas. Therefore, it is preferable to use virus antigens derived from Japanese for diagnosis in Japan.

b) Preparation of RNA from liver tissue of patients Patients with liver cancer considered to have had infection with non-A, non-B hepatitis in the past can be obtained from liver tissue resected from patients with liver cancer using standard methods [Exp. 1,7]. RNA can be prepared.

(3) Construction of cDNA library

Using the obtained RNA as type III, a double-stranded cDNA is synthesized by the Gubler-Hoffman method [Gene, 25, 263 (1983)] using a random hexamer as a primer. The cDNA is inserted into a gtll phage vector by a conventional method. Agtll is a vector that has been developed and cloned for cloning the gene of an antigen corresponding to a specific antibody [Young, RA and Davis, RW, Proc. Natl. Acad. Sci. USA, 80, 1194 (1983) ), Experiment 2, p49-78] ₀ or vectors other than λ gtll such as ZAP, λ ΖΑΡΙΙ, pUC19, pUEXl may be used.

(4) Imno screening of cDNA library with patient serum

A recombinant infected with Escherichia coli (eg, strain Y1090); gtll grows under certain conditions according to the growth mechanism of pacteriophage, Along with this, the previously integrated cDNA is also amplified. Proliferated bacteriophages lyse host E. coli under certain conditions and form plaques that are visually recognizable. Since the cDNA inserted into the gtl phage vector is translated as a fusion antigen with ^ -galactosidase (^ -gal), a large number of recombinant antisera raised against specific antigens By screening the body gtll, cDNAs encoding specific antigens can be identified by immunochemical methods. In the present invention, the cDNA library prepared in (3) above is screened with pooled sera derived from non-A non-B hepatitis patients. Specifically, the fusion antigen expressed from the culture dish forming the plaque was transferred to a ditrocellulose membrane, and the pooled serum described above was reacted as a primary antibody, and the peroxidase-labeled goat anti- Human IgG) IgG is reacted as a secondary antibody, and plaques that are positive for color reaction are isolated.

(5) Determination of base sequence E

The thus obtained nucleotide sequence of the antigen gene of the present invention can be obtained by subjecting the recombinant DNA and the subcloned plasmid DNA to type III by the Sanger method [Sanger, F., et aL. Proc. Natl. Acad. USA., 74,5463 (1977), Experiments 1, 13], and can be determined using a commercially available sequence kit.

(6) A method for detecting the viral genome in patient serum using the nucleotide sequence of the antigen gene

Polynucleotides containing a continuous sequence of 8 bases or more in length derived from the complementary strand of the non-A non-B hepatitis virus polynucleotide of the present invention hybridize with the virus genome of the present invention, so that serum And useful as a probe for detecting the virus genome in tissues. These polynucleotides can be synthesized by a phosphoramidite method [Hunkapiller, M. et al. Nature 310, 105-111 (1984)] or a commercially available DNA synthesizer (for example, Applied Biosystems (ABI), 380AM DNA synthesizer). In addition, the non-A non-B hepatitis virus polynucleotide of the present invention can be synthesized by using the polynucleotide as a type II and subjecting it to a double translation method, a random primer labeling method, and the like. When the polynucleotide is used for diagnosis, it is used as a labeling probe such as a radioactive probe, a biotin fluorescent probe, and a chemiluminescent probe in a usual manner, but the hybridization method described above is used. The method of labeling, and the method of signal amplification and detection are all known, and can be carried out with reference to, for example, Toyozo Takatachika.

So far the non-A, non-B hepatitis virus (1/10 ⁵ in the case of hepatitis B virus) that only exist in trace amounts in the patient serum was known, the base K columns of the present invention One part or two or more primer pairs for PCR (polymerase-chain-reaction) are created using a part of the primers, and reverse transcription-PCR method [Kawasaki, ES, et al "Proc. Natl. Acad. Sci. USA, 85, 5698 (1988); Garson. JA et al., Lancet, 335, 1419 (1990); Enomoto, N "et al" BBRC, 170, 1021 (1990); Okamoto, H., et al., Japan J. Exp. Med., 60, 215 (1990)] to amplify the target viral genome and then detect it.

(7) Preparation of non-A non-B hepatitis virus antigen polypeptide and fusion antigen polypeptide

a) Expression of recombinant antigen polypeptide and recombinant fusion antigen polypeptide using gene recombination technology:

The antigen polypeptide and the polypeptide constituting the epitope and the fusion antigen polypeptide containing the sequence thereof can be expressed using a commercially available expression vector. For example, for expression in E. coli, pKK233-12, pKK223-3, pPL-lambda, pRIT5, pRIT2T, pMC1871 (Pharmacia), pMAL-c, pMAL-p (New England Biolabs), pEX 1-3 (BM), pGEX (AMRAD) or the like can be used. Experimental book 1, Chapter 17; Experimental book 3, p59-88; Experimental book 6, pl 1-128; Tabor, S. and Richardson, CC 'Pro Natl. Acad. Sci. USA, 82, 1074 (1985) This can be done with reference to the above. When the antigen polypeptide of the present invention is expressed in Escherichia coli, Met derived from the initiation codon is added to the N-terminus, and the linker and adapter used in constructing the expression vector, as well as the multicloning site, are used. In some cases, about 1 to 20 amino acids derived from the base sequence of the amino acid may be added to the N-terminal of the polypeptide (for example, in the examples of the present specification, the N-terminal of the Y19 antigen is Met-Ala However, Met-Ala-Ala-Ala is added to the N-terminal of the Y22-19 antigen). When expressed as a fusion antigen, all or part of 3-gal, Protein A, T7 gene lO, maltose binding protein, glutathione S-transferase, etc. is added to the N-terminal and / or C-terminal of the antigen polypeptide. Is done. In each case, if the reactivity to the non-A non-B patient serum is not essentially changed, it can be basically regarded as the same as the non-A non-B hepatitis virus antigen of the present invention. However, if an antibody that reacts with the protein added to the antigen is contained in human serum, it may cause a false positive. Therefore, it is preferable to minimize the addition of another type of protein. Expression in yeast is described in Experiment 3, pl 41-161, Experiment 6, p231-484, and expression in mammalian cells is described in Chapter 1, Chapter 16 of Experiment 1, Experiment 3 , pl 63-212, Experiment 6, p485-598, etc. Expression using a baculovirus vector is described in Susumu Maeda, Experimental Medicine, 7, 146 (1989); Yoshiharu Matsuura, Experimental Medicine, 8, 281 (1990), or commercially available MaxBac (Invitrogen, K822-03). This can be done using

Expressed recombinant antigen polypeptides and recombinant fusion antigen polypeptides can be purified from lysed cells or medium by protein purification methods known in the art (ultrafiltration, centrifugation, dialysis, ion exchange chromatography). , Hydrophobic chromatography, Gel 'neglect, Electrophoresis, Affinity Purification can be carried out by using chromatography, HPLC or the like; see, for example, Methods in Enzymology, Vol. 182 (Edited by Deutscher, MP), Academic Press (1990).

b) Preparation of the antigen polypeptide by peptide synthesis:

The antigen polypeptide can be prepared by chemical synthesis based on the amino acid sequence determined in the present invention. Preferably, the entire sequence is divided into fragments (10 to 50 residues) that are relatively easy to synthesize, and further divided so that adjacent fragments overlap by about 5 to 10 residues, and synthesized. After baking, it is preferable to use only the fragments containing the epitope alone or in combination.

Various polypeptides containing the amino acid sequence of the antigen polypeptide are chemically synthesized manually by a liquid phase method or a solid phase method, or by an automatic synthesizer (for example, ABI 430A type peptide synthesizer). be able to. Polypeptides synthesized by the solid-phase method according to the t-Boc method or the f-moc method are cut out of the resin using trifluormethanesulfonic acid, hydrogen fluoride, or trifluoroacetic acid. These synthetic peptides can be purified by preparative reversed-phase high-performance liquid chromatography (HPLC) to increase the degree of purification.

