WO1998044130A1 - RECOMBINANT MICROORGANISMS EXPRESSING ANTIGENIC PROTEINS OF $i(HELICOBACTER PYLORI) - Google Patents

RECOMBINANT MICROORGANISMS EXPRESSING ANTIGENIC PROTEINS OF $i(HELICOBACTER PYLORI) Download PDF

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WO1998044130A1
WO1998044130A1 PCT/KR1998/000073 KR9800073W WO9844130A1 WO 1998044130 A1 WO1998044130 A1 WO 1998044130A1 KR 9800073 W KR9800073 W KR 9800073W WO 9844130 A1 WO9844130 A1 WO 9844130A1
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ctxa2b
gene
chimeric protein
pylori
vibrio cholerae
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PCT/KR1998/000073
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French (fr)
Inventor
Byung-O Kim
Sung-Seup Shin
Young-Hyo Yu
Myung-Hwan Park
Deok-Joon Choi
Hyung-Jin Jung
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Daewoong Pharmaceutical Co., Ltd.
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Priority claimed from KR1019970011951A external-priority patent/KR19980075705A/en
Priority claimed from KR1019970011950A external-priority patent/KR19980075704A/en
Application filed by Daewoong Pharmaceutical Co., Ltd. filed Critical Daewoong Pharmaceutical Co., Ltd.
Priority to AU65248/98A priority Critical patent/AU6524898A/en
Publication of WO1998044130A1 publication Critical patent/WO1998044130A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to chimeric proteins consisting of antigenic proteins of Helicobacter pylori and A2 andB subunits of Vibrio cholerae toxin, more specifically, to recombinant DNAs coding for antigenic proteins of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin, recombinant expression vectors containing the genes, a process for preparing the chimeric proteins employing the recombinant microorganisms transformed with the said expression vectors, and preventive and therapeutic vaccines comprising the chimeric proteins for Helicobacter pylori -associated diseases.
  • gastritis-associated diseases such as gastritis, gastric ulcer and duodenal ulcer are caused by various etiological factors, they are mainly caused by Helicobacter pylori (hereinafter, referred to as 'H. pylori ' ) colonizing m the junctional region of epithelial cells of stomach mucous membrane. It has been reported that 90% or more of Asians and 60% or more of Europeans are infected with H. pylori though there are local differences. Also, it has been known that recurrence of gastritis, gastric ulcer or duodenal ulcer is caused by drug-resistant H. pylori , which may give rise to the occurrence of gastric cancer (see : Timothy, et al., ASM News, 61:21(1995)).
  • H. pylori genes coding for antigenic determinants of H. pylori , e.g., urease gene (see : Timothy, et al . , Infection and Immunity, 59:1264(1991)), flagella gene (see : Leying, et al . , Molecular Microbiology, 6:2863(1992)), adhesin gene (see: Evans, et al .
  • a vaccine employing an urease gene has poor immunogenicity
  • a vaccine employing a vacuolating cytotoxin gene may have toxicity of cytotoxin itself, though it provides excellent immunogenicity
  • a vaccine employing a non-toxic varient gene of the vacuolating cytotoxin gene does not have effects on all over the strains of H. pylori , since the non-toxic varient gene does not appear in all H. pylori
  • a vaccine employing adhesin gene despite its excellent immunogenicity, does not have good efficacy, since it does not stimulate production of secretory IgAC'sIgA" .
  • H. pylori is controlled by slgA not by serum IgG, since it colonizes in the junctional region of epithelial cells of stomach mucous membrane.
  • the aforesaid vaccines cannot penetrate the mucous membrane of intestines easily, they are not able to stimulate mucosal immune system, which, in turn, results in decreased production of slgA.
  • serious problems have occurred that immunological effects of the vaccines against H. pylori decrease and the vaccines are easily denaturated by gastric acid(pH 1-2) to provide poor activities.
  • the present inventors first, prepared chimeric proteins expressed from recombinant DNAs which contain genes coding for antigenic determinants of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin. Also, they discovered that the chimeric proteins successfully solve the problems of the conventional vaccines and can be used as effective vaccines for H. pylori , based on their excellent immunogenicity for H. pylori , stability under stomach environment, and penetrating property through intestinal membrane to stimulate slgA production.
  • the first object of the invention is, therefore, to provide a series of DNA sequences prepared by ligating antigenic determinant coding genes of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin, and amino acid sequences translated therefrom.
  • the second object of the invention is to provide expression vectors comprising the said DNA sequences and recombinant microorganisms transformed therewith.
  • the third object of the invention is to provide a process for preparing chimeric proteins consisting antigen proteins of H. pylori and A2 and B subunits of Vibrio cholerae toxin from the said microorganisms.
  • the fourth object of the invention is to provide preventive and therapeutic vaccines for H. pylori -associated diseases employing the chimeric proteins prepared above.
  • Figure 1 shows a DNA sequence of a fusion gene prepared by ligating ureB gene of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin.
  • Figure 2 shows an amino acid sequence translated from the DNA sequence of Figure 1.
  • Figure 3 shows a DNA sequence of a fusion gene prepared by ligating cagA gene of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin.
  • Figure 4 shows an amino acid sequence translated from the DNA sequence of Figure 3.
  • Figure 5 is a schematic diagram showing construction strategy of an expression vector for UreB/CTXA2B, pHU044.
  • Figure 6 is a schematic diagram showing construction strategy of an expression vector for
  • FIG. 7 is a photograph showing 15% SDS-PAGE pattern of whole cell lysate of E. coli transformed with pHU044 expression vector.
  • Figure 8 is a photograph showing 15% SDS-PAGE pattern of whole cell lysate of E. coli transformed with pHC033 expression vector.
  • Figure 9 is a chromatogram showing comparison of serum IgG production of mice immunized with UreB/CTXA2B chimeric protein and UreB, respectively.
  • Figure 10 is a chromatogram showing comparison of secretory IgA production of mice immunized with UreB/CTXA2B chimeric protein and UreB, respectively.
  • Figure 11 is a chromatogram showing comparison of serum IgG production of mice immunized with CagA/CTXA2B chimeric protein and CagA, respectively.
  • Figure 12 is a chromatogram showing comparison of secretory IgG production of mice immunized with CagA/CTXA2B chimeric protein and
  • a gene of Vibrio cholerae toxin consists of genes coding for three subunits of Al , A2 and B.
  • Al subunit has a toxic activity of the toxin, and A2 and B subunits bind to host cell to stimulate production of slgA and guarantee stability of the protein under a surrounding environment.
  • vaccines employing A2 and B subunit genes of Vibrio cholerae toxin can be applied to human body, due to their tolerable characteristics, while various vaccines employing intact cholera toxin gene as a fusion partner, owing to toxic property of Al subunit, can not be used directly for human body.
  • the present inventors prepared chimeric proteins employing A2 and B subunit genes of Vibrio cholerae toxin and antigenic determinant coding genes of H. pylori whose products have excellent immunogenicity, in order to stimulate production of antibody to H. pylori .
  • pylori include ureB, cagA, alpA, alpB, fliQ, babAl , babA2 , ureC, ureD, ureA, sodB, urel, ureE, ureF, ureG, ureH, flaA, flaB, catA, vacA, and babB .
  • the antigenic determinant coding genes of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin were prepared by employing polymerase chain reaction (PCR) technique, respectively. Then, each gene was cleaved with EcoRI and said two genes were ligated with T DNA ligase . The fusion genes thus prepared were cleaved with restriction enzymes, and inserted into plasmid vectors to prepare respective recombinant expression vectors. Then, E. coli was transformed with each of expression vector, the recombinant E. coli was cultured, and chimeric proteins of antigenic proteins of H. pylori and A2 and B subunits of Vibrio cholerae toxin were obtained.
  • PCR polymerase chain reaction
  • chimeric proteins may be used as active ingredients of vaccines for prevention and treatment of H. pylori - associated diseases, diagnostic kits for H. pylori infection, and used in the production of anti-H. pylori antibody.
  • the chimeric proteins of the invention induce mucosal immune response to bring about infiltration of IgA antibodies and/or lymphocytes into gastric mucosa.
  • prevention of H. pylori infection or removal of H. pylori already infected can be accomplished.
  • the chimeric proteins can be administered for the prevention of H. pylori infection of normal people or for the removal of H. pylori and the treatment of H. pylori -associated diseases of H. pylori - infected patients.
  • the chimeric proteins of the invention can be manufactured in a medicament for the conventional oral administration such as solutions, tablets, capsules and granules, and administered orally.
  • the said medicament for the oral administration can be manufactured by formulating them with pharmaceutically acceptable buffering agents such as sodium bicarbonate, potassium bicarbonate and sodium phosphate to protect the chimeric proteins stably by increasing pH of gastric juice or neutralizing the gastric juice, and manufactured by formulating them with various pharmaceutically acceptable carriers such as stabilizers and sweeteners.
  • the medicament can be mixed with other antibiotics , etc. for effective prevention of H. pylori infection and removal of H. pylori , and with various anti-ulcer agents for shortening of period required for the treatment of gastritis, gastric ulcer or duodenal ulcer.
  • the chimeric proteins in case of an adult of 60kg body weight, may be administered preferably in one dose of lO ⁇ g to 1, OOOmg per day, and the dosage may be changed by the conventionally skilled in the art. If necessary, re-administration may be performed at 1-week or 2 -week intervals to induce a booster reaction, and a booster dose may be the same as or lower than that during the primary administration.
  • the present invention further provides preventive and therapeutic vaccines for H. pyroli-associated diseases which comprise the chemeric proteins' functional equivalents.
  • the term 'functional equivalents' is employed to mean all proteins substituted by the combinations such as Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and, Phe, Tyr among the amino acid sequences of the chimeric protein, and all genes comprising nucleotide sequences coding for all the said combinations among the nucleotide sequences of the fusion gene, respectively.
  • Example 1 Isolation of chromosomal DNA from H. pylori
  • H. pylori 11637 RPH 13487 (ATCC 43504) was cultured in the BHI (brain heart infusion) liquid medium (consisting of lOmg/ml vancomycin, 5mg/ml trimetofrim and 4mg/ml amphotericin B) containing 5% horse serum, and incubated for 72 hours under an environment of 10% (v/v) C0 2 . Then, chromosomal DNA was isolated from the cultured cells by the conventional method in the art.
  • Example 2 Synthesis of oligonucleotides for amplification of antigenic determinant coding genes
  • Example 2-1 Synthesis of oligonucleotides for ureB gene amplification - -
  • the oligonucleotides were synthesized employing an automatic nucleotide synthesizer (Pharmacia-LKB Biotechnology, Uppsala, Sweden).
  • the solutions were left to stand at 50°C for 12 hours, and concentrated under a reduced pressure with gas removal to reach a final volume of 0.5ml. And then, using the oligonucleotides thus concentrated, primary purification was carried out with acetonitrile/triethylamine buffer employing a SEP-PAK cartridge (Waters Inc., USA), and electrophoresis was performed using 15% polyacrylamide gel (in TE-borate, pH 8.3 ) .
  • oligonucleotides were visualized under shortwave ultraviolet rays, and only the gel fragments corresponding to the oligonucleotides were cleaved. Then, oligonucleotides were electroeluted from the gel fragments, while remaining salts with acetonitrile/triethylamine buffer employing SEP-PAK cartridge connected with a syringe to purify each oligonucleotide.
  • Oligonucleotides thus purified were labelled with ⁇ -[ 32 P]-ATP employing T 4 polynucleotide kinase (New England Biolabs, #201S, USA) and the nucleotide sequences were determined by Maxam and Gilbert's nucleotide sequencing method (see : Maxam, A.M. & Gilbert, W. , Proc. Natl. Acad. Sci. , USA, 74 :560-564 (1977) ) .
  • Example 2-2 Synthesis of oligonucleotides for cagA gene amplification
  • Example 2-4 Synthesis of oligonucleotides for alpB gene amplification
  • Example 2-5 Synthesis of oligonucleotides for fliQ gene amplification Two oligonucleotides of 24-mer and 21-mer as followings , were synthesized to amplify fliQ gene of H. pylori in an analogous manner as in Example 2-1:
  • Example 2-6 Synthesis of oligonucleotides for babAl gene amplification
  • Example 2-7 Synthesis of oligonucleotides for babA2 gene amplification
  • Example 2-8 Synthesis of oligonucleotides for ureC gene amplification
  • Example 2-10 Synthesis of oligonucleotides for ureA gene amplification
  • Example 2-11 Synthesis of oligonucleotides for sodB gene amplification
  • Example 2-12 Synthesis of oligonucleotides for urel gene amplification
  • Example 2-13 Synthesis of oligonucleotides for ureE gene amplification
  • Example 2-14 Synthesis of oligonucleotides for ureF gene amplification
  • Example 2-15 Synthesis of oligonucleotides for ureG gene amplification
  • Example 2-16 Synthesis of oligonucleotides for ureH gene amplification
  • Example 2-17 Synthesis of oligonucleotides for flaA gene amplification
  • Example 2-18 Synthesis of oligonucleotides for flaB gene amplification
  • Example 2-19 Synthesis of oligonucleotides for catA gene amplification
  • Example 2-20 Synthesis of oligonucleotides for vacA gene amplification
  • Example 2-21 Synthesis of oligonucleotides for babB gene amplification
  • Example 3 Amplification of antigenic determinant genes
  • Example 3-1 Amplification of ureB gene and A2/B subunit genes of Vibrio cholerae
  • lO ⁇ l of lOx Taq polymerase buffer (lOmM Tris-HCl (pH 8.3) containing 500mM KC1, 15mM MgCl 2 and 0.1% (v/v) gelatin)
  • lO ⁇ l of dNTP's mixture (containing an equimolar concentration of 1.25mM dGTP, dATP, dTTP and dCTP)
  • 2 ⁇ g of each primer oligonucleotides synthesized in Example 2-1
  • Ampli Taq DNA polymerase Perkin-Elmer Cetus, USA
  • chromosomal DNA of H. pylori isolated in Example 1 was used as a template DNA, and oligonucleotides synthesized in Example 2-1, i.e., 37-mer and 30-mer , were used as primers ; and, in case of amplification of A2 and B subunit genes of Vibrio cholerae toxin, chromosomal DNA of Vibrio cholerae was used as a template DNA, and oligonucleotides synthesized in Example 2-1, i.e., 28 -mer and 27 -mer, were used as primers .
  • TM (Cetus/Perkin-Elmer, USA) , and final reaction was carried out at 72°C for 10 minutes. And then, in order to remove polymerase, the equal volume of phenol/chloroform mixture (1 : 1 (v/v) ) was added to the reaction mixture, mixed well, and subsequently centrifuged. The supernatant thus obtained was transferred to a fresh tube. Then, 1/10 volume of 3M sodium acetate and 2 volume of 100% ethanol was added to the supernatant, mixed and centrifuged to obtain double-stranded nucleic acid. The nucleic acid was dissolved in 20 ⁇ l of TE buffer for later use.
  • Example 3-2 Amplification of cagA gene and A2/B subunit genes of Vibrio cholerae
  • lO ⁇ l of lOx Taq polymerase buffer (lOmM Tris-HCl (pH 8.3) containing 500mM KCl, 15mM MgCl 2 and 0.1% (v/v) gelatin), lO ⁇ l of dNTP's mixture (containing an equimolar concentration of 1.25mM dGTP, dATP, dTTP and dCTP) , 2 ⁇ g of each primer (oligonucleotides synthesized in Example 2-2) and 0.5 ⁇ l of Ampli Taq DNA polymerase (Perkin-Elmer Cetus , USA), was added distilled water to be a final volume of lOO ⁇ l .
  • lOmM Tris-HCl (pH 8.3) containing 500mM KCl, 15mM MgCl 2 and 0.1% (v/v) gelatin
  • dNTP's mixture containing an equimolar concentration of 1.25mM dGTP, dATP, dTTP
  • chromosomal DNA of H. pylori isolated in Example 1 was used as a template DNA, and oligonucleotides synthesized in Example 2-2 , i.e., 37-mer and 30-mer, were used as primers ; and, in case of amplification of A2 and B subunit genes of Vibrio cholerae toxin, chromosomal DNA of Vibrio cholerae was used as a template DNA, and oligonucleotides synthesized in Example 2-1 , i.e., 28 -mer and 27 -mer, were used as primers .
  • Denaturation (95°C, 1 minute) , annealing (55°C, 1 minute) , and extension (72°C, 2 minute) were carried out for 30 cycles in a serial manner, using Thermal Cycler TM (Cetus/Perkin-Elmer, USA) , and final reaction was carried out at 72°C for 10 minutes. And then, in order to remove polymerase, the equal volume of phenol/chloroform ' mixture (1 : 1 (v/v) ) was added to the reaction mixture, mixed well, and subsequently centrifuged. The supernatant thus obtained was transferred to a fresh tube.
  • nucleic acid was dissolved in 20 ⁇ l of TE buffer for later use.
  • Example 3-3 Amplification of alpA gene and A2/B subunit genes of Vibrio cholerae
  • alpA gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of I _ pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 23 -mer and 21-mer synthesized in Example 2-3 were employed as primer.
  • Example 3-4 Amplification of alpB gene and A2/B subunit genes of Vibrio cholerae
  • Example 3-1 alpB gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that : chromosomal DNA of E . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 25-mer and 21-mer synthesized in Example 2-4 were employed as primer.
  • Example 3-5 Amplification of fliQ gene and A2/B subunit genes of Vibrio cholerae
  • fliQ gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL_ pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 21-mer synthesized in Example 2-5 were employed as primer.
  • Example 3-6 Amplification of babAl gene and A2/B subunit genes of Vibrio cholerae
  • babAl gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of E . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 21-mer and 21-mer synthesized in Example 2-6 were employed as primer.
  • Example 3-7 Amplification of babA2 gene and A2/B subunit genes of Vibrio cholerae
  • babA2 gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of EL_ pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 22 -mer and 21-mer synthesized in Example 2-7 were employed as primer.
  • Example 3-8 Amplification of ureC gene and A2/B subunit genes of Vibrio cholerae - -
  • ureC gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 21-mer and 23 -mer synthesized in Example 2-8 were employed as primer.
  • Example 3-9 Amplification of ureD gene and A2/B subunit genes of Vibrio cholerae
  • ureD gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 23 -mer synthesized in Example 2-9 were employed as primer.
  • Example 3-10 Amplification of ureA gene and A2/B subunit genes of Vibrio cholerae
  • ureA gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 25-mer and 22 -mer synthesized in Example 2-10 were employed as primer.
  • Example 3-11 Amplification of sodB gene and A2/B subunit genes of Vibrio cholerae
  • sodB gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that : chromosomal DNA of IL . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 25-mer synthesized in Example 2-11 were employed as primer.
  • Example 3-12 Amplification of urel gene and A2/B subunit genes of Vibrio cholerae urel gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 26-mer and 24-mer synthesized in Example 2-12 were employed as primer.
  • Example 3 -13 Amplification of ureE gene and A2/B subunit genes of Vibrio cholerae
  • ureE gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that : chromosomal DNA of IL . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 22-mer and 21-mer synthesized in Example 2-13 were employed as primer .
  • Example 3-14 Amplification of ureF gene and A2/B subunit genes of Vibrio cholerae
  • ureF gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 25-mer and 23 -mer synthesized in Example 2-14 were employed as primer.
  • Example 3-15 Amplification of ureG gene and A2/B subunit genes of Vibrio cholerae
  • ureG gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 25-mer and 25-mer synthesized in Example 2-15 were employed as primer.
  • Example 3-16 Amplification of ureH gene and A2/B subunit genes of Vibrio cholerae
  • ureH gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that : chromosomal DNA of IL . pylori isolated m Example 1 was employed as template DNA, and oligonucleotides of 22 -mer and 20 -mer synthesized m Example 2-16 were employed as primer.
  • Example 3-17 Amplification of flaA gene and A2/B subunit genes of Vibrio cholerae
  • flaA gene and A2/B subunit gene of Vibrio cholerae were amplified m an analogus manner as m Example 3-1, with the exceptions that : chromosomal DNA of EL . pylori isolated m Example 1 was employed as template DNA, and oligonucleotides of 21-mer and 20-mer synthesized m Example 2-17 were employed as primer.
  • Example 3-18 Amplification of flaB gene and A2/B subunit genes of Vibrio cholerae
  • flaB gene and A2/B subunit gene of Vibrio cholerae were amplified m an analogus manner as m Example 3-1, with the exceptions that: chromosomal DNA of IL . pylori isolated m Example 1 was employed as template DNA, and oligonucleotides of 23 -mer and 21-mer synthesized m Example 2-18 were employed as primer.
  • Example 3-19 Amplification of catA gene and A2/B subunit genes of Vibrio cholerae
  • catA gene and A2/B subunit gene of Vibrio cholerae were amplified m an analogus manner as m Example 3-1, with the exceptions that: chromosomal DNA of IL . pylori isolated m
  • Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 27 -mer synthesized in Example 2-19 were employed as primer.
  • Example 3 -20 Amplification of vacA gene and A2/B subunit genes of Vibrio cholerae
  • vacA gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 26-mer synthesized in Example 2-20 were employed as primer.
  • Example 3-21 Amplification of babB gene and A2/B subunit genes of Vibrio cholerae
  • babB gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 23 -mer and 21-mer synthesized in Example 2-21 were employed as primer.
  • Example 4-1 Construction of an expression vector, pHU044
  • nucleotide sequence of base position 1 to 1679 corresponds to signal peptide sequence of the ureB
  • nucleotide sequence of base position 1680 to 1712 corresponds to signal peptide sequence of the B subunit of Vibrio cholerae toxin.
  • Figure 2 shows an amino acid sequence translated from the DNA sequence of Figure 1.
  • the fusion gene having the nucleotide sequence thus determined was double-digested with Dsal and PstI, and inserted into pTED plasmid vector double-digested with the said restriction enzymes to prepare a circular plasmid which was designated as 'pHU044' .
  • the said plasmid pTED is 2.95kb plasmid which was created Dsal restriction enzyme recognition site to pTE105, isolated from E. coli JM 101 (DW/BT-2042) transformed with pTE105 (KCCM-10027) .
  • Figure 5 is a schematic diagram showing the construction strategy of pHU044. Further, treatment of pHU044 with restriction enzyme and 1% agarose gel electrophoresis revealed that: the pHU044 expression vector has unique restriction site for each restriction enzyme; and the fusion gene was correctly inserted .
  • Example 4-2 Construction of an expression vector, pHC033
  • nucleotide sequence of base position 1 to 3444 corresponds to signal peptide sequence of the cagA, and nucleotide sequence of base position
  • 3445 to 3477 corresponds to signal peptide sequence of the
  • Figure 4 shows an amino acid sequence translated from the DNA sequence of Figure 3.
  • the fusion gene having the nucleotide sequence thus determined was double-digested with Dsal and PstI, and inserted into pTED plasmid vector double-digested with the said restriction enzymes to prepare a circular plasmid which was designated as 'pHC033' .
  • the said plasmid pTED is 2.95kb plasmid which was created Dsal restriction enzyme recognition site to pTE105 isolated from E. coli JM101 (DW/BT-2042) transformed with pTE105 (KCCM-10027) .
  • Figure 6 is a schematic diagram showing the construction strategy of pHC033.
  • pHC033 treatment pHC033 with restriction enzyme and 1% agarose gel electrophoresis revealed that: the pHC033 expression vector has unique restriction site for each restriction enzyme; and the fusion gene was correctly inserted.
  • Example 4-3 Construction of an expression vector containing alpA gene
  • a fusion gene of about 2.4kb was obtained and its nucleotide sequence was determined.
  • the fusion gene having the nucleotide sequence thus determined was double-digested with Dsal and PstI, and inserted into pTED plasmid vector double-digested with the said restriction enzymes to prepare an expression vector containing a chimeric gene of alpA gene and A2/B subunit gene of Vibrio cholerae toxin.
  • Example 4-4 Construction of an expression vector containing alpB gene
  • Expression vector containing a chimeric gene of alpB gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of alpB gene and A2/B subunit gene of Vibrio cholerae toxin of 2.3kb, with an exception of employing alpB gene of IL . pylori amplified in Example 3-4.