(8) Imnoassay detection method for viral antibodies

The non-A, non-B hepatitis virus antigen polypeptide obtained by the present invention can be used to prepare, for example, a biological sample such as blood, plasma, serum, cerebrospinal fluid, and a biological product such as a human immunoglobulin preparation by using Imnoassay. It can be used to detect the presence of non-A, non-B hepatitis virus antibodies contained in E. coli. In the immunoassay, the antigen polypeptide and the sample are subjected to conditions under which an immunological conjugate (antigen antibody conjugate) can be formed between the antigen polypeptide and the non-A non-B hepatitis virus antibody in the sample. Make contact. If any antigen-antibody conjugate is formed, the presence of non-A non-B hepatitis virus antibody in the sample can be detected and measured by appropriate means. With such detection methods And immunoprecipitation method, agglutination method, Radioimnoassy (RIA), EIA, ELISA. ETijssen, P. Eiji Ishikawa's translation, Enzymimnoattsie, Biochemical Experiment Vol.ll, Tokyo Chemical Dojin (1989)] , CEDIA [Henderson; DR et al., Clin. Chem., 32, 1637 (1986)], and Western blot atsey or binding imnoatsay.

In Examples 4 and 6, the antigen obtained according to the present invention was expressed as a fusion protein with, for example, 3-gal, and the nitrocellulose membrane-produced antigen was immobilized on a solid phase to obtain an antigen. Methods for detecting non-A, non-B hepatitis virus antibodies (eg, immunoplaque assay, western plot assay, etc.) were presented. In addition, Examples 11, 12, and 13 show a method for detecting non-A non-B hepatitis virus antibody by ELISA.

(9) Diagnosis of non-A non-B hepatitis using two or more antigens

As mentioned in (1), in order to increase the detection rate of non-A non-B hepatitis, it is necessary to obtain a highly comprehensive antigen that reacts widely with patient sera. The purpose is to create an antibody measurement system using a mixed antigen in which two or more antigens having different reaction spectra with respect to the serum of non-hepatitis B patients are combined. In Example 13, a method was described in which a titer plate was coated with a mixture of Y19, # 22-3 and # 22-16 antigens, and the virus antibody was measured at a high rate. In addition to the combination of Y19 antigen and Υ22 antigen, increase the detection rate by combining Y19 antigen and / or Υ22 antigen with one or more non-A non-B hepatitis-related antigens such as HCVcore antigen, clone 14 antigen, and GOR antigen. It is also possible.

In addition to the case where these antigens are used in combination, it is also possible to prepare a composite antigen obtained by fusing two or more antigens using genetic recombination technology and use it for diagnosis. By using this method, not only can two or more antigens be synthesized at the same time, but also each antigen can be immobilized on a plate bead at the same ratio, and the production and quality control of kits can be improved. Above is convenient. When the antigen is fused, It is necessary to add an appropriate spacer sequence between the antigens (Gly Gly Ala Met Ala is used as a spacer in Example 9) so as not to impair the function of the epitope. Example 9 shows an example in which a Y22-19 complex antigen was expressed. However, other examples include a complex antigen with a non-A non-B hepatitis-related antigen polypeptide, such as Y19-core, Y19-core. —Clone 14, Y19—Y22—Clone 14, Y19—Y22—core, Y19—Y22-CP9 / CP10-GOR, Y19-GOR-core and other complex antigens can be produced. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a histogram of absorbance distribution in a healthy human sample by the Y19 ELISA method.

FIG. 2 shows the absorbance distribution of the serum of a non-A non-B chronic hepatitis patient by the Y19 ELISA method and the C100 ELISA method.

FIG. 3 shows the results obtained by examining the reactivity of various synthetic polypeptide fragments containing the sequence of the Y22 antigen of the present invention with pooled sera of non-A and non-B type patients by ELISA.

FIG. 4 shows a histogram of absorbance distribution in a healthy human sample by the Y19 + 22 ELISA method.

FIG. 5 shows the absorbance distribution of the serum of a non-A non-B chronic hepatitis patient by Y19 + 22 ELISA and C100 ELISA. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the scope of the present invention. Example 1: Preparation of plasma-derived RNA

After thawing at room temperature 2000 ml of frozen plasma with abnormally high ALT activity (> 70ΐυ / Ί) and negative for HBsAg and HBV-DNA, Centrifugation was performed at 5000 rpm (Hitachi, RPR9-2 rotor) at 4 ° C for 20 minutes to remove fibrin. 80 g of polyethylene glycol 4000 was added to the supernatant and dissolved. After cooling on ice for 30 minutes, the mixture was centrifuged under the same conditions, and the precipitate was recovered. Extraction of RNA from the precipitate was performed according to the AGPC method using guanidine 'thiosineate [Chomczynski. P. and Sacchi. N .: Analytical Biochemistry, 162, 156 (1987)]. That is, 2 times the amount of the precipitate (converted to 1 ml of the precipitate lg) of a 6D solution (6 M guanidine. Thiosinate, 25 mM sodium citrate, ρΗ7.0, 0.5% sarcosil, 0.1 M 2-mercaptoethanol) After dissolving in water, 0.1 volume of sodium 2-sodium sulphate (ρΗ4.0) was added, and then an equal volume of phenol and 0.2 volume of a mixture of porcine: isoamyl alcohol (49: 1) were added and suspended. After cooling on ice for 15 minutes, the mixture was centrifuged, the upper layer was separated, and an equal volume of 2-propanol was added to precipitate RNA. After centrifugation, dissolve the precipitate in 0.5 ml of 4D solution (4 M guanidine thiosinate, 25 mM sodium citrate pH 7.0, 0.5% sarcosil, 0.1 M 2-mercaptoethanol), and add 2 volumes of ethanol. The RNA was precipitated again. After centrifugation, the sediment was washed with 75% ethanol and dried. The precipitate was dissolved in 501 sterile distilled water to obtain an RNA solution. Example 2: Creation of cDNA library

CDNA synthesis was performed using the RNA solution of 101 as a starting material. Double-stranded cDNA was prepared from RNA using a commercially available cDNA synthesis system, Brass (Amersham, RPN-1256Z). The conversion rate from RNA to single-stranded cDNA was about 2%, and the conversion rate from single-stranded cDNA to double-stranded cDNA was about 100%. The synthesized double-stranded cDNA was subjected to ethanol precipitation, and then dissolved in a 100 1 TE solution (10 mM Tris-CI, pH 8.0, ImM EDTA). Next, using a commercially available cDNA synthesis kit (Pharmacia LKB, 27-9260-01), The reaction was performed. The buffer was exchanged with the 1X T4 ligase solution by passing the cDNA solution through the Sephacryl S-300 span column attached to the kit. Next, an EcoRI adapter addition reaction and a T4 kinase reaction were performed according to the instructions. The peptide that did not bind to the cDNA was removed using a Sephacryl S-300 span column. The cDNA in the eluate was ethanol precipitated with 5 μg of carrier tRNA. The precipitate was washed with 75% ethanol and air-dried.

Next, the following reaction was carried out using a commercially available cDNA cloning system igtll * adapter method (Amersham, RPN1280). The precipitate was dissolved by adding 51 of sterile distilled water, 41 (2 ag) of Sgtll EcoRI arm, and 11 of LZK buffer.Then, 11 (2.5 U) of T4 ligase was added, and the mixture was added at 15 ° C. I left it. Next, GIGAPACK

Using a Gold λ DNA caging kit (Stratagene, 200216), the above reaction product (recombinant; containing Igtll DNA) was subjected to an in vitro caging reaction to prepare a cDNA library (Lot. B). Example 3: Cloning of non-A non-B hepatitis virus antigen gene The reaction between plaques in the cDNA library (Lot.B) and the primary and secondary antibodies was determined by using a commercially available screen 'Immunsk Cleaning System (Amersham). , RPN 1281Z). The primary antibody used for screening was bull serum from patients with non-A and non-B hepatitis (5 acute hepatitis recovery periods and 5 chronic hepatitis) in E. coli lysate (Bio'Rad, After absorbing with 17-3205) (2 mg lysate Zml serum), this was diluted 20-fold with solution A (10 mM Tris-CI, pH 7.5, 150 mM NaCl, 2% (W / V) BSA). Peroxidase-labeled goat anti-human (IgG) IgG (Kappel, 3201-0081), diluted 500-fold with solution A, was used as the secondary antibody. Color reaction is commercially available The detection was performed using the GAR-HRP (Bio'Rad, 17-3556). From about 500,000 plaques, 10 positive plaques (colored blue-purple on a nitrocellulose filter) were isolated. Two clones having a stronger relative signal were isolated from them (named Y19 and Y22), and the following analysis was continued.

Example 4: Antigen specificity test of clone Y19

(1) Imnoplaque Atsushi

A mixture of Y19 clones and negative clones (; gt11 parent strain) mixed at a ratio of 2: 8 was plated, and non-A non-B chronic chronic hepatitis patients (previously transfused) using the above-mentioned Imnosk cleaning method. Serum (5), non-A non-B acute hepatitis patient (no transfusion history) Serum (6), non-A non-B chronic hepatitis patient (no transfusion history) Serum (5), normal control serum (26 ), Sera of chronic hepatitis B patients (9), and sera of acute hepatitis A patients (2). The results are shown in Table 1.

table 1

The results showed that clone Y19 specifically reacted only with non-A, non-B hepatitis patient serum. That is, it was confirmed that clone Y19 encodes an antigen (epitope) that is immunologically recognized by serum derived from a non-A non-B hepatitis patient. Also, this antigen is in the acute phase It was suggested that the sera of non-A non-B hepatitis patients also reacted with a high rate. (2) Western Blot Atsey

λ gtll (parent strain) Lysogen and recombinant; igtllY19 Lysogen preparation and expression of 3-gal and / 3-gal fusion antigen polypeptides were performed according to the method described in Experiment 3, p76. Was. IgtllY19 lysogen is named Escherichia coli Y1089 (λgtllY19), and the strain has been deposited as follows.

(B) Name of the depositary organization · Address

Name: Research Institute of Microbial Technology, Ministry of International Trade and Industry Address: 1-3-1, Higashi 1-3-chome, Tsukuba, Ibaraki, Japan

(Postal code 305)

(Mouth) Deposit (Original deposit date) July 16, 1990

(C) Accession No. Jiken-Jojo No. 3454 (FERM BP-3454) After induction of expression, lysogens were supersonically disrupted, and the protein concentration of the lysate was measured using Protein Assay (Bio-Rad, 500-0001 ). A protein equivalent to 5 zg from each lysate was applied to SDS-PAG plate 4-20 (Daiichi Kagaku, 120476), and after electrophoresis, Western blotting was performed using a semi-dry electroplotter (Intergrated Separation Systems: ISS). became. The sera listed in Table 1 were used as the primary antibodies, and the reactivity of the sera to the S-gal and / 3-gal fusion antigens was determined using the immunoscreening method described in Example 3. Examined. As a result, a color reaction was observed only in the combination of the / 3-gal fusion antigen polypeptide (about 10 OKd) and non-A, non-B hepatitis patient serum (positive rate is the same as the result in Table 1). The antigen specificity of the polypeptide was confirmed. Example 5: Determination of the nucleotide sequence of cDNA derived from clone Y19 and the sequence of the antigen polypeptide encoded in the sequence

The clone Y19 was propagated by the plate lysate method described in 2.118 of Experiment 1, and then phage DNA was prepared. 5 μg fur After the DNA was digested with EcoRI, the DNA was purified by phenol extraction and ethanol precipitation. 3 after the g of EcoRI digest was treated alkaline phosphatase, were performed 5 'end labeled with T4 kinase and 7 ³² P- ATP. After purifying the DNA fragment by phenol extraction and ethanol precipitation, 5.0% polyacrylamide gel electrophoresis was performed, and the length of the cDNA excised by EcoRI digestion was examined by autoradiography. As a result, the size of the cDNA was found to be about 400 base pairs. Next, a Τ4 ligase reaction was performed with the above-mentioned EcoRI digest and pTZ19 (Pharmacia LKB, 27-4986-01) dephosphorylated after EcoRI digestion. The reaction solution was transfused into Escherichia coli JM109 (Takara Shuzo, 9052), and a plasmid having an insert of about 400 base pairs was isolated from the transformants (designated ρΤΖ19-Y19). The nucleotide sequence of the cDNA incorporated into ρΤΖ19-Y19 was determined by performing a sequence reaction using a DyeDeoxy Terminator Taq Sequencing Kit (ABI, 401070) and running with a DNA sequencer (ABI.373A). The sequence was determined using both M13 primer M4 (Takara Shuzo, 3832), M13 primer RV (Takara Shuzo, 3830) and synthetic primer E 1 (5'-GGAGACGTCGTTGTTGT-3 '), E2 (5'-GCCTGTCATCAGAGCGT-1 3'). The chains were read and determined. In addition, synthetic oligonucleotides were prepared using a 380A DNA synthesizer (ABI) and purified according to the attached instructions.

In addition, in order to confirm the orientation of the cDNA on the recombinant Agtll, the base sequence was determined by directly transforming the phage DNA into a 鍀 type using the primers and the reverse primers (New England Biolabsl218, 1222). Was. Based on these results, the nucleotide sequence encoding the antigen that specifically reacts with the serum of non-A non-B hepatitis patients is shown in Formula (IX), and the amino acid sequence of the antigen is shown in Formula (I). Was decided. The amino acid sequence is obtained by translating the base sequence of formula (IX) from the first position. Example 6: Antigen specificity test of clone Y22

(1) Imnoplaque Atsushi

A mixture of clone Y22 and negative clones at a ratio of 2: 8 was plated, and non-A non-B chronic chronic hepatitis (previous ring blood) serum (5 blood samples) was applied using the immunoscreening method described above. ), Non-A non-B acute hepatitis patients (no transfusion history) Serum (6 patients), non-A non-B chronic hepatitis patients (no transfusion history) sera (5 patients), normal control serum (26 patients) ), Sera from chronic hepatitis B (9 patients) and sera from acute hepatitis A (2 patients) were examined. Table 2 shows the results. The results showed that clone Y22 specifically reacted only with non-A, non-B hepatitis patient serum.

Table 2

That is, it was confirmed that clone Y22 encodes an antigen (epitope) that is immunologically recognized by serum from a non-A non-B hepatitis patient.

(2) Western plot technology

λ gtll (parent strain) lysogen and recombinant λ gtll Y22 lysogen preparation, cultivation of the lysogen described above, and induction of expression of the ^ -gal and 3-gal fusion antigen polypeptides were described in Experiment 3, _p . Performed according to the method. The λ gtllY22 lysogen was named Escherichia coli Y 1089 (λ gtllY22), and Deposited as follows.

(B) Name of the depositary organization · Address

Name: Research Institute of Microbial Technology, Ministry of International Trade and Industry Address: 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan (Postal code: 305)

(Mouth) Deposits (Original deposits) July 16, 1990

(C) Accession No. Jiken No. 3453 (FERM BP-3453) After induction of expression, lysogens were sonicated and the protein concentration of the lysate was measured using Protein Assay. A protein equivalent to 5 // g from each crushed solution was subjected to SDS-PAGE, and after electrophoresis, nitrocellulose (Sehlicher & Schuell, BA85) Western blot was performed using a semi-dry electroplotter (Integated Separation Systems). Various sera listed in Table 2 were used as primary antibodies, and the reactivity of the various sera to the iS-gal, -gal fusion antigen was examined by the immunoscreening method described in Example 3. As a result, a color reaction was observed only in the combination of the ^ -gal fusion antigen polypeptide (about 10 OOKd) and non-A, non-B hepatitis patient serum (positive rate is the same as the result in Table 2). The antigen specificity of the polypeptide was confirmed. Example 7: Determination of nucleotide sequence of cDNA derived from clone Y22 and antigen polypeptide sequence encoded in the sequence

After subcloning the cDNA region of clone Y22 into pTZ 19 according to the method of Example 5 (ρΤΖ19-Υ22), the nucleotide sequence was determined according to the method of Example 5. For determination, both strands were read using M13 Primer 1-4 (Takara Shuzo, 3832) and M13 Primer RV (Takara Shuzo, 3830) to determine.