  • Example 4-5 Construction of an expression vector containing fliQ gene
  • Expression vector containing a chimeric gene of fliQ gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of fliQ gene and A2/B subunit gene of Vibrio cholerae toxin of 0.9kb, with an exception of employing fliQ gene of IL . pylori amplified in Example 3-5.
  • Example 4-6 Construction of an expression vector containing babAl gene
  • Expression vector containing a chimeric gene of babAl gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of babAl gene and A2/B subunit gene of Vibrio cholerae toxin of 2.8kb, with an exception of employing babAl gene of EL . pylori amplified in Example 3-6.
  • Example 4-7 Construction of an expression vector containing babA2 gene
  • Expression vector containing a chimeric gene of babA2 gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of babA2 gene and A2/B subunit gene of Vibrio cholerae toxin of 2.9kb, with an exception of employing babA2 gene of IL .
  • pylori amplified m Example 3-7 Example 3-7.
  • Example 4-8 Construction of an expression vector containing ureC gene
  • Expression vector containing a chimeric gene of ureC gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureC gene and A2/B subunit gene of Vibrio cholerae toxin of 2.0kb, with an exception of employing ureC gene of EL . pylori amplified in Example 3-8.
  • Example 4-9 Construction of an expression vector containing ureD gene
  • Expression vector containing a chimeric gene of ureD gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureD gene and A2/B subunit gene of Vibrio cholerae toxin of l.lkb, with an exception of employing ureD gene of IL . pylori amplified in Example 3-9.
  • Example 4-10 Construction of an expression vector containing ureA gene
  • Expression vector containing a chimeric gene of ureA gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureA gene and A2/B subunit gene of Vibrio cholerae toxin of 1.4kb, with an exception of employing ureA gene of IL. pylori amplified in Example 3-10.
  • Example 4-11 Construction of an expression vector containing sodB gene
  • Expression vector containing a chimeric gene of sodB gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of sodB gene and A2/B subunit gene of Vibrio cholerae toxin of 1.3kb, with an exception of employing sodB gene of IL. pylori amplified in Example 3-11.
  • Example 4-12 Construction of an expression vector containing urel gene
  • Expression vector containing a chimeric gene of urel gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of urel gene and A2/B subunit gene of Vibrio cholerae toxin of 1.3kb, with an exception of employing urel gene of EL. pylori amplified in Example 3-12.
  • Example 4-13 Construction of an expression vector containing ureE gene Expression vector containing a chimeric gene of ureE gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureE gene and A2/B subunit gene of Vibrio cholerae toxin of 1.2kb, with an exception of employing ureE gene of EL . pylori amplified in Example 3-13.
  • Example 4-14 Construction of an expression vector containing ureF gene
  • Expression vector containing a chimeric gene of ureP gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureF gene and A2/B subunit gene of Vibrio cholerae toxin of 1.5kb, with an exception of employing ureF gene of IL . pylori amplified in Example 3-14.
  • Example 4-15 Construction of an expression vector containing ureG gene
  • Expression vector containing a chimeric gene of ureG gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureG gene and A2/B subunit gene of Vibrio cholerae toxin of 1.3kb, with an exception of employing ureG gene of EL . pylori amplified in Example 3-15.
  • Example 4-16 Construction of an expression vector containing ureH gene - -
  • Expression vector containing a chimeric gene of ureH gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureH gene and
  • Example 4-17 Construction of an expression vector containing flaA gene
  • Expression vector containing a chimeric gene of flaA gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of flaA gene and A2/B subunit gene of Vibrio cholerae toxin of 2.2kb, with an exception of employing flaA gene of EL . pylori amplified in Example 3-17.
  • Example 4-18 Construction of an expression vector containing flaB gene
  • Expression vector containing a chimeric gene of flaB gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of flaB gene and A2/B subunit gene of Vibrio cholerae toxin of 2.2kb, with an exception of employing flaB gene of EL . pylori amplified in Example 3-18.
  • Example 4-19 Construction of an expression vector containing catA gene
  • Expression vector containing a chimeric gene of catA gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3-,- after sequence determination of the fused gene of catA gene and A2/B subunit gene of Vibrio cholerae toxin of 2.2kb, with an exception of employing catA gene of IL. pylori amplified in Example 3-19.
  • Example 4-20 Construction of an expression vector containing vacA gene
  • Expression vector containing a chimeric gene of vacA gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of vacA gene and A2/B subunit gene of Vibrio cholerae toxin of 4.5kb, with an exception of employing vacA gene of IL . pylori amplified in Example 3-20.
  • Example 4-21 Construction of an expression vector containing ureF gene
  • Expression vector containing a chimeric gene of babB gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of babB gene and A2/B subunit gene of Vibrio cholerae toxin of 1.4kb, with an exception of employing babB gene of EL_ pylori amplified in Example 3-21.
  • Example 5-1 Preparation of a transformant containing pHU044
  • E. coli JMlOl was first inoculated in liquid LB medium, cultured at 37°C until absorbance at 600nm reached to a level of 0.25 to 0.5, and harvested, which was subsequently washed with 0.1M MgCl 2 , and centrifuged.- - To the precipitate thus obtained were added solution containing 0.1M CaCl 2 and 0.05M MgCl 2 , and the pHU044 expression vector prepared in Example 4-1 , and incubated on ice . The cells were centrifuged again, and dispersed uniformly in the same solution (see : DNA Cloning Vol . I, A Practical Approach, IRL
  • Example 5-2 Preparation of a transformant containing pHC033
  • E. coli JMlOl was first inoculated in liquid LB medium, cultured at 37°C until absorbance at 600nm reached to a level of 0.25 to 0.5, and harvested, which was subsequently washed with 0.1M MgCl 2 , and centrifuged. To the precipitate thus obtained were added solution containing 0.1M CaCl 2 and 0.05M MgCl 2 , and the pHC033 expression vector prepared in Example 4-2 , and incubated on ice . The cellswere centrifuged again, and dispersed uniformly in the same solution (see : DNA Cloning Vol . I, A Practical Approach, IRL press, 1985) .
  • Example 5-3 Preparation of a transformant expressing alpA-fused gene
  • Example 5-1 with an exception of employing the expression vector containing a fused gene of alpA gene and A2/B subunit gene of Vibrio cholerae toxin which was prepared in Example
  • Example 5-4 Preparation of a transformant expressing alpB-fused gene
  • Example 5-1 with an exception of employing the expression vector containing a fused gene of alpB gene and A2/B subunit gene of Vibrio cholerae toxin which was prepared in Example 4-4.
  • Example 5-5 Preparation of a transformant expressing fliQ- fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of fliQ gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-5.
  • Example 5-6 Preparation of a transformant expressing babAl-fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of babAl gene and A2/B- subunit gene of Vibrio cholerae toxin prepared in Example 4-6.
  • Example 5-7 Preparation of a transformant expressing babA2- fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of babA2 gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-7.
  • Example 5-8 Preparation of a transformant expressing ureC- fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of ureC gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-8.
  • Example 5-9 Preparation of a transformant expressing ureD- fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of ureD gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-9.
  • Example 5-10 Preparation of a transformant expressing ureA- fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of ureA gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-10.
  • Example 5-11 Preparation of a transformant expressing sodB- fused gene Transformant was prepared in an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of sodB gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-11.
  • Example 5-12 Preparation of a transformant expressing urel -fused gene
  • Transformant was prepared m an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of urel gene and A2/B subunit gene of Vibrio cholerae toxin prepared m Example 4-12.
  • Example 5-13 Preparation of a transformant expressing ureE- fused gene
  • Transformant was prepared in an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of ureE gene and A2/B subunit gene of Vibrio cholerae toxin prepared m Example 4-13.
  • Example 5-14 Preparation of a transformant expressing ureF- fused gene
  • Transformant was prepared in an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of ureF gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-14.
  • Example 5-15 Preparation of a transformant expressing ureG- fused gene
  • Transformant was prepared in an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of ureG gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-15.
  • Example 5-16 Preparation of a transformant expressing ureH-fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of ureH gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-16.
  • Example 5-17 Preparation of a transformant expressing flaA- fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of flaA gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-17.
  • Example 5-18 Preparation of a transformant expressing flaB- fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of flaB gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-18.
  • Example 5-19 Preparation of a transformant expressing catA- fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of catA gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-19.
  • Example 5-20 Preparation of a transformant expressing vacA- fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of vacA gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-20.
  • Example 5-21 Preparation of a transformant expressing babB-fused gene
  • Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of babB gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-21.
  • Example 6-1 Expression of a chimeric protein in transformant
  • a transformant E. coli DW/HU-044 was inoculated in about 3ml of a medium which is disclosed in Table 1 below, and overnight cultured at 37°C and 250rpm, and 0.5ml of the culture was inoculated in about 50 ml of the same medium and cultured at 37°C while shaking at 250rpm to reach 1.8 to 2.0 of the absorbance at 600nm.
  • lane M shows molecular size-marker
  • lane 1 shows cell lysate before IPTG induction
  • lane 2 shows cell lysate of 24hrs cultured cells after IPTG induction
  • top arrow indicates locus of a chimeric protein containing ureB of H. pylori and A2 subunit of Vibrio cholerae toxin
  • bottom arrow indicates locus of B subunit of Vibrio cholerae toxin.
  • Example 6-2 Expression of a chimeric protein in transformant
  • a transformant E. coli DW/HC-033 was cultured similarly as m Example 6-1, and harvested after cetrifugation, suspended in a buffer solution (lOmM Tris-HCl (pH 8.0) containing 0.1% Triton X-100, 2mM EDTA and liriM PMSF) to lyse cells, and electrophoresed on 15% SDS-PAGE (see : Figure 8) .
  • a buffer solution lOmM Tris-HCl (pH 8.0) containing 0.1% Triton X-100, 2mM EDTA and liriM PMSF
  • lane M shows molecular size marker
  • lane 1 shows cell lysate before IPTG induction
  • lane 2 shows cell lysate of 24hrs cultured cells after IPTG induction
  • top arrow indicates locus of a chimeric protein containing cagA of H. pylori and A2 subunit of Vibrio cholerae toxin
  • bottom arrow indicates locus of B subunit of Vibrio cholerae toxin.
  • Example 6-3 Expression of AlpA/CTXA2B in transformant
  • Example 5-3 The transformant E. coli prepared in Example 5-3 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'AlpA/CTXA2B' .
  • Example 6-4 Expression of AlpB/CTXA2B in transformant
  • Example 5-4 The transformant E. coli prepared in Example 5-4 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'AlpB/CTXA2B' .
  • Example 5-5 The transformant E. coli prepared in Example 5-5 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'FliQ/CTXA2B' .
  • Example 6-6 Expression of BabAl/CTXA2B in transformant
  • Example 6-7 Expression of BabA2/CTXA2B in transformant
  • Example 5-7 The transformant E. coli prepared in Example 5-7 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'BabA2/CTXA2B' .
  • Example 5-8 The transformant E. coli prepared in Example 5-8 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreC/CTXA2B' .
  • Example 5-9 The transformant E. coli prepared in Example 5-9 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreD/CTXA2B' .
  • Example 5-10 The transformant E. coli prepared in Example 5-10 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreA/CTXA2B' .
  • Example 5-11 The transformant E. coli prepared in Example 5-11 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'SodB/CTXA2B' .
  • Example 6-12 Expression of Urel/CTXA2B in transformant
  • the transformant E. coli prepared in Example 5-12 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'Urel/CTXA2B' .
  • Example 5-13 The transformant E. coli prepared in Example 5-13 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreE/CTXA2B' .
  • Example 5-14 The transformant E. coli prepared in Example 5-14 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreF/CTXA2B' .
  • Example 5-15 The transformant E. coli prepared in Example 5-15 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreG/CTXA2B' .
  • Example 5-16 The transformant E. coli prepared in Example 5-16 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreH/CTXA2B' .
  • Example 5-17 The transformant E. coli prepared in Example 5-17 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as
  • Example 5-18 The transformant E. coli prepared in Example 5-18 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'FlaB/CTXA2B' .
  • Example 5-19 The transformant E. coli prepared in Example 5-19 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'CatA/CTXA2B' .
  • Example 6-20 Expression of VacA/CTXA2B in transformant
  • Example 5-20 The transformant E. coli prepared in Example 5-20 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'VacA/CTXA2B' .