In addition, recombination; the orientation of the cDNA on Igtl was determined using the EcoRI site primer forward and reverse (New England Biolabs, 1218, 1222); Array The decision was made and decided. Based on these results, the nucleotide sequence encoding an antigen that specifically reacts with the serum of a non-A non-B hepatitis patient is represented by formula (X), and the amino acid sequence of the antigen is represented by formula (Π) ). Example 8: Expression of Y19 antigen in E. coli

(1) Preparation of DNA fragment encoding Y19 antigen

Based on the determined nucleotide sequence of the Y19 antigen, a pair of PCR primers was prepared that hybridize to the N-terminal and C-terminal [19-η: 5 '-1c: 5'-AAAAGCTTGGATCCTTAGCGTTCCCCTGGAGTCACATACCT] 19-η Primers have BamHI and Ncol sites, and the 19-c primer has stop codons, BamHI and Hindlll sites. Heat treatment (95 minutes, 10 minutes); PCR was performed using GeneAmp DNA amplification reagent kit (PERKIN ELMER CETUS, N801-0055) with 40 pmol of each primer for PCR added to the aqueous solution of IgtllY19 phage (94 °). C, 1 minute, 45 ° C. 2 minutes, 72. C, 3 minutes 35 cycles). The reaction mixture was extracted with phenol-X-form, and ethanol was added to the aqueous phase to sediment DNA fragments.

(2) Create pK2-Y19

The PCR product obtained in (1) was treated with Τ4 kinase by a conventional method, and then ligated using Τ4 ligase. The reaction solution was extracted with a phenol micropore form, and ethanol was added to the aqueous phase to precipitate DNA fragments. Next, the DNA was double-digested with Ncol and Hindlll, and the digest was electrophoresed on 1.2% agarose (SeaKem GTG Agarose: Takara Shuzo, 5160). A band of about 400 base pairs was cut out, and DNA was recovered using SUPREC-01 (Takara Shuzo 9040). Next, the 400 base pair DNA fragment was ligated to pKK233-2 expression vector (Pharmacia, 27-5005-01), which had been double digested with Ncol and Hindlll, using Τ4 ligase. E. coli JM109 strain Was transformed. From the transformants that appeared on the ampicillin plate, a clone containing pKK233 (pK2-Y19) having an insert of 400 bases was obtained (JM109 / pk2-Y19: hereinafter referred to as strain K19). A clone containing pKK233-2 was also prepared for control (JM109 / pKK233: hereinafter referred to as Kc strain).

(3) Expression of Y19 antigen in E. coli

Culture the K19 and Kc strains obtained in (2) in a 2xYT medium (containing 50ug / ml Amp) (37 ° C, 160rpm) so that when OD600 reaches about 1.0, IPTG will reach a final concentration of 0.5mM. Was added. After continuing the culture for 4 hours, the cells were collected by centrifugation. Next, the collected bacteria were suspended in an L solution (50 mM Tris-HC1, pH 8.0, 2 mM EDTA, 100 mM NaCl) at an OD600 of 20 / ml, and sonicated. The protein concentration in the lysate was measured using Protein Assay. The lysate containing 10 zg of protein and a molecular weight marker were electrophoresed on two sets of SDS-PAGE plates 10Z20 (Daiichi Kagaku, 120483). One was stained with CBB by a conventional method, and the other was Western-blotted to a nitrocellulose filter (Schleicher & Schuell, BA85) using an ISS semi-dry electroplotter (Daiichi Kagaku, SS110201). The ditrocellulose filter was reacted with a pool serum of a non-A non-B hepatitis patient according to the method described in Example 3. Peroxidase-labeled goat anti-human (IgG) IgG (Kappel, 3201-0081) was used as the secondary antibody. As a result, in the Western blot, coloring was observed only at the K19 strain at a site with a molecular weight of 17,000. In CBB staining, a new band, which was not observed in the Kc strain, was detected at a site with a molecular weight of 17,000 in the K19 strain. That is, the K19 strain expressed an antigen (Y19 antigen) that retained reactivity with patient serum.

In addition, using a similar method, the Y19 antigen was converted to pED3d (Experiment 6, p60-89) using the T7 promoter, pGEMEX-11 (Promega, P2211), and pMAL-c (New England Biolabs, # 800) can be used to express in E. coli cells. Example 9: Expression of Y22-19 complex antigen in E. coli

(1) Preparation of DNA fragment encoding Y22 antigen

Based on the determined nucleotide sequence of the Y22 antigen, a pair of PCR primers was prepared that hybridize to the N-terminal and C-terminal [22-n: 5'-

22-c: 5'-AGCCATGGCGCCACCGTCCTTCCAGGGCTCTAACAGTGG] The 22-n primer has a BamHI site and a Pstl site, and the 22-c primer has an Ncol site. To the heat-treated (95 ° C, 10 minutes) λgtllY22 phage aqueous solution, 40 pmol of each primer for PCR was added, and PCR was performed using the GeneAmp DNA amplification reagent kit (94 ° C, 1 minute, 45 ° C, 35 cycles of 2 minutes, 72 ° C, 3 minutes). The reaction solution was extracted with phenol / chloroform, and ethanol was added to the aqueous phase to precipitate DNA fragments.

(2) Creating pKY22-19

After the PCR product obtained in (1) was digested with Ncol, the reaction solution was extracted with form of funinol in the mouth, and ethanol was added to the aqueous phase to precipitate DNA fragments. Similarly, after digesting the PCR product obtained in Example 8 (1) with Ncol, the reaction solution was extracted with phenol Z chloroform, and ethanol was added to the aqueous phase to precipitate fragments. Next, after both were ligated with T4 ligase, the reaction solution was extracted with phenol Z chloroform, and ethanol was added to the aqueous phase to precipitate the ligated product. Next, the ligated product was subjected to double digestion with Pstl and Hindlll, and the digest was electrophoresed on 1.2% agarose (SeaKem GTG Agarose: Takara Shuzo, 5160). A band of about 550 base pairs was cut out, and DNA was recovered using SUPREC-01 (Takara Shuzo, 9040). Next, this 550 bp DNA fragment was double digested with Pstl and Hindlll to obtain a pKK233-2 expression vector (Pharmacia, 27-5005). Ligation was performed using T4 ligase. Escherichia coli JM109 strain was transformed using this ligation product. A clone containing ρ に 233 (ρΚΥ22-19) with an insert of 550 bases was obtained from the transformants that appeared on the ampicillin plate (JM109 / pKY22-19: hereinafter referred to as strain K2219).

(3) Expression of Υ22-19 complex antigen in E. coli

When the K2219 strain obtained in (2) and the Kc strain obtained in Example 8 (2) were cultured (37 ° C, 160 rpm) in a 2χΥΤ medium (containing 50 ug / ml Amp), the OD600 reached about 1.0. Was added to a final concentration of 0.5 mM. After continuing the culture for 4 hours, the cells were collected by centrifugation. Next, each of the collected bacteria was suspended in an L solution (50 mM Tris-HC1, pH 8.0, 2 mM EDTA, 100 mM NaCl) so as to have an OD600 of 20 / ml, and ultrasonically disrupted. The protein concentration in the lysate was measured using the Protein Assay. The lysate containing 10 / g of protein and the molecular weight marker were electrophoresed on two sets of SDS-PAGE plates 10/20 (Daiichi Shigaku, 120483). One was stained with CBB by a conventional method, and the other was subjected to Western blotting in the same manner as in Example 8 (3) to react with non-A, non-B hepatitis patient pool serum. As a result, in the Western blot, color development was observed only at the K2219 strain at a site with a molecular weight of 26,000. In CBB staining, a new band was detected at a site with a molecular weight of 26,000 in the K2219 strain, which was not observed in the Kc strain. That is, the K2219 strain expressed an antigen (Y22-19 antigen) that retained the reactivity with the patient's serum.

Also, using a similar method, the Y22-19 antigen can be expressed using pED3d, pGEMEX-11, pMAL-c, and the like. Example 10: Purification of Y19 antigen from E. coli

6 L of E. coli transformed with the expression vector containing the Y19 antigen gene prepared in Example 8 was cultured. IPTG was added to a final concentration of ImM at an OD600 of 1.0, and the cells were collected 20 hours later. Fungus The cells were suspended in lysozyme-containing (lmg / ml) L solution to an OD600 of 10 / ml, and then freeze-thawed twice. The melt was sonicated, and the lysate was centrifuged (9000 × _g , 10 minutes) to collect the precipitated fraction. The sedimented fraction was again suspended in an L solution (without lysozyme), and the precipitated fraction was collected by centrifugation. After repeating this operation twice, the precipitate fraction was dissolved in 50 mM Tris-HC1, ρΗ8.4, ImM EDTA, and 6 M guanidine hydrochloride, and the centrifuged supernatant (200,000 × g, 30 minutes) was added to the C solution (0.1). M Tris-HC1, pH 8.4, ImM EDTA, 2M Urea). After dialysis, the mixture was centrifuged again, and the supernatant was applied to a Q Sepharose column (30 ml gel volume). The unbound fraction was then applied to an S Sepharose column (30 ml gel volume) and eluted with a 0.0 to 0.5 M salt gradient. The peak fraction eluted at about 0.2M salt concentration was collected, and ammonium sulfate was added to a final concentration of 1M. This solution was then poured onto a Phenyl Sepharose column. The adsorbed fraction eluted with a C solution containing 0.05M ammonium sulfate. The eluted fraction was dialyzed against the C solution and concentrated on a S Sepharose column. About 7 mg of purified Y19 antigen was obtained from 6 L of culture. The purity of the purified protein was estimated to be 98% or more based on the results of SDS-PAGE CBB staining. In addition, 1 g of the purified protein was subjected to Western blotting after SDS-PAGE, and reacted with the pooled serum of non-A non-B hepatitis patients and the pooled serum of normal subjects according to the method described in Example 3. As a result, no contamination of E. coli-derived protein that could react with normal human sera was observed in the purified protein. When reacted with pooled sera from non-A, non-B hepatitis patients, only a molecular weight of about 17,000 band developed. Example 11: Detection of non-A non-B hepatitis virus antibody by Y19 ELISA

A non-A, non-B hepatitis virus antibody (Y19 antibody) in the serum was detected by a Y19 ELISA method (Y19 ELISA method) using the Y19 antigen obtained in Example 10. Coating mouthwash containing Y19 antigen (1 Z g / ml) [0. 1M sodium bicarbonate (PH9.5)] was added at 200 / zl to the immobilized plate (Nunc, 439454) overnight 4. After incubating with C, the solution was removed. Add 250 1 of blocking buffer [PBS (10 mM phosphate buffer, 137 mM sodium chloride, 2.7 mM potassium chloride, pH 7.4), 1% bovine serum albumin (BSA)] to the gel, and add 4% Placed. The solution was removed and washed three times with a washing buffer [PBS, 0.05% Tween 20]. 37. To the washed well, add the sample 200 1 diluted 20-fold with the sample diluent [5.5 volumes PBS, 0.5% BSA, 1% Triton X-100]. C, incubated for 1 hour. Next, the gel washed 5 times with the washing buffer was washed with 10,000 labeled antibody solution (peroxidase-labeled goat anti-human (IgG) IgG using a labeled antibody diluent [wash buffer containing 0.5% BSA] in 10,000 buffer). (Diluted 1: 2 solution) at 37 for 1 hour. After washing 5 times with washing buffer, substrate reaction solution [0.4mgr / ml 0-phenylene Renjiamin'ni hydrochloride and 0.01% H ₂ 0 ₂ containing phosphoric acid-Kuen acid lOOmM slow銜液, PH5. 0] 200 1 was added, and the mixture was incubated at room temperature for 30 minutes in the dark. The reaction was stopped by adding 501 of 4.5 M sulfuric acid, and the absorbance was measured at 492 nm.

(1) Reactivity with healthy human serum

Specimens obtained from healthy subjects with normal liver function (27 subjects) were examined using the Y19 ELISA method described above. The results obtained with these samples are shown in a histogram showing the absorbance distribution (Fig. 1). As can be seen from FIG. 1, all samples showed absorbances between 0.1 and 0.5. The average value of the absorbance of the 27 healthy subjects was 0.274 and the standard deviation was 0.086. All of these samples were included between the mean ± 3-fold standard deviation.

(2) Reactivity in patients' sera by liver disease

Using the above-mentioned Y19 ELISA method, non-A non-B acute hepatitis patients (no transfusion history) sera (6 patients), non-A non-B chronic hepatitis patients (no transfusion history) sera (5 patients), non-A Non-chronic hepatitis B serum (previous ring blood) serum (5 subjects) was examined. Table 3 shows the results of comparison of the number of positive samples with the C100 antibody. C100 Antibodies were measured using a commercially available HCV antibody ELISA test (Autho Diagnostics). Table 3

As is clear from Table 3, the Y19 antigen showed a strong reactivity (mean 1.639, standard deviation 0.206) with all non-A non-B liver disease samples. The C100 antigen did not react with two sera of patients with non-A non-B liver disease (one serum of non-A non-B acute hepatitis patients and one serum of non-A non-B chronic hepatitis patients). Measurement of non-A, non-B hepatitis virus antibodies using the Y19 antigen resulted in a higher detection rate than the C100 antigen.

(3) Y19 antibody measurement in non-A non-B chronic hepatitis patient serum

Using the Y19 ELISA method described above, a preliminary clinical study was conducted on 122 sera of patients with non-A non-B chronic hepatitis. The results are shown graphically in comparison with the C100 antibody (Figure 2). As is evident from FIG. 2, there are quite a few samples with high absorbance of Y19 antibody activity (absorbance of 1.0 or more) despite low absorbance of C100 antibody activity (absorbance of 0.5 or less). On the other hand, only two samples showed high absorbance of C100 antibody and low absorbance of Y19 antibody activity. This indicates that the Y19 antigen is more useful than the C100 antigen in diagnosing non-A, non-B hepatitis patients. Example 12: Detection of non-A non-B hepatitis virus antibody by ELISA using synthetic polypeptide

(1) Synthesis of polypeptide

Based on the amino acid sequence determined in Example 7 (see formula (II)), the following formulas (ΙΠ) (VII) and (XII) selected to include a relatively hydrophilic region and Example 7 The control polypeptide represented by the formula (II) not containing the amino acid sequence (see formula (II)) determined in the above was synthesized as follows. (III)

Leu Asp Ser Phe Asp Pro Leu Arg Ala Glu

Glu Asp Glu Arg Glu Val Ser Val A la Ala

Glu lie Leu Arg Arg Ser Arg Lys

(Y22-3 antigen)

(IV)

Ser Val Ala Ala Glu lie Leu Arg Arg Ser

Arg Lys Phe Pro Ala Ala Leu Pro lie Trp

Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu

Glu Pro Trp Lys Asp (Y22-7 antigen)

(V)

Glu lie Leu Arg Arg Ser Arg Lys Phe Pro

Ala Ala Leu Pro lie Trp Ala Arg Pro Asp

Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys

Asp (Y22-6 antigen) (VI)

Arg Arg Ser Arg Lys Phe Pro Ala Ala Leu

Pro lie Trp Ala Arg Pro Asp Tyr Asn Pro

Pro Leu Leu Glu Pro Trp Lys Asp

(Y22-5 antigen)

(VII)

Ser Arg Lys Phe Pro Ala Ala Leu Pro lie

Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp (Y22-4 antigen)

(XII)

Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp

(XIII)