  • Example 5-21 The transformant E. coli prepared in Example 5-21 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'BabB/CTXA2B' .
  • Example 7 Purification of chimeric proteins from the culture
  • Example 7-1 Purification of UreB/CTXA2B chimeric protein
  • the E. coli DW/HU-044 (KCCM-10124) was cultured in a LB medium and further cultured for 4 hours after IPTG induction.
  • the cultured cells were harvested by centrifugation and lysed with lysozyme.
  • the lysed cells were washed several times with 0.5% Triton X-100, and washed with 8M urea to remove contaminated proteins.
  • inclusion bodies were dissolved in 8M urea and 0. IM DTT, diluted with glutathione redox buffer to refold the UreB/CTXA2B protein.
  • Example 7-2 Purification of CagA/CTXA2B chimeric protein
  • the E. coli DW/HC-033 (KCCM-10123) was cultured in a LB medium and further cultured for 4 hours after IPTG induction.
  • the cultured cells were harvested by centrifugation and lysed with lysozyme.
  • the lysed cells were washed several times with 0.5% Triton X-100, and washed with 8M urea to remove contaminated proteins. Then, inclusion bodies were dissolved in 8M urea and 0.
  • IM DTT diluted with glutathione redox buffer to refold the CagA/CTXA2B protein Centrifugation was carried out to obtain the refolded chimeric protein, and size-exclusion chromatography was performed to obtain the CagA/CTXA2B chimeric protein only. SDS-PAGE, Western-blot and GMi-ganglioside analysis confirmed that the obtained protein is CagA/CTXA2B chimeric protein.
  • Example 7-3 Purification of AlpA/CTXA2B chimeric protein
  • Example 6-3 Chimeric protein was expressed in Example 6-3, which was prepared and identified as AlpA/CTXA2B in accordance with the method described in Example 7-1.
  • Example 7-4 Purification of AlpB/CTXA2B chimeric protein
  • Example 6-4 Chimeric protein was expressed in Example 6-4, which was prepared and identified as AlpB/CTXA2B in accordance with the method described in Example 7-1.
  • Example 7-5 Purification of FliQ/CTXA2B chimeric protein
  • Example 6-5 Chimeric protein was expressed in Example 6-5, which was prepared and identified as FHQ/CTXA2B in accordance with the method described in Example 7-1.
  • Example 7-6 Purification of BabAl/CTXA2B chimeric protein
  • Example 6-6 Chimeric protein was expressed in Example 6-6, which was prepared and identified as BAbAl/CTXA2B in accordance with the method described in Example 7-1.
  • Example 7-7 Purification of BabA2/CTXA2B chimeric protein
  • Example 6-7 Chimeric protein was expressed in Example 6-7, which was prepared and identified as BabA2/CTXA2B in accordance with the method described in Example 7-1.
  • Example 7-8 Purification of UreC/CTXA2B chimeric protein
  • Example 6-8 Chimeric protein was expressed in Example 6-8, which was prepared and identified as UreC/CTXA2B in accordance with the method described in Example 7-1.
  • Example 7-9 Purification of UreD/CTXA2B chimeric protein
  • Example 6-9 Chimeric protein was expressed in Example 6-9, which was prepared and identified as UreD/CTXA2B in accordance with the method described in Example 7-1.
  • Example 6-10 Chimeric protein was expressed in Example 6-10, which was prepared and identified as UreA/CTXA2B in accordance with the method described in Example 7-1.
  • Example 7-11 Purification of SodB/CTXA2B chimeric protein
  • Example 6-11 Chimeric protein was expressed in Example 6-11, which was prepared and identified as SodB/CTXA2B in accordance with the method described in Example 7-1.
  • Example 6-12 Chimeric protein was expressed in Example 6-12, which was prepared and identified as Urel/CTXA2B in accordance with the method described in Example 7-1.
  • Example 7-13 Purification of UreE/CTXA2B chimeric protein
  • Example 6-13 Chimeric protein was expressed in Example 6-13, which was prepared and identified as UreE/CTXA2B in accordance with the method described in Example 7-1.
  • Example 6-14 Chimeric protein was expressed in Example 6-14, which was prepared and identified as UreF/CTXA2B in accordance with the method described in Example 7-1.
  • Example 6-15 Chimeric protein was expressed in Example 6-15, which was prepared and identified as UreG/CTXA2B in accordance with the method described in Example 7-1.
  • Example 6-16 Chimeric protein was expressed in Example 6-16., .. which was prepared and identified as UreH/CTXA2B in accordance with the method described in Example 7-1.
  • Example 7-17 Purification of FlaA/CTXA2B chimeric protein Chimeric protein was expressed in Example 6-17, which was prepared and identified as FlaA/CTXA2B m accordance with the method described in Example 7-1.
  • Example 6-18 Chimeric protein was expressed m Example 6-18, which was prepared and identified as FlaB/CTXA2B m accordance with the method described in Example 7-1.
  • Example 7-19 Purification of CatA/CTXA2B chimeric protein
  • Example 6-19 Chimeric protein was expressed m Example 6-19, which was prepared and identified as CatA/CTXA2B m accordance with the method described m Example 7-1.
  • Example 7-20 Purification of VacA/CTXA2B chimeric protein
  • Example 6-20 Chimeric protein was expressed m Example 6-20, which was prepared and identified as VacA/CTXA2B m accordance with the method described m Example 7-1.
  • Example 7-21 Purification of BabB/CTXA2B chimeric protein
  • Example 6-21 Chimeric protein was expressed m Example 6-21, which was prepared and identified as BabB/CTXA2B m accordance with the method described in Example 7-1.
  • Example 8-1 Immunological reaction of the chimeric protein (UreB/CTXA2B)
  • test animals were starved for 2 hours before the oral administration and for 1 hour after the oral administration.
  • Sera were obtained by tail bleeding at 1 day before immunization (0 -day) and every week after immunization (8 , 18, 28-day) .
  • Antibodies of extract of gastric juice were prepared by administering 0.5ml of a lavage solution (containing of 25mM NaCl, 40mMNa 2 SO , lOm KCl, 20mM NaHC0 3 and 48.5mM polyethyleneglycol) four times at 15-minute intervals into mice, injecting 0.2ml of pilocarpine (0.5mg/ml) peritoneally at 30 minutes after the last administration and obtaining extracts of gastric juice from mice at 30 minutes after injection.
  • a lavage solution containing of 25mM NaCl, 40mMNa 2 SO , lOm KCl, 20mM NaHC0 3 and 48.5mM polyethyleneglycol
  • Quantitation of the antibody produced by UreB/CTXA2B was carried out using ELISA as followings: That is, after sera and extract of gastric juice were treated into a 96-well plate treated with goat anti -mouse IgG and IgA antibodies, goat peroxidase-conjugated antibodies against each isotype of mouse antibody as secondary antibodies were treated. Absorbance at 405nm was measured using p-nitrophenyl phosphate as substrates of peroxidase to determine an antibody production rate.
  • Example 8-2 Immunological reaction of the chimeric - protein (CatA/CTXA2B)
  • Example 7-2 In order to determine an antibody production rate of the CatA/CTXA2B chimeric protein obtained in Example 7-2, an animal experiment was carried out, in accordance with a protocol of the National Institutes of Health (NIH) : That is, taking 4 Balb/C mice of 11 to 12 -week as one experimental group, lOO ⁇ g of the CatA/CTXA2B chimeric protein dissolved in 0.5ml of 350mM NaHC0 3 , lOO ⁇ g of CatA dissolved in 0.5ml of 350mM NaHC0 3 , and only 0.5ml of 350mM NaHC0 3 as a control were administered orally into stomach three times at 10- day intervals for immunization, respectively. The test animals were starved for 2 hours before the oral administration and for 1 hour after the oral administration.
  • NASH National Institutes of Health
  • Sera were obtained by tail bleeding at 1 day before immunization (0 -day) and every week after immunization (8 , 18,
  • Antibodies of extract of gastric juice were prepared by administering 0.5ml of a lavage solution (containing of 25mM NaCl, 40mMNa 2 SO , lOmMKCl, 20mM NaHCO ⁇ and 48.5mM polyethyleneglycol ) four times at 15 -minute intervals into mice, injecting 0.2ml of pilocarpine (0.5mg/ml) peritoneally at 30 minutes after the last administration and obtaining extracts of gastric juice from mice at 30 minutes after injection.
  • a lavage solution containing of 25mM NaCl, 40mMNa 2 SO , lOmMKCl, 20mM NaHCO ⁇ and 48.5mM polyethyleneglycol
  • Quantitation of the antibody produced by CatA/CTXA2B was carried out using ELISA as followings: That is, after sera and extract of gastric juice were treated into a 96-well plate treated with goat anti -mouse IgG and IgA antibodies, goat peroxidase-conjugated antibodies against each isotype of mouse antibody as secondary antibodies were treated. Absorbance at 405nm was measured using p-nitrophenyl phosphate as substrates of peroxidase to determine an antibody production rate.
  • Example 8-3 Immunological reaction of the chimeric protein (AlpA/CTXA2B) The animal experiment and antibody quantitation were carried out m an analogous manner as in Example 8-1, with an exception that AlpA/CTXA2B chimeric protein was employed for the determination of antibody productivity of AlpA/CTXA2B prepared m Example 7-3.
  • AlpA/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only AlpA. Also, it was revealed that amount of IgA m extract of gastric juice increased compared with mice administered with only AlpA.
  • Example 8-4 Immunological reaction of the chimeric protein (AlpB/CTXA2B)
  • Example 8-5 Immunological reaction of the chimeric protein (Fl ⁇ Q/CTXA2B)
  • Example 8-6 Immunological reaction of the chimeric protein (BabAl/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that BabAl/CTXA2B chimeric protein was employed for the determination of antibody productivity of BabAl/CTXA2B prepared in Example 7-6. As a result, it was found that: when the BabAl/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only BabAl .
  • Example 8-7 Immunological reaction of the chimeric protein (BabA2/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that BabA2/CTXA2B chimeric protein was employed for the determination of antibody productivity of BabA2/CTXA2B prepared in Example 7-7.
  • BabA2/CTXA2B chimeric protein was employed for the determination of antibody productivity of BabA2/CTXA2B prepared in Example 7-7.
  • Example 8-8 Immunological reaction of the chimeric protein (UreC/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreC/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreC/CTXA2B prepared in Example 7-8.
  • UreC/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreC/CTXA2B prepared in Example 7-8.
  • the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreC.
  • amount of IgA in extract of gastric juice increased compared with mice administered with only UreC.
  • Example 8-9 Immunological reaction of the chimeric protein (UreD/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreD/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreD/CTXA2B prepared in Example 7-9.
  • UreD/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreD/CTXA2B prepared in Example 7-9.
  • the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreD .
  • amount of IgA in extract of gastric juice increased compared with mice administered with only UreD.
  • Example 8-10 Immunological reaction of the chimeric protein (UreA/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreA/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreA/CTXA2B prepared in Example 7-10.
  • UreA/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreA/CTXA2B prepared in Example 7-10.
  • Example 8-11 Immunological reaction of the chimeric protein (SodB/CTXA2B) The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that SodB/CTXA2B chimeric protein was employed for the determination of antibody productivity of SodB/CTXA2B prepared in Example 7-11. As a result, it was found that : when the SodB/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only SodB. Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only SodB.
  • Example 8-12 Immunological reaction of the chimeric protein (Urel/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that Urel/CTXA2B chimeric protein was employed for the determination of antibody productivity of Urel/CTXA2B prepared in Example 7-12.
  • Urel/CTXA2B chimeric protein was employed for the determination of antibody productivity of Urel/CTXA2B prepared in Example 7-12.
  • the amount of serum IgG increased remarkably after 18 days compared with mice administered with only Urel .
  • amount of IgA in extract of gastric juice increased compared with mice administered with only Urel.
  • Example 8-13 Immunological reaction of the chimeric protein (UreE/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreE/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreE/CTXA2B prepared in Example 7-13. As a result, it was found that: when the UreE/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreE .
  • Example 8-15 Immunological reaction of the chimeric protein (UreG/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreG/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreG/CTXA2B prepared in Example 7-15. As a result, it was found that: when the UreG/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreG.