Ala Asn Gly Val Gin Leu Arg Asp Asn Gin

Leu Val Val Thr Ser Glu Gly Leu Tyr Leu lie Tyr Ser Gin Val Leu Ser Lys Gly Gin

Using a Gly Applied Biosystems, Inc. peptide synthesizer Model 430A, and using reagents and solvents marketed by the company, solid phase synthesis was performed according to the standard operating program of the machine. The t-Boc amino acids used were all L-type and the protecting group was Mts (mesitylenesulfonyl group) for Arg, OBzl (benzyl ester) for Asp or Glu, and C1—Z (p— Benzoyloxycarbonyl group), Bzl (0-benzyl group) for Ser, Br—Z (bromo-benzyloxycarbonyl group) for Tyr, and unprotected other t-Boc amino acids ¾ Used. Polypeptides of the formulas (III) and (XIII) are prepared by using triammonium phenylacetamidomethyl (Pam) resin obtained by force-pulping t-Boc-Lys and t-Boc-Gly, respectively. The removal of the t-Boc group, the activation of t-Boc amino acid to be linked with Ν, Ν-dicyclohexylcarbodiimide, and the coupling were sequentially repeated to synthesize. However, for the activation of t-Boc-Arg, Ν, N-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole were used in equal amounts. The peptides of the formulas (IV) to (XII) were synthesized in the same manner using a Pam resin obtained by force-pulping t-Boc-Asp as a starting material. However, a mixed solution of trifluoracetic acid: triethylamine: dichloromethane (35: 1: 9) was used to remove the t-Boc group. Each polypeptide was released from the resin with trifluorosulfonic acid methanesulfonic acid according to the method described in the user's manual. The released polypeptide was freeze-dried to obtain a white or pale yellow-white solid.

These polypeptides were analyzed by high performance liquid chromatography. Using an R— ODS—5 column (4.6 ø X 250 mm, Yamamura Chemical), from solution A (¾0, 0.1% trifluorosulfonic acid) 0% to solution B [acetonitrinol: 0 (70:30) The elution time of the main peaks of the polypeptides of formulas (III) to (VII) and (ΧΠ) and (XIII) was increased by linear gradient elution of 35% over 100% of 0.1% trifluoroacetic acid. Approximately 26.2 minutes, 30.7 minutes, 29.2 minutes, 33.0 minutes, 29.7 minutes, 28.4 minutes, and 29.1 minutes, respectively.

b) Detection of non-A non-B hepatitis virus antibody by ELISA

Non-A, non-B hepatitis virus antibodies were detected in the serum using an ELISA method using the synthetic polypeptide as an antigen. Coating buffer [0.1 M sodium bicarbonate (pH 9.5)] containing each synthetic polypeptide (10 g / ml) was added to immobilized plate (Nunc, 439454) in 100/1 ratio. Then 4 overnight. After incubation with C, the solution was removed by suction. Add 250 // 1 blocking buffer [PBS (l OmM Phosphate buffer, 137 mM sodium chloride, 2.7 mM chloride, pH 7.4) and 1% bovine serum albumin] were added, and the mixture was kept at room temperature for 3 hours. The solution was removed and washed three times with a washing buffer (PBS, 0.05% Tween 20). To the washed genil, add bovine serum from a non-A, non-B hepatitis patient diluted 30 times with a washing buffer containing 0.25% BSA or 100/1 bull serum from a healthy person as a control at 4 ° C overnight. Incubated. Then, the gel washed 5 times with the washing buffer was added to a 100/1 secondary antibody solution (a solution of peroxidase-labeled goat anti-human (IgG) IgG diluted 3000-fold with the washing buffer). Treated at room temperature for hours. After washing 5 times with wash loose衢液, substrate reaction solution (0.4 mg / ml 0-Fuwenirenjiami Nni hydrochloride and phosphoric acid-Kuen acid buffer l OOmM containing ¾0 ₂ of 0.01%, pH 5.0 ) 200/1 was added and incubated at room temperature in the dark for 10 minutes. The reaction was stopped by adding 501 of 4.5 M sulfuric acid, and the absorbance was measured at 492 nm.

Using pooled sera of non-A non-B hepatitis patients and pooled sera of healthy individuals, the polypeptide formulas (III) to (VII) and (VII) derived from the amino acid sequence of the polypeptide determined in Example 7 XII) was compared with the reactivity of the polypeptide of formula (XIII) containing no amino acid sequence determined in Example 7. The results are shown in a bar graph represented by absorbance (Fig. 3). As can be seen from FIG. 3, although the synthetic peptide of formula (XII) showed relatively low reactivity with pooled sera of non-A non-B hepatitis patients, the synthesis of formulas (III) to (VII) All peptides showed significantly higher (p <0.001, n = 3) reactivity with pooled sera from patients with non-A, non-B hepatitis compared to pooled sera from healthy individuals. On the other hand, the synthetic peptide of the formula (XIII) used as a control did not react with the pooled serum of the non-A non-B hepatitis patient serum. This indicates that the synthetic polypeptides of formulas (III) to (XII) are useful for specifically detecting serum antibodies of non-A non-B hepatitis patients. In addition, whether the formula (III) and the polypeptide of the formula (ΧΠ) are amino acid sequences of different regions of the antigenic peptide represented by the formula (Π) Thus, it is considered that at least two epitopes are present in the antigenic polypeptide represented by the formula (II) determined in the present invention. Example 13: Detection of non-A non-B hepatitis virus antibody by Y19 + 22 ELISA

An ELISA method using a triple antigen mixture in which a polypeptide (Y19 antigen) expressed in E. coli and purified by ion exchange chromatography etc. and a synthetic polypeptide (Y22-3 antigen, Y22-6 antigen) were mixed. Y19 + 22 ELISA method) was used to detect non-A non-B hepatitis virus antibodies in serum or plasma.

(1) Y19 + 22 ELISA assay

200 ml of a coated mouth bite solution [0.1 M sodium bicarbonate (pH9.5)] containing Y19 antigen (1 ^ gZml), Y22-3 antigen (1 / gZml), and Y22-16 antigen (10 // gXml) / zl was added to the wells of an immunoplate (Nunc, 439454), incubated at 4 ° C overnight, and the solution was removed. Add 250 μl of blocking buffer [PBS (10 mM phosphate buffer, 137 mM sodium chloride, 2.7 mM chloride, pH 7.4), 1% bovine serum albumin (BSA)] to the gel at 4 ° C overnight. Put it in C. Thereafter, after reacting with the secondary antibody according to the method described in Example 11, the color was similarly developed, the reaction was stopped by adding 50/1 4.5 M sulfuric acid, and the absorbance was measured at 492 nm.

(2) Reactivity with healthy human serum

Specimens obtained from healthy subjects with normal liver function (29 subjects) were examined using the Y19 + 22 ELISA method described above. The results obtained with these samples are shown in histograms showing the distribution of OD values (Fig. 4). As can be seen in FIG. 4, all samples showed absorbances between 0.1 and 0.4. The average value of the absorbance of the 29 healthy subjects was 0.201, and the standard deviation was 0.072. All of these samples were included between the mean ± 3-fold standard deviation.

(3) Reactivity in patient serum by liver disease Using the above-mentioned Y19 + 22 ELISA method, non-A non-B acute hepatitis patients (no transfusion history) Serum (6 patients), non-A non-B chronic hepatitis patients (no ring blood history) Serum (5 people) The sera of non-A and non-B chronic hepatitis (previously transfused) sera (5 patients) and the sera of patients with chronic hepatitis B (5 patients) were examined. The results are shown by comparing the number of positive samples with the C100 antibody (Table 4). The cut-off value of the Y19 + 22 ELISA was 492 nm absorbance 0.5. This value is 4.15 times the standard deviation of the average value of absorbance of healthy persons measured in Example 13 (2).

Table 4

As is clear from Table 4, the Y19 + 22 antigen showed strong reactivity (mean 1.763, standard deviation 0.061) with all non-A non-B liver disease samples. The C100 antigen did not react with two sera of patients with non-A non-B liver disease (one serum of non-A non-B acute hepatitis patients and one serum of non-A non-B chronic hepatitis patients). Measurement of HCV antibody using Y19 + 22 showed a higher detection rate than C100 antibody. That is, it is useful for the detection of non-A non-B hepatitis virus antibodies that could not be detected conventionally.

(4) Measurement of Y19 + 22 antibody in sera of non-A non-B chronic hepatitis patients Using the above-mentioned Y19 + 22 ELISA, a preliminary clinical study was conducted on 122 sera of non-A non-B chronic hepatitis patients. . The results are shown graphically in comparison with the C100 antibody (Figure 5). As is clear from Fig. 5, although the absorbance of C100 antibody activity is low (absorbance of 0.5 or less), the absorbance of Y19 + 22 antibody activity is high (absorbance of 1.0 or more). It turns out that there exists quite a. On the other hand, only three samples showed high absorbance of C100 antibody and low absorbance of Y19 + 22 antibody activity. This indicates that the Y 19 +22 antigen is effective for specifically detecting non-A non-B hepatitis virus antibody.

(5) Comparison of Y19 + 22 antibody measurement ELISA method and core antibody measurement ELISA method

a) Polypeptide synthesis of core antigen (CP-9, CP-10)

CP-9 and CP-10 likely to contain the HCV core region epitope [Hiroaki Okamoto Liver 31 suppl. 40 1990, H. Okamoto et.al. Japan J. Exp. Med., 60.223 (1990)] Was synthesized according to the method of Example 12.

CP-9

Arg Arg Gly Pro Arg Leu Gly Val Arg Ala

Thr Arg Lys Thr Ser Glu Arg Ser Gin Pro

Arg Gly Arg Arg Gin Pro lie Pro Lys Val

Arg Arg Pro Glu Gly Arg

CP-1 10

Pro Lys Pro Gin Arg Lys Thr Lys Arg Asn

Thr Asn Arg Arg Pro Gin Asp Val Lys The elution times of the main peaks of the above two polypeptides were about 20.0 minutes and 16.7 minutes, respectively.

b) Measurement method of CP9 + 10 ELISA

Add coating buffer containing CP-9 (1 μg / ml) and CP-10 (5 βg / ml) to the immunoplate. The subsequent operation was performed in the same manner as in Example 13 (1).

c) Measurement of Y19 + 22 antibody in serum of non-A non-B chronic hepatitis patients Comparison of CP9 + 10 antibody measurement

Further, CP9 + 10 antibody measurement was performed on 122 sera of patients with non-A non-B chronic hepatitis using the above-mentioned CP9 + 10 ELISA method. The Y19 + 22 antibody measurement results were obtained in Example 13 (4). Table 5 shows the results. Table 5

The Y19 + 22 antibody positive rate was 89.3%, and the CP9 + 10 antibody positive rate was 79.5%.丫 The HCV antibody positive rate was higher in the 19 + 22 ELISA method than in the CP9 + 10 ELISA method. The agreement between the Y19 + 22 antibody measurement and the CP9 + 10 antibody measurement was + Z + 93 cases (76.2%). Some specimens were positive only for Y19 + 22 or only CP9 + 10. The discordant cases were +/- 16 cases (13.1%) and one Z + 4 cases (3.3%). The above results indicate that the Y19 + 22 antigen is useful for specifically detecting serum antibodies of non-A non-B chronic hepatitis patients. In addition, it shows that the detection rate can be further increased by further mixing the CP9 + 10 antigen with the Y19 + 22 antigen.

Example 14 Measurement of Y19 + 22 antibody in serum of patients with non-A and non-B cirrhosis

A preliminary clinical study of non-A non-B type cirrhosis serum (38 cases) was performed using the Y19 + 22 ELISA method of Example 13 (1). The results are shown in Table 6 together with the C100 antibody. Table 6

The positive rate of the Y19 + 22 antibody was 73.7%, and the positive rate of the C100 antibody was 68.4%. Y19 + 22 antibody measurement has been shown to be useful in diagnosing non-A non-B cirrhosis. Industrial applicability

The antigen polypeptides of the present invention and the method for measuring non-A non-B hepatitis virus antibodies using a mixture or conjugate thereof have a higher detection rate than the conventional antigen-based test method, and the existing antigens have a higher detection rate. It is useful for diagnosing non-A, non-B hepatitis virus-related diseases that could not be diagnosed (acute hepatitis, chronic hepatitis, liver cirrhosis, liver cancer, etc.), and helps determine treatment policies. In addition, the antigen polypeptide can supplement non-A non-B hepatitis virus healthy human carriers, which could not be detected with the existing antigen, and is useful for preventing the development of post-ring blood hepatitis. Further, using the antigen polypeptide, polyclonal antibodies and monoclonal antibodies that can be used for the detection of the virus antigen and the treatment of non-A, non-B hepatitis can be prepared. In addition, the antigen polypeptide can be used for production of a vaccine for prevention of virus infection and treatment after infection. The non-A, non-B hepatitis virus polynucleotide of the present invention is useful as a gene for producing the antigen. In addition, the presence or absence of the virus genome (particles) can be examined using the nucleotide sequence.

Claims

The scope of the claims

1. Antigen polypeptide Y19 group, antigen polypeptide Y22 group or its fragment group, composite antigen polypeptide group and antigen polypeptide Y19 group in which two or more antigens selected from these polypeptide groups are fused. Or a single antigen selected from the group of antigenic polypeptides having an epitope that can be regarded as immunologically identical to the epitope of the antigenic polypeptide contained in the group Y22, or a mixed antigen and a mixture of two or more antigens The presence of non-A non-B hepatitis virus antibody in a biological sample is determined by measuring the immunological conjugate formed by the reaction with the non-A non-B hepatitis virus antibody present in the biological sample. How to detect non-A non-B hepatitis virus antibodies

2. A single antigen or a mixed antigen obtained by mixing two or more antigens described in claim 1, and a mixed antigen or antigen polypeptide obtained by further mixing a non-A non-B hepatitis-associated antigen polypeptide. Group Y 19, antigen polypeptide Group Y22 or fragments thereof, and one or more antigens selected from group Y22 and non-A, non-B hepatitis-associated antigen polypeptide fused to antigen polypeptides and biology Immunological conjugate formed by reaction with non-A, non-B hepatitis virus antibody present in biological sample to confirm the presence of non-A, non-B hepatitis virus antibody in biological sample For detecting non-A non-B hepatitis virus antibodies

3. The detection method according to claim 1 or 2, wherein the method for measuring the immunological conjugate is an ELISA method.

4. Kit for analyzing the presence of antibodies of antigen polypeptides Y19 and Z or antigen polypeptide Y22, comprising, in a suitable container, antigen polypeptide Y19 and antigen polypeptide Beptide Y22 group or its fragment Group, a complex antigen polypeptide group in which two or more antigens selected from these polypeptide groups are fused, and an antigen polypeptide antigen polypeptide included in the Y19 group or the Y22 group Kits containing a single antigen selected from the group of antigenic polypeptides having epitopes that can be considered to be chemically identical or a mixed antigen obtained by mixing two or more antigens

5. A kit for analyzing the presence of antibodies of the antigen polypeptide Y19 group and Z group or the antigen polypeptide Y22 group, wherein the single antigen according to claim 1 is placed in a suitable container. Alternatively, a mixed antigen obtained by mixing two or more antigens and a non-A non-B hepatitis-related antigen polypeptide, or a mixed antigen or antigen polypeptide Y19 group, antigen polypeptide Y22 group Or a kit having a complex antigen polypeptide obtained by fusing one or more antigens selected from the fragment group with a non-A non-B hepatitis-related antigen polypeptide.

6. Non-A non-Hepatitis B virus antigen polypeptide Y19 group represented by the following amino acid sequence

N-Xi lie Pro lie Glu Al a lie Ly s G l y G ly

Arg His Leu lie Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Ser Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser lie lie Pro Thr Ser G ly Asp Val Val Val Val Ala Thr Asp

Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val lie Asp Cys Asn Thr Cys Val Thr Gin Thr Val Asp Phe Ser Leu Asp Pro

Thr Phe Thr lie Glu Thr Thr Thr Val Pro Gin Asp Ala Val Ser Arg Ser Gin Arg Arg Gly Arg Thr Gly Arg Gly Arg Gly Gly lie Tyr Arg Tyr Val Thr Pro Gly Glu Arg (wherein, X, is a hydrogen atom at the N-terminus or any of 1 to 20 It means amic acid.)