  • Example 8-16 Immunological reaction of the chimeric protein (UreH/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreH/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreH/CTXA2B prepared in Example 7-16. As a result, it was found that: when the UreH/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreH.
  • Example 8-17 Immunological reaction of the chimeric protein (FlaA/CTXA2B) The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that FlaA/CTXA2B chimeric protein was employed for the determination of antibody productivity of FlaA/CTXA2B prepared in Example 7-17. As a result, it was found that : when the FlaA/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only FlaA. Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only FlaA.
  • Example 8-18 Immunological reaction of the chimeric protein (FlaB/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that FlaB/CTXA2B chimeric protein was employed for the determination of antibody productivity of FlaB/CTXA2B prepared in Example 7-18.
  • FlaB/CTXA2B chimeric protein was employed for the determination of antibody productivity of FlaB/CTXA2B prepared in Example 7-18.
  • the amount of serum IgG increased remarkably after 18 days compared with mice administered with only FlaB.
  • amount of IgA in extract of gastric juice increased compared with mice administered with only FlaB.
  • Example 8-19 Immunological reaction of the chimeric protein (CatA/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that CatA/CTXA2B chimeric protein was employed for the determination of antibody productivity of CatA/CTXA2B prepared in Example 7-19.
  • CatA/CTXA2B chimeric protein was employed for the determination of antibody productivity of CatA/CTXA2B prepared in Example 7-19.
  • Example 8-20 Immunological reaction of the chimeric protein (VacA/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that VacA/CTXA2B chimeric protein was employed for the determination of antibody productivity of VacA/CTXA2B prepared in Example 7-20.
  • VacA/CTXA2B chimeric protein was employed for the determination of antibody productivity of VacA/CTXA2B prepared in Example 7-20.
  • Example 8-21 Immunological reaction of the chimeric protein (BabB/CTXA2B)
  • Example 8-1 The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that BabB/CTXA2B chimeric protein was employed for the determination of antibody productivity of BabB/CTXA2B prepared in Example 7-8.
  • BabB/CTXA2B chimeric protein was employed for the determination of antibody productivity of BabB/CTXA2B prepared in Example 7-8.
  • the amount of serum IgG increased remarkably after 18 days compared with mice administered with only BabB .
  • amount of IgA in extract of gastric juice increased compared with mice administered with only BabB.
  • Example 9 Effect of the chimeric proteins as a vaccine for H. pylori-associated disease
  • Example 9-1 Effect of the UreB/CTXA2B chimeric protein as a vaccine
  • pylori Q- 35 (obtainable from the College of Medicine, Kyungsang National University, Korea) strain was suspended in 0.1ml of a physiological saline in a concentration of 10 7 CFU and administered into mice three times at 2 -day intervals using polyethylene catecher.
  • horse serum horse serum, lOmg/ml vancomycin, 5mg/ml trimethoprim and 4mg/ml amphotericin B) and cultured at 37°C for 5 days in a C0 2 incubator (10% C0 2 , humidity of 90% or more). After cultivation, number of colonies showing appearance of H. pylori was measured and the corresponding colonies were transferred onto a fresh medium and cultured for 3 days .
  • the cultured strains were suspended in 500ml of a physiological saline and catalase, oxidase and " Urease reactions were carried out as followings: First, lOOul of each sample was added to 1ml of an urease-detecting reagent (2 Og/1 urea, 0.05% (w/v) phenolred, 0.044g/l NaH 2 P0H 2 0, 1.02g/l Na 2 HP0 4 , 0.2g/l NaN 3 ) , vortexed well , and incubated at room temperature for 4 hours, and its absorbance at 550nm was measured. In this connection, a sample having a value of 0.1 or more higher than a control without a sample was considered as a sample showing a positive reaction.
  • an urease-detecting reagent 2 Og/1 urea, 0.05% (w/v) phenolred, 0.044g/l NaH 2 P0H 2 0, 1.02g/l Na 2 HP0 4 , 0.2g/l Na
  • Example 9-2 Effect of the CagA/CTXA2B chimeric protein as a vaccine
  • mice After 8 to 11 C57BL/6 mice were taken as one experimental group, lOOug of the CagA/CTXA2B chimeric protein dissolved m 0.5ml of a physiological saline, lOOug of CagA dissolved m 0.5ml of a physiological saline, and only 0.5ml of a physiological saline as a control were administered orally into stomach three times at 1-week intervals using polyethylene catecher, respectively.
  • H. pylori (Q-35 , ATCC 11637) strain was suspended m 0.1ml of a physiological saline m a concentration of 10 7 CFU and administered into mice three times at 2 -day intervals using polyethylene catecher.
  • H. pylori of stomachs of all mice were cut m a size of 0.5cm x 0.5cm and soaked in 1ml of a sterilized Brain Heart Infusion broth(D ⁇ fco, U.S.A.).
  • the cultured strains were suspended m 500ml of a physiological saline and catalase, oxidase and urease reactions were carried out as followings : First, lOOul of each sample was added to 1ml of an urease-detectmg reagent (20g/l urea, 0.05% (w/v) phenolred, 0.044g/l NaH 2 P0 H O, 1.02g/l Na 2 HP0 4 , 0.2g/l NaN 3 ) , vortexed well, and incubated at room temperature for 4 hours, and its absorbance at 550nm was measured.
  • an urease-detectmg reagent (20g/l urea, 0.05% (w/v) phenolred, 0.044g/l NaH 2 P0 H O, 1.02g/l Na 2 HP0 4 , 0.2g/l NaN 3 )
  • a sample having a value of 0.1 or more higher than a control without a sample was considered as a sample showing a positive reaction.
  • one drop of a sample was added onto a slide glass and one drop of 3% H 2 0 2 was dropped onto it.
  • a reaction showing generation of gas and bubbles was considered as a positive reaction.
  • one drop of a sample was added onto a filter paper and one drop of 1% N,N'- tetramethyl-p-phenylenediamine dissolved in isoamylalcohol was dropped onto it.
  • a reaction showing a purple color within several minutes was considered as a positive reaction.
  • Example 9-3 Effect of the AlpA/CTXA2B chimeric protein as a vaccine
  • Example 9-4 Effect of the AlpB/CTXA2B chimeric protein as a vaccine
  • Example 9-5 Effect of the FUQ/CTXA2B chimeric protein as a vaccine . .
  • Example 9-6 Effect of the BabAl/CTXA2B chimeric protein as a vaccine
  • Example 9-7 Effect of the BabA2/CTXA2B chimeric protein as a vaccine
  • Example 9-8 Effect of the UreC/CTXA2B chimeric protein as a vaccine
  • Example 9-9 Effect of the UreD/CTXA2B chimeric protein as a vaccine
  • Example 9-10 Effect of the UreA/CTXA2B chimeric protein as a vaccine
  • Example 9-11 Effect of the SodB/CTXA2B chimeric protein as a vaccine
  • Example 9-12 Effect of the Urel/CTXA2B chimeric protein as a vaccine
  • Example 9-13 Effect of the UreE/CTXA2B chimeric protein as a vaccine
  • Example 9-14 Effect of the UreF/CTXA2B chimeric protein as a vaccine
  • Example 9-15 Effect of the UreG/CTXA2B chimeric protein as a vaccine
  • Example 9-16 Effect of the UreH/CTXA2B chimeric protein as a vaccine
  • UreH/CTXA2B and UreH showed prevention rate of 65% and 45%, respectively.
  • all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
  • Example 9-17 Effect of the FlaA/CTXA2B chimeric protein as a vaccine
  • Example 9-18 Effect of the FlaB/CTXA2B chimeric protein as a vaccine
  • Example 9-19 Effect of the CatA/CTXA2B chimeric protein as a vaccine
  • Example 9-20 Effect of the VacA/CTXA2B chimeric protein as a vaccine
  • Example 9-21 Effect of the BabB/CTXA2B chimeric protein as a vaccine
  • a solution containing CagA/CTXA2B chimeric protein was prepared as described above.
  • a solution containing FliQ/CTXA2B chimeric protein was prepared as described above.
  • a solution containing BabAl/CTXA2B chimeric protein was prepared as described above.
  • a solution containing BabA2/CTXA2B chimeric protein was prepared as described above.
  • SodB/CTXA2B chimeric protein was prepared as described above .
  • a solution containing UreF/CTXA2B chimeric protein was prepared as described above .
  • a solution containing FlaA/CTXA2B chimeric protein was prepared as described above .
  • a solution containing FlaB/CTXA2B chimeric protein was prepared as described above .
  • a solution containing VacA/CTXA2B chimeric protein was prepared as described above .
  • a solution containing BabB/CTXA2B chimeric protein was prepared as described above .
  • the present invention provides a series of recombinant DNAs which are prepared by ligating antigenic determinant coding genes of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin, and a process for preparing the chimeric proteins of antigenic proteins of H. pylori and A2 and B subunits of Vibrio cholerae toxin, employing recombinant microorganisms transformed with the recombinant expression vectors comprising the recombinant DNAs.
  • the recombinant DNAs which are designed for convenient expression and gene manipulation, can express chimeric proteins having excellent immunogenicity to H.
  • the chimeric proteins expressed from the recombinant DNAs may be used as an active ingredient of the diagnostic kit for H. pylori infection and preventive or therapeutic vaccine for H. pylori -associated diseases , and may be used in the production of anti-H. pylori antibody.

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Abstract

The present invention relates to chimeric proteins consisting of antigenic proteins of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin, more specifically, to recombinant DNAs coding for antigenic proteins Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin, recombinant expression vectors containing the genes, a process for preparing the chimeric proteins employing the recombinant microorganisms transformed with the said expression vectors, and preventive and therapeutic vaccines comprising the chimeric proteins for Helicobacter pylori-associated diseases. The recombinant DNAs which are designed for convenient expression and gene manipulation, can express chimeric proteins having excellent immunogenicity to H. pylori, which are stable in stomach, and penetrate mucous membrane of intestines easily, finally to stimulate production of sIgA. Accordingly, the chimeric proteins expressed from the recombinant DNAs may be used as an active ingredient of the diagnostic kit for H. pylori infection and preventive or therapeutic vaccine for H. pylori-associated diseases, and may be used in the production of anti-H. pylori antibody.

Description

RECOMBINANT MICROORGANISMS EXPRESSING ANTIGENIC PROTEINS OF Helicobacter pylori
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to chimeric proteins consisting of antigenic proteins of Helicobacter pylori and A2 andB subunits of Vibrio cholerae toxin, more specifically, to recombinant DNAs coding for antigenic proteins of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin, recombinant expression vectors containing the genes, a process for preparing the chimeric proteins employing the recombinant microorganisms transformed with the said expression vectors, and preventive and therapeutic vaccines comprising the chimeric proteins for Helicobacter pylori -associated diseases.
Description of the Prior Art
Although gastritis-associated diseases such as gastritis, gastric ulcer and duodenal ulcer are caused by various etiological factors, they are mainly caused by Helicobacter pylori (hereinafter, referred to as 'H. pylori ' ) colonizing m the junctional region of epithelial cells of stomach mucous membrane. It has been reported that 90% or more of Asians and 60% or more of Europeans are infected with H. pylori though there are local differences. Also, it has been known that recurrence of gastritis, gastric ulcer or duodenal ulcer is caused by drug-resistant H. pylori , which may give rise to the occurrence of gastric cancer (see : Timothy, et al., ASM News, 61:21(1995)).
So far, a variety of chemical therapeutic agents such as antibiotics and anti-ulcer agents have been used, m order to treat the gastritis-associated diseases caused by H. pylori . However, these drugs have revealed some drawbacks as followings : limitation in penetrating the mucous membrane of intestines, emergence of drug-resistant microorganisms, occurrence of reinfection and untoward effects of the drugs. Under the circumstances, there are strong reasons for exploring and developing alternative drugs for the control of H. pylori employing new therapeutic approach, e.g., immunological therapy which can substitute for chemical therapy.
Recently, in order to solve the said problems, studies on the development of vaccines to H. pylori have been carried out. As a result, diagnostic agents of H. pylori infection and preventive vaccines for H. pylori -associated diseases have been developed, employing genes coding for antigenic determinants of H. pylori , e.g., urease gene (see : Timothy, et al . , Infection and Immunity, 59:1264(1991)), flagella gene (see : Leying, et al . , Molecular Microbiology, 6:2863(1992)), adhesin gene (see: Evans, et al . , Journal of Bacteriology, 175:674(1993)), superoxide dismutase gene (see : Christiane, et al . , Infection and Immunity, 61:5315(1993)), catalase gene and vacuolating cytotoxin gene (see : Timothy, et al . , Infection and Immunity, 58 : 603 (1990) ) , some of which have been tested in preclinical phase .
However, they have not been manufactured up to now, owing to the following disadvantages : a vaccine employing an urease gene has poor immunogenicity; a vaccine employing a vacuolating cytotoxin gene may have toxicity of cytotoxin itself, though it provides excellent immunogenicity; a vaccine employing a non-toxic varient gene of the vacuolating cytotoxin gene does not have effects on all over the strains of H. pylori , since the non-toxic varient gene does not appear in all H. pylori ; and, a vaccine employing adhesin gene, despite its excellent immunogenicity, does not have good efficacy, since it does not stimulate production of secretory IgAC'sIgA") .
In general, H. pylori is controlled by slgA not by serum IgG, since it colonizes in the junctional region of epithelial cells of stomach mucous membrane. However, since the aforesaid vaccines cannot penetrate the mucous membrane of intestines easily, they are not able to stimulate mucosal immune system, which, in turn, results in decreased production of slgA. Thus, serious problems have occurred that immunological effects of the vaccines against H. pylori decrease and the vaccines are easily denaturated by gastric acid(pH 1-2) to provide poor activities.
SUMMARY OF THE INVENTION
Since vaccines employing H. pylori gene alone have the said various disadvantages, the present inventors have made an effort to prepare a chimeric protein fused with a protein which can penetrate mucous membrane of intestines easily and stimulate mucosal immune system to produce slgA, as a fusion partner, for the purpose of using the chimeric protein as a potential vaccine for H. pylori .
Thus, the present inventors, first, prepared chimeric proteins expressed from recombinant DNAs which contain genes coding for antigenic determinants of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin. Also, they discovered that the chimeric proteins successfully solve the problems of the conventional vaccines and can be used as effective vaccines for H. pylori , based on their excellent immunogenicity for H. pylori , stability under stomach environment, and penetrating property through intestinal membrane to stimulate slgA production.
The first object of the invention is, therefore, to provide a series of DNA sequences prepared by ligating antigenic determinant coding genes of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin, and amino acid sequences translated therefrom. The second object of the invention is to provide expression vectors comprising the said DNA sequences and recombinant microorganisms transformed therewith.^ The third object of the invention is to provide a process for preparing chimeric proteins consisting antigen proteins of H. pylori and A2 and B subunits of Vibrio cholerae toxin from the said microorganisms. The fourth object of the invention is to provide preventive and therapeutic vaccines for H. pylori -associated diseases employing the chimeric proteins prepared above.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and the other objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which:
Figure 1 shows a DNA sequence of a fusion gene prepared by ligating ureB gene of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin. Figure 2 shows an amino acid sequence translated from the DNA sequence of Figure 1.
Figure 3 shows a DNA sequence of a fusion gene prepared by ligating cagA gene of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin. Figure 4 shows an amino acid sequence translated from the DNA sequence of Figure 3.
Figure 5 is a schematic diagram showing construction strategy of an expression vector for UreB/CTXA2B, pHU044. Figure 6 is a schematic diagram showing construction strategy of an expression vector for
CagA/CTXA2B, pHC033. Figure 7 is a photograph showing 15% SDS-PAGE pattern of whole cell lysate of E. coli transformed with pHU044 expression vector. Figure 8 is a photograph showing 15% SDS-PAGE pattern of whole cell lysate of E. coli transformed with pHC033 expression vector. Figure 9 is a chromatogram showing comparison of serum IgG production of mice immunized with UreB/CTXA2B chimeric protein and UreB, respectively. Figure 10 is a chromatogram showing comparison of secretory IgA production of mice immunized with UreB/CTXA2B chimeric protein and UreB, respectively. Figure 11 is a chromatogram showing comparison of serum IgG production of mice immunized with CagA/CTXA2B chimeric protein and CagA, respectively. Figure 12 is a chromatogram showing comparison of secretory IgG production of mice immunized with CagA/CTXA2B chimeric protein and
CagA, respectively.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors first gave an attention to the following characteristics of a toxin gene of Vibrio cholerae in the course of searching for a fusion partner of H. pylori gene : A gene of Vibrio cholerae toxin consists of genes coding for three subunits of Al , A2 and B. Al subunit has a toxic activity of the toxin, and A2 and B subunits bind to host cell to stimulate production of slgA and guarantee stability of the protein under a surrounding environment. Also, vaccines employing A2 and B subunit genes of Vibrio cholerae toxin can be applied to human body, due to their tolerable characteristics, while various vaccines employing intact cholera toxin gene as a fusion partner, owing to toxic property of Al subunit, can not be used directly for human body. Further, studies on a vaccine employing A2 and B subunits of cholera toxin as a fusion partner, have revealed that production of slgA and serum IgG is stimulated when a chimeric protein prepared by ligating A2 and B subunit genes of cholera toxin with adhesin gene of Streptococcus mutans is orally administrated (see : Haj ishengallis, et al . , The Journal of Immunology, 154:4322(1995)).
Grounded on the afore-mentioned knowledges , the present inventors prepared chimeric proteins employing A2 and B subunit genes of Vibrio cholerae toxin and antigenic determinant coding genes of H. pylori whose products have excellent immunogenicity, in order to stimulate production of antibody to H. pylori . The antigenic determinant coding genes of H. pylori include ureB, cagA, alpA, alpB, fliQ, babAl , babA2 , ureC, ureD, ureA, sodB, urel, ureE, ureF, ureG, ureH, flaA, flaB, catA, vacA, and babB .
First, the antigenic determinant coding genes of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin were prepared by employing polymerase chain reaction (PCR) technique, respectively. Then, each gene was cleaved with EcoRI and said two genes were ligated with T DNA ligase . The fusion genes thus prepared were cleaved with restriction enzymes, and inserted into plasmid vectors to prepare respective recombinant expression vectors. Then, E. coli was transformed with each of expression vector, the recombinant E. coli was cultured, and chimeric proteins of antigenic proteins of H. pylori and A2 and B subunits of Vibrio cholerae toxin were obtained.
The antibody production rates and their effects as potential vaccines against H. pylori of the said chimeric proteins were examined. As a result, it was found that use of the said chimeric proteins permit to solve the problem of the conventional vaccines using the antigenic proteins, i.e. , no stimulation of slgA production, and mice immunized with the said chimeric proteins produced considerable amount of slgA compared to mice administered with the antigenic protein alone. Also, it was revealed that mice immunized with the chimeric proteins showed prevention rate against infection with H. pylori of 75% while control group and mice immunized with only antigenic protein showed lower than 55% of prevention rate, respectively, which clearly demonstrates that the chimeric proteins may be used as active ingredients of vaccines for prevention and treatment of H. pylori - associated diseases, diagnostic kits for H. pylori infection, and used in the production of anti-H. pylori antibody.
The chimeric proteins of the invention induce mucosal immune response to bring about infiltration of IgA antibodies and/or lymphocytes into gastric mucosa. Thus, prevention of H. pylori infection or removal of H. pylori already infected can be accomplished. Accordingly, the chimeric proteins can be administered for the prevention of H. pylori infection of normal people or for the removal of H. pylori and the treatment of H. pylori -associated diseases of H. pylori - infected patients.
The chimeric proteins of the invention can be manufactured in a medicament for the conventional oral administration such as solutions, tablets, capsules and granules, and administered orally. The said medicament for the oral administration can be manufactured by formulating them with pharmaceutically acceptable buffering agents such as sodium bicarbonate, potassium bicarbonate and sodium phosphate to protect the chimeric proteins stably by increasing pH of gastric juice or neutralizing the gastric juice, and manufactured by formulating them with various pharmaceutically acceptable carriers such as stabilizers and sweeteners.
Also, the medicament can be mixed with other antibiotics , etc. for effective prevention of H. pylori infection and removal of H. pylori , and with various anti-ulcer agents for shortening of period required for the treatment of gastritis, gastric ulcer or duodenal ulcer.
In general, the chimeric proteins, in case of an adult of 60kg body weight, may be administered preferably in one dose of lOμg to 1, OOOmg per day, and the dosage may be changed by the conventionally skilled in the art. If necessary, re-administration may be performed at 1-week or 2 -week intervals to induce a booster reaction, and a booster dose may be the same as or lower than that during the primary administration.
As a result of oral administration of the chimeric proteins into 10 mice, it was found that all of the proteins have LD50 of 4g/kg or more, which shows that the chimeric proteins are sufficiently safe in the range of effective dose .
The present invention further provides preventive and therapeutic vaccines for H. pyroli-associated diseases which comprise the chemeric proteins' functional equivalents.
In describing the amino acid sequence and the nucleotide sequence of the present invention, the term 'functional equivalents' is employed to mean all proteins substituted by the combinations such as Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and, Phe, Tyr among the amino acid sequences of the chimeric protein, and all genes comprising nucleotide sequences coding for all the said combinations among the nucleotide sequences of the fusion gene, respectively.
The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention.
Example 1 : Isolation of chromosomal DNA from H. pylori
H. pylori 11637 RPH 13487 (ATCC 43504) was cultured in the BHI (brain heart infusion) liquid medium (consisting of lOmg/ml vancomycin, 5mg/ml trimetofrim and 4mg/ml amphotericin B) containing 5% horse serum, and incubated for 72 hours under an environment of 10% (v/v) C02. Then, chromosomal DNA was isolated from the cultured cells by the conventional method in the art.
Example 2 : Synthesis of oligonucleotides for amplification of antigenic determinant coding genes
Example 2-1: Synthesis of oligonucleotides for ureB gene amplification - -
Two oligonucleotides of 37-mer and 30-mer as followings , were synthesized to amplify ureB gene of H. pylori by PCR technique described in Example 3 below: 5' -CCGTG GATGA AAAAG ATTAG CAGAA AAGAA TATGC TT-3' 5'-AGAAT TCTCA CTTTA TTGGC TGGTT TAGAG-3'
In an analogous manner, two oligonucleotides of 28- mer and 27-mer as followings, were synthesized to amplify A2 and B subunit genes of Vibrio cholerae toxin:
5'-AGAAT TCGAA GAGCC GTGGA TTCAT CAT-3' 5'-ACTGC AGCAC ATAAT ACGCA CTAAG GA-3'
In this connection, the oligonucleotides were synthesized employing an automatic nucleotide synthesizer (Pharmacia-LKB Biotechnology, Uppsala, Sweden). The oligonucleotides thus prepared were reacted with TTD (thiophenol/triethylamine/dioxane=l/2/2 , v/v/v) solution in a silica matrix, washed with methanol and ethanol sufficiently, and treated with strong ammonia water to separate the synthesized oligonucleotides from the silica matrix. To the oligonucleotide solutions thus obtained was further added strong ammonia water . Then, the solutions were left to stand at 50°C for 12 hours, and concentrated under a reduced pressure with gas removal to reach a final volume of 0.5ml. And then, using the oligonucleotides thus concentrated, primary purification was carried out with acetonitrile/triethylamine buffer employing a SEP-PAK cartridge (Waters Inc., USA), and electrophoresis was performed using 15% polyacrylamide gel (in TE-borate, pH 8.3 ) .
After electrophoresis, oligonucleotides were visualized under shortwave ultraviolet rays, and only the gel fragments corresponding to the oligonucleotides were cleaved. Then, oligonucleotides were electroeluted from the gel fragments, while remaining salts with acetonitrile/triethylamine buffer employing SEP-PAK cartridge connected with a syringe to purify each oligonucleotide. Oligonucleotides thus purified were labelled with γ-[32P]-ATP employing T4 polynucleotide kinase (New England Biolabs, #201S, USA) and the nucleotide sequences were determined by Maxam and Gilbert's nucleotide sequencing method (see : Maxam, A.M. & Gilbert, W. , Proc. Natl. Acad. Sci. , USA, 74 :560-564 (1977) ) .
Example 2-2 : Synthesis of oligonucleotides for cagA gene amplification
Two oligonucleotides of 37-mer and 30-mer as followings , were synthesized to amplify cagA gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGA CTAAC GAAAC CATTG ACCAA CAACC AC- 3' 5'-AGAAT TCTTA AGATT TTTGG AAACC ACCTT-3'
Example 2-3 : Synthesis of oligonucleotides for alpA gene amplification
Two oligonucleotides of 23-mer and 21-mer as followings , were synthesized to amplify alpA gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGA TAAAA AAGAA TAG-3' 5'-GAATT CTTAG AATGA ATACC C-3 '
Example 2-4: Synthesis of oligonucleotides for alpB gene amplification
Two oligonucleotides of 25-mer and 21-mer as followings, were synthesized to amplify alpB gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGA CACAA TCTCA AAAAG-3' 5'-GAATT CTTAG AAGGC GTAGC C-3'
Example 2-5: Synthesis of oligonucleotides for fliQ gene amplification Two oligonucleotides of 24-mer and 21-mer as followings , were synthesized to amplify fliQ gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGG AATCA CAACT CATG-3' 5'-GAATT CGCCT ATGAT TTTGG G-3'
Example 2-6 : Synthesis of oligonucleotides for babAl gene amplification
Two oligonucleotides of 21-mer and 21-mer as followings , were synthesized to amplify babAl gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGG TAACA AACAC C-3' 5'-GAATT CTTAG TAAGC GAACA C-3'
Example 2-7: Synthesis of oligonucleotides for babA2 gene amplification
Two oligonucleotides of 22-mer and 21-mer as followings , were synthesized to amplify babA2 gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGA AAAAA CACAT CC-3' 5'-GAATT CTTAA TAAGC GAACA C-3'
Example 2-8: Synthesis of oligonucleotides for ureC gene amplification
Two oligonucleotides of 21-mer and 23 -mer as followings , were synthesized to amplify ureC gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGA AAATT TTTGG G-3'
5'-GAATT CTTAG CACAA ATGCC CTT-3' Example 2-9 : Synthesis of oligonucleotides for ureD gene amplification
Two oligonucleotides of 24-mer and 23-mer as followings, were synthesized to amplify ureD gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GGTGC TAAAA ACCAC TAAA-3' 5'-GAATT CTCAT GACAT CAGCG AAG-3'
Example 2-10: Synthesis of oligonucleotides for ureA gene amplification
Two oligonucleotides of 25-mer and 22 -mer as followings, were synthesized to amplify ureA gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGA AACTC ACCCC AAAAG-3' 5'-GAATT CTTAC TCCTT AATTG TT-3'
Example 2-11: Synthesis of oligonucleotides for sodB gene amplification
Two oligonucleotides of 24 -mer and 25-mer as followings , were synthesized to amplify sodB gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGT TTACA TTACG AGAG-3' 5'-GAATT CTCAT TCAAG CTTTT TATGC-3'
Example 2-12 : Synthesis of oligonucleotides for urel gene amplification
Two oligonucleotides of 26 -mer and 24-mer as followings , were synthesized to amplify urel gene of H. pylori in an analogous manner as in Example 2-1: 5'-CCGTG GATGC TAGGA CTTGT ATTGT T-3'
5'-GAATT CTCAC ACCCA GTGTT GGAT-3'
Example 2-13 : Synthesis of oligonucleotides for ureE gene amplification
Two oligonucleotides of 22 -mer and 21-mer as followings, were synthesized to amplify ureE gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGA TCATA GAGCG TT-3' 5'-GAATT CCTAT TTCAT GACCA C-3'
Example 2-14 : Synthesis of oligonucleotides for ureF gene amplification
Two oligonucleotides of 25-mer and 23 -mer as followings , were synthesized to amplify ureF gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGG ATAAA GGAAA AAGCG-3'
5'-GAATT CTCAA GACAT ATAAA GGC- 3'
Example 2-15 : Synthesis of oligonucleotides for ureG gene amplification
Two oligonucleotides of 25-mer and 25-mer as followings, were synthesized to amplify ureG gene of H. pylori in an analogous manner as in Example 2-1:
5'-CCGTG GATGG TAAAA ATTGG AGTTT-3'
5'-GAATT CTCAA TCTTC CAATA AAGCG-3'
Example 2-16: Synthesis of oligonucleotides for ureH gene amplification
Two oligonucleotides of 22 -mer and 20 -mer as fol'lowings , were synthesized to amplify ureH gene of H. pylori in an analogous manner as in Example 2-1:
5' CCGTG GATGA ACACT TACGC TC-3' 5' GAATT CTTAA ACCTT TGGCG-3'
Example 2-17: Synthesis of oligonucleotides for flaA gene amplification
Two oligonucleotides of 21-mer and 20 -mer as followings, were synthesized to amplify flaA gene of H. pylori in an analogous manner as in Example 2-1:
5' -CCGTG GATGG CTTTT CAGGT C-3' 5' -GAATT CCTAA GTTAA AAGCC-3'
Example 2-18: Synthesis of oligonucleotides for flaB gene amplification
Two oligonucleotides of 23 -mer and 21-mer as followings, were synthesized to amplify flaB gene of H. pylori m an analogous manner as in Example 2-1:
5' -CCGTG GATGA GTTTT AGGAT AAA-3' 5 '-GAATT CTTAT TGTAA AAGCC T-3'
Example 2-19: Synthesis of oligonucleotides for catA gene amplification
Two oligonucleotides of 24-mer and 27 -mer as followings, were synthesized to amplify catA gene of H. pylori in an analogous manner as in Example 2-1:
5' -CCGTG GATGG TTAAT AAAGA TGTG-3' 5 '-GAATT CTTAC TTTTT CTTTT TTGTG TG-3'
Example 2-20: Synthesis of oligonucleotides for vacA gene amplification
Two oligonucleotides of 24-mer and 26 -mer as followings, were synthesized to amplify vacA gene of H. pylori in an analogous manner as in Example 2-1:
5' -CCGTG GGCCT TTTTT ACAAC CGTG-3'
5' -GAATT CTTAG AAACT ATACC TCAGG C-3'
Example 2-21: Synthesis of oligonucleotides for babB gene amplification
Two oligonucleotides of 23 -mer and 21-mer as followings , were synthesized to amplify babB gene of H. pylori in axi analogous manner as in Example 2-1:
5' -CCGTG GATGA AAAAA AACCC TTT-3' 5' -GAATT CCTAG TAAGC GAACA C-3'
Example 3 : Amplification of antigenic determinant genes
Example 3-1 : Amplification of ureB gene and A2/B subunit genes of Vibrio cholerae
To the solution containing template DNA(lOng) , lOμl of lOx Taq polymerase buffer (lOmM Tris-HCl (pH 8.3) containing 500mM KC1, 15mM MgCl2 and 0.1% (v/v) gelatin) , lOμl of dNTP's mixture (containing an equimolar concentration of 1.25mM dGTP, dATP, dTTP and dCTP) , 2μg of each primer (oligonucleotides synthesized in Example 2-1) and 0.5μl of Ampli Taq DNA polymerase (Perkin-Elmer Cetus, USA), was added distilled water to be a final volume of lOOμl. In order to ^prevent evaporation of the solution, 50μl of mineral oil was added to the solution. In case of amplification of ureB gene of H. pylori , chromosomal DNA of H. pylori isolated in Example 1 was used as a template DNA, and oligonucleotides synthesized in Example 2-1, i.e., 37-mer and 30-mer , were used as primers ; and, in case of amplification of A2 and B subunit genes of Vibrio cholerae toxin, chromosomal DNA of Vibrio cholerae was used as a template DNA, and oligonucleotides synthesized in Example 2-1, i.e., 28 -mer and 27 -mer, were used as primers .
Denaturation (95°C, 1 minute) , annealing (55°C, 1 minute) , and extension (72°C, 2 minute) were carried out for 30 cycles in a serial manner, using Thermal Cycler
TM (Cetus/Perkin-Elmer, USA) , and final reaction was carried out at 72°C for 10 minutes. And then, in order to remove polymerase, the equal volume of phenol/chloroform mixture (1 : 1 (v/v) ) was added to the reaction mixture, mixed well, and subsequently centrifuged. The supernatant thus obtained was transferred to a fresh tube. Then, 1/10 volume of 3M sodium acetate and 2 volume of 100% ethanol was added to the supernatant, mixed and centrifuged to obtain double-stranded nucleic acid. The nucleic acid was dissolved in 20μl of TE buffer for later use.
Example 3-2 : Amplification of cagA gene and A2/B subunit genes of Vibrio cholerae
To the solution containing template DNA(lOng) , lOμl of lOx Taq polymerase buffer (lOmM Tris-HCl (pH 8.3) containing 500mM KCl, 15mM MgCl2 and 0.1% (v/v) gelatin), lOμl of dNTP's mixture (containing an equimolar concentration of 1.25mM dGTP, dATP, dTTP and dCTP) , 2μg of each primer (oligonucleotides synthesized in Example 2-2) and 0.5μl of Ampli Taq DNA polymerase (Perkin-Elmer Cetus , USA), was added distilled water to be a final volume of lOOμl . In order to prevent evaporation of the solution, 50μl of mineral oil was added to the solution. In case of amplification of cagA "gene of H. pylori , chromosomal DNA of H. pylori isolated in Example 1 was used as a template DNA, and oligonucleotides synthesized in Example 2-2 , i.e., 37-mer and 30-mer, were used as primers ; and, in case of amplification of A2 and B subunit genes of Vibrio cholerae toxin, chromosomal DNA of Vibrio cholerae was used as a template DNA, and oligonucleotides synthesized in Example 2-1 , i.e., 28 -mer and 27 -mer, were used as primers .
Denaturation (95°C, 1 minute) , annealing (55°C, 1 minute) , and extension (72°C, 2 minute) were carried out for 30 cycles in a serial manner, using Thermal Cycler TM (Cetus/Perkin-Elmer, USA) , and final reaction was carried out at 72°C for 10 minutes. And then, in order to remove polymerase, the equal volume of phenol/chloroform' mixture (1 : 1 (v/v) ) was added to the reaction mixture, mixed well, and subsequently centrifuged. The supernatant thus obtained was transferred to a fresh tube. Then, 1/10 volume of 3M sodium acetate and 2 volume of 100% ethanol was added to the supernatant, mixed and centrifuged to obtain double-stranded nucleic acid. The nucleic acid was dissolved in 20μl of TE buffer for later use.
Example 3-3 : Amplification of alpA gene and A2/B subunit genes of Vibrio cholerae
alpA gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of I _ pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 23 -mer and 21-mer synthesized in Example 2-3 were employed as primer.
Example 3-4 : Amplification of alpB gene and A2/B subunit genes of Vibrio cholerae
alpB gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that : chromosomal DNA of E . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 25-mer and 21-mer synthesized in Example 2-4 were employed as primer. Example 3-5 : Amplification of fliQ gene and A2/B subunit genes of Vibrio cholerae
fliQ gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL_ pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 21-mer synthesized in Example 2-5 were employed as primer.
Example 3-6: Amplification of babAl gene and A2/B subunit genes of Vibrio cholerae
babAl gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of E . pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 21-mer and 21-mer synthesized in Example 2-6 were employed as primer.
Example 3-7: Amplification of babA2 gene and A2/B subunit genes of Vibrio cholerae
babA2 gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of EL_ pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 22 -mer and 21-mer synthesized in Example 2-7 were employed as primer.
Example 3-8 : Amplification of ureC gene and A2/B subunit genes of Vibrio cholerae - -
ureC gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 21-mer and 23 -mer synthesized in Example 2-8 were employed as primer.
Example 3-9 : Amplification of ureD gene and A2/B subunit genes of Vibrio cholerae
ureD gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 23 -mer synthesized in Example 2-9 were employed as primer.
Example 3-10: Amplification of ureA gene and A2/B subunit genes of Vibrio cholerae
ureA gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 25-mer and 22 -mer synthesized in Example 2-10 were employed as primer.
Example 3-11: Amplification of sodB gene and A2/B subunit genes of Vibrio cholerae
sodB gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that : chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 25-mer synthesized in Example 2-11 were employed as primer.
Example 3-12: Amplification of urel gene and A2/B subunit genes of Vibrio cholerae urel gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 26-mer and 24-mer synthesized in Example 2-12 were employed as primer.
Example 3 -13 : Amplification of ureE gene and A2/B subunit genes of Vibrio cholerae
ureE gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that : chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 22-mer and 21-mer synthesized in Example 2-13 were employed as primer .
Example 3-14: Amplification of ureF gene and A2/B subunit genes of Vibrio cholerae
ureF gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 25-mer and 23 -mer synthesized in Example 2-14 were employed as primer.
Example 3-15: Amplification of ureG gene and A2/B subunit genes of Vibrio cholerae
ureG gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 25-mer and 25-mer synthesized in Example 2-15 were employed as primer. Example 3-16: Amplification of ureH gene and A2/B subunit genes of Vibrio cholerae
ureH gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that : chromosomal DNA of IL. pylori isolated m Example 1 was employed as template DNA, and oligonucleotides of 22 -mer and 20 -mer synthesized m Example 2-16 were employed as primer.
Example 3-17: Amplification of flaA gene and A2/B subunit genes of Vibrio cholerae
flaA gene and A2/B subunit gene of Vibrio cholerae were amplified m an analogus manner as m Example 3-1, with the exceptions that : chromosomal DNA of EL. pylori isolated m Example 1 was employed as template DNA, and oligonucleotides of 21-mer and 20-mer synthesized m Example 2-17 were employed as primer.
Example 3-18: Amplification of flaB gene and A2/B subunit genes of Vibrio cholerae
flaB gene and A2/B subunit gene of Vibrio cholerae were amplified m an analogus manner as m Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated m Example 1 was employed as template DNA, and oligonucleotides of 23 -mer and 21-mer synthesized m Example 2-18 were employed as primer.
Example 3-19: Amplification of catA gene and A2/B subunit genes of Vibrio cholerae
catA gene and A2/B subunit gene of Vibrio cholerae were amplified m an analogus manner as m Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated m
Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 27 -mer synthesized in Example 2-19 were employed as primer.
Example 3 -20 : Amplification of vacA gene and A2/B subunit genes of Vibrio cholerae
vacA gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 24-mer and 26-mer synthesized in Example 2-20 were employed as primer.
Example 3-21: Amplification of babB gene and A2/B subunit genes of Vibrio cholerae
babB gene and A2/B subunit gene of Vibrio cholerae were amplified in an analogus manner as in Example 3-1, with the exceptions that: chromosomal DNA of IL. pylori isolated in Example 1 was employed as template DNA, and oligonucleotides of 23 -mer and 21-mer synthesized in Example 2-21 were employed as primer.
Example 4 : Construction of expression vectors expressing chimeric proteins
Example 4-1: Construction of an expression vector, pHU044
The ureB gene of H. pylori and the A2 and B subunit genes of Vibrio cholerae toxin amplified in Example 3-1, respectively, were digested with EcoRI , respectively. Each of lμg of H. pylori DNA and Vibrio cholerae DNA was mixed. Then, 3μl of lOx concentrated solution for fusio"n"(600mM Tris-HCl buffer (pH 7.5) containing lOmM DTT and lOOmM MgCl.) , lμl of lOmM ATP and 10 unit of T4 DNA ligase were added to the DNA mixture to reach a final reaction volume of 30μl. and held at 14°C for 16 hours. After 1% agarose gel electrophoresis of the reaction product, a fusion gene of about 2.4kb was obtained and its nucleotide sequence was determined (see : Figure 1) . In Figure 1, nucleotide sequence of base position 1 to 1679 corresponds to signal peptide sequence of the ureB, and nucleotide sequence of base position 1680 to 1712 corresponds to signal peptide sequence of the B subunit of Vibrio cholerae toxin. Figure 2 shows an amino acid sequence translated from the DNA sequence of Figure 1. The fusion gene having the nucleotide sequence thus determined was double-digested with Dsal and PstI, and inserted into pTED plasmid vector double-digested with the said restriction enzymes to prepare a circular plasmid which was designated as 'pHU044' . The said plasmid pTED is 2.95kb plasmid which was created Dsal restriction enzyme recognition site to pTE105, isolated from E. coli JM 101 (DW/BT-2042) transformed with pTE105 (KCCM-10027) . Figure 5 is a schematic diagram showing the construction strategy of pHU044. Further, treatment of pHU044 with restriction enzyme and 1% agarose gel electrophoresis revealed that: the pHU044 expression vector has unique restriction site for each restriction enzyme; and the fusion gene was correctly inserted .
Example 4-2: Construction of an expression vector, pHC033
The cagA gene of H. pylori and the A2 and B subunit genes of Vibrio cholerae toxin amplified in Example 3-2, respectively, were digested with EcoRI , respectively. Each of lμg of H. pylori DNA and Vibrio cholerae DNA was mixed. Then, 3μl of lOx concentrated solution for fusion (600mM Tris-HCl buffer (pH 7.5) containing lOmM DTT and lOOmM MgCl2) , Iμl of lOmM ATP and 10 unit of T4 DNA ligase were added to the DNA mixture to reach a final reaction volume of 30μl, and held at 14°C for 16 hours. After 1% agarose gel electrophoresis of the reaction product, a fusion gene of about 4. lkb was obtained and its nucleotide sequence was determined (see : Figure 3) . In Figure 3, nucleotide sequence of base position 1 to 3444 corresponds to signal peptide sequence of the cagA, and nucleotide sequence of base position
3445 to 3477 corresponds to signal peptide sequence of the
B subunit of Vibrio cholerae toxin. Figure 4 shows an amino acid sequence translated from the DNA sequence of Figure 3.
The fusion gene having the nucleotide sequence thus determined was double-digested with Dsal and PstI, and inserted into pTED plasmid vector double-digested with the said restriction enzymes to prepare a circular plasmid which was designated as 'pHC033' . The said plasmid pTED is 2.95kb plasmid which was created Dsal restriction enzyme recognition site to pTE105 isolated from E. coli JM101 (DW/BT-2042) transformed with pTE105 (KCCM-10027) . Figure 6 is a schematic diagram showing the construction strategy of pHC033.
Further, treatment pHC033 with restriction enzyme and 1% agarose gel electrophoresis revealed that: the pHC033 expression vector has unique restriction site for each restriction enzyme; and the fusion gene was correctly inserted.
Example 4-3 : Construction of an expression vector containing alpA gene
The alpA gene of H. pylori and the A2 and B subunit genes of Vibrio cholerae toxin amplified in Example 3-3, respectively, were digested with EcoRI , respectively. Each of lμg of H. pylori DNA and Vibrio cholerae DNA was mixed. Then, 3μl of lOx concentrated solution for fusion (600mM Tris-HCl buffer (pH 7.5) containing lOmM DTT and lOOmM MgCl2) , lμl of lOmM ATP and 10 unit of T4 DNA ligase were added to the DNA mixture to reach a final reaction volume of 30μl, and held at 14°C for 16 hours. After 1% agarose gel electrophoresis of the reaction product, a fusion gene of about 2.4kb was obtained and its nucleotide sequence was determined. The fusion gene having the nucleotide sequence thus determined was double-digested with Dsal and PstI, and inserted into pTED plasmid vector double-digested with the said restriction enzymes to prepare an expression vector containing a chimeric gene of alpA gene and A2/B subunit gene of Vibrio cholerae toxin.
Example 4-4 : Construction of an expression vector containing alpB gene
Expression vector containing a chimeric gene of alpB gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of alpB gene and A2/B subunit gene of Vibrio cholerae toxin of 2.3kb, with an exception of employing alpB gene of IL. pylori amplified in Example 3-4.
Example 4-5 : Construction of an expression vector containing fliQ gene
Expression vector containing a chimeric gene of fliQ gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of fliQ gene and A2/B subunit gene of Vibrio cholerae toxin of 0.9kb, with an exception of employing fliQ gene of IL. pylori amplified in Example 3-5.
Example 4-6 : Construction of an expression vector containing babAl gene
Expression vector containing a chimeric gene of babAl gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of babAl gene and A2/B subunit gene of Vibrio cholerae toxin of 2.8kb, with an exception of employing babAl gene of EL. pylori amplified in Example 3-6.
Example 4-7 : Construction of an expression vector containing babA2 gene
Expression vector containing a chimeric gene of babA2 gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of babA2 gene and A2/B subunit gene of Vibrio cholerae toxin of 2.9kb, with an exception of employing babA2 gene of IL. pylori amplified m Example 3-7.
Example 4-8 : Construction of an expression vector containing ureC gene
Expression vector containing a chimeric gene of ureC gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureC gene and A2/B subunit gene of Vibrio cholerae toxin of 2.0kb, with an exception of employing ureC gene of EL. pylori amplified in Example 3-8.
Example 4-9 : Construction of an expression vector containing ureD gene
Expression vector containing a chimeric gene of ureD gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureD gene and A2/B subunit gene of Vibrio cholerae toxin of l.lkb, with an exception of employing ureD gene of IL. pylori amplified in Example 3-9. Example 4-10 : Construction of an expression vector containing ureA gene
Expression vector containing a chimeric gene of ureA gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureA gene and A2/B subunit gene of Vibrio cholerae toxin of 1.4kb, with an exception of employing ureA gene of IL. pylori amplified in Example 3-10.
Example 4-11 : Construction of an expression vector containing sodB gene
Expression vector containing a chimeric gene of sodB gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of sodB gene and A2/B subunit gene of Vibrio cholerae toxin of 1.3kb, with an exception of employing sodB gene of IL. pylori amplified in Example 3-11.
Example 4-12 : Construction of an expression vector containing urel gene
Expression vector containing a chimeric gene of urel gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of urel gene and A2/B subunit gene of Vibrio cholerae toxin of 1.3kb, with an exception of employing urel gene of EL. pylori amplified in Example 3-12.
Example 4-13 : Construction of an expression vector containing ureE gene Expression vector containing a chimeric gene of ureE gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureE gene and A2/B subunit gene of Vibrio cholerae toxin of 1.2kb, with an exception of employing ureE gene of EL. pylori amplified in Example 3-13.
Example 4-14: Construction of an expression vector containing ureF gene
Expression vector containing a chimeric gene of ureP gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureF gene and A2/B subunit gene of Vibrio cholerae toxin of 1.5kb, with an exception of employing ureF gene of IL. pylori amplified in Example 3-14.
Example 4-15: Construction of an expression vector containing ureG gene
Expression vector containing a chimeric gene of ureG gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureG gene and A2/B subunit gene of Vibrio cholerae toxin of 1.3kb, with an exception of employing ureG gene of EL. pylori amplified in Example 3-15.
Example 4-16: Construction of an expression vector containing ureH gene - -
Expression vector containing a chimeric gene of ureH gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of ureH gene and
A2/B subunit gene of Vibrio cholerae toxin of 1.5kb, with an exception of employing ureH gene of EL. pylori amplified in Example 3-16.
Example 4-17: Construction of an expression vector containing flaA gene
Expression vector containing a chimeric gene of flaA gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of flaA gene and A2/B subunit gene of Vibrio cholerae toxin of 2.2kb, with an exception of employing flaA gene of EL. pylori amplified in Example 3-17.
Example 4-18 : Construction of an expression vector containing flaB gene
Expression vector containing a chimeric gene of flaB gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of flaB gene and A2/B subunit gene of Vibrio cholerae toxin of 2.2kb, with an exception of employing flaB gene of EL. pylori amplified in Example 3-18.
Example 4-19: Construction of an expression vector containing catA gene
Expression vector containing a chimeric gene of catA gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3-,- after sequence determination of the fused gene of catA gene and A2/B subunit gene of Vibrio cholerae toxin of 2.2kb, with an exception of employing catA gene of IL. pylori amplified in Example 3-19. Example 4-20: Construction of an expression vector containing vacA gene
Expression vector containing a chimeric gene of vacA gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of vacA gene and A2/B subunit gene of Vibrio cholerae toxin of 4.5kb, with an exception of employing vacA gene of IL. pylori amplified in Example 3-20.
Example 4-21 : Construction of an expression vector containing ureF gene
Expression vector containing a chimeric gene