7. Non-A non-B hepatitis virus antigen polypeptide Y22 group represented by amino acid sequence below

N-X ₂ Leu Asp Ser Phe Asp Pro Leu Arg Ala

Glu Glu Asp Glu Arg Glu Val Ser Val Ala Ala Glu lie Leu Arg Arg Ser Arg Lys Phe Pro Ala Ala Leu Pro lie Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys A sp

(In the above formula, X ₂ represents a hydrogen atom at the N-terminus or any 1 to 20 amino acids.)

8. A fragment group of the polypeptide Y22 group according to claim 7, which is any one of non-A non-B hepatitis virus antigen polypeptides selected from the group consisting of amino acid sequences described below.

Leu Asp Ser Phe Asp Pro Leu Arg Ala Glu Glu A sp Glu Arg Gl u Val Ser Val A la Ala G lu lie Leu Arg Arg Ser Arg Lys

(Y22-3 antigen) Ser Val Ala Ala Glu lie Leu Arg Arg Ser

Arg Lys Phe Pro Ala Ala Leu Pro lie Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp (Y22-7 antigen)

Glu lie Leu Arg Arg Ser Arg Lys Phe Pro Ala Ala Leu Pro He Trp Ala Arg Pro Asp

Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp (Y22-6 antigen)

Arg Arg Ser Arg Lys Phe Pro Ala Al a Leu Pro lie Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp

(Y22-5 antigen)

Ser Arg Lys Phe Pro Ala Al a Leu Pro lie

Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp Lys Asp

(Y22-4 antigen)

9. A fusion antigen polypeptide having the amino acid sequence according to claim 6 or 7.

10. A fusion antigen polypeptide having the amino acid sequence according to claim 6 or 7, and fused to ^ -galactosidase (^ -gal)

1 1. A polypeptide having an epitope that can be considered to be immunologically identical to the epitope contained in the polypeptide according to claim 6 or 7.

12. A complex antigen polypeptide obtained by fusing two or more antigens selected from the group consisting of the polypeptide Y19 group, the polypeptide Y22 group, the fragment group of the polypeptide Y22 group, and the non-A non-B hepatitis-related antigen polypeptide group. Do

1 3. Non-A non-Hepatitis B virus antigen polypeptide Y22—19 group represented by the following amino acid sequence

N-X ₃ Leu Asp Ser Phe Asp Pro Leu Arg Ala

Glu Glu Asp Glu Arg Glu Val Ser Val Ala Ala Glu lie Leu Arg Arg Ser Arg Lys Phe

Pro Ala Ala Leu Pro lie Trp A la Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Pro Trp

Lys Asp Y lie Pro lie G lu A l a l i e Lys Gly G ly Arg His Leu lie Phe Cys Hi s Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys

Leu Ser Ser Leu Gly Val Asn A la Va A la

Tyr Tyr Arg Gly Leu Asp Val Ser lie lie Pro Thr Ser Gly Asp Val Val Val Val Ala

Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly

Asp Phe Asp Ser Val lie Asp Cys Asn Thr

Cys Val Thr Gin Thr Val Asp Phe Ser Leu

Asp Pro Thr Phe Thr He Glu Thr Thr Thr

Val Pro Gin Asp Ala Val Ser Arg Ser Gin

Arg Arg Gly Arg Thr Gly Arg Gly Arg Gly

Gly lie Tyr Arg Tyr Val Thr Pro Gly Glu

Arg

(In the above formula, X ₃ represents a hydrogen atom at the N-terminus or 1 to 20 arbitrary amino acids, and Y represents 1 to 20 arbitrary amino acids.)

14. A polynucleotide encoding the antigen polypeptide according to any one of claims 6 to 13.

15. Non-A non-Hepatitis B virus polynucleotide Y19 represented by the following nucleotide sequence

ATT CCC ATC GAG GCC ATC AAG GGG GGG AGG CAC CTC ATC TTC TGC CAT TCC AAG AAG AAG TGT GAC GAG CTC GCC GCG AAG CTG TCG TCC CTC GGA GTC AAT GCT GTA GCA TAC TAC CGG GGT CTT GAC GTG TCC ATC GCGA GGA GAC GTC GTT GTT GTG GCA ACA GAC GCT CTG ATG ACA GGC TAT ACC GGC GAC TTC GAC TCA GTG ATC GAC TGT AAC ACA TGT GTC ACC CAG ACA GTC GAT TTC AGC TTG GAC CCT ACA TTC ACC ATT GAG ACG ACA ACC GCC GAC GCG GTG TCG CGC TCG CAG CGG CGA GG C AGG ACT GGT AGG GGC AGA GGG GGC ATA TAC A GG TAT GTG ACT CCA GGG GAA CG G

16. Non-A non-Hepatitis B virus polynucleotide Y22 represented by the following nucleotide sequence:

CTG GAC TCT TTC GAC CCG CTT CGA GCG GAG

GAG GAT GAG AGG GAA GTA TCC GTT GCG GCG

GAG ATC CTG CGA AGA TCC AGG AAG TTC CCC

GCA GCA CTG CCC ATA TGG G CG CGG CCG GA T

TAC AAC CCT CCA CTG TTA GAG CCC TG G AAG GAC

17. Non-A non-B hepatitis virus polynucleotide Y22-19 represented by the following nucleotide sequence

C TG GAC TCT TTC GAC CCG CT T CGA GCG GA G GAG GAT GAG AGG GAA GTA TCC GTT GCG GCG GAG ATC CTG CGA AGA TCC AGG A AG TTC CCC GCA GCA CTG CC C ATA TG G GCG CGG CCG GAT TAC AAC CCT CCA CTG TTA GAG CCC TGG AAG GAC GGT GGC GCC ATG GCT ATT CCC ATC GAG GCC ATC AAG GGG GGG AGG CAC CTC ATC TTC TGC CAT TCC AAG AAG AAG AAG TGT GAC GAG CTC GCC GCG AAG CTG TCG TCC CTC GGA GTC AAT GTAC GCA GTC TAC CGG GGT CTT GAC GTG TCC ATC ATA CCG ACA AGC GGA GAC GTC G TT GTT GTG GCA ACA GAC GCT CTG ATG ACA GGC TAT ACC GGC GAC TTC GAC TCA GTG ATC GAC TGT AAC ACA TGT GTC ACC CAG ACA GTC GAT TTC AGC GAC CCT ACA TTC ACC ATT GAG ACG ACA ACC GTG CCC CAA GAC GCG GTG TCG CGC TCG CAG CGG CGA GGC AGG ACT GGT AGG GGC AGA GGG GGC ATA TAC AGG TAT GTG ACT CCA G GG GAA CGG

18. A polynucleotide probe for detecting a polynucleotide derived from a non-A, non-B hepatitis virus, comprising eight consecutive nucleotides derived from the sequence of the phase capture chain according to claim 15 or 16, or Polynucleotide probes containing longer sequences

19. A method for detecting a polynucleotide derived from non-A non-B hepatitis virus using the polynucleotide probe according to claim 18.

20. A pair of polynucleotide primers for detecting a polynucleotide derived from a non-A non-B hepatitis virus by reverse transcription-PCR, comprising the sequence according to claim 15 or 16 and a complementary strand thereof. A pair of polynucleotide primers containing a sequence consisting of 8 or more consecutive bases derived from

21. A method for detecting a polynucleotide derived from non-A and non-B hepatitis virus by reverse transcription-PCR using the pair of polynucleotide primers according to claim 20.

22. culturing a host cell transformed with a recombinant expression vector encoding the antigen or the complex antigen polypeptide according to claims 6 to 13 under conditions for expressing the polypeptide; Special production method of antigen polypeptide

23. The production method according to claim 22, wherein the expression vector is an expression vector for Escherichia coli and the host is Escherichia coli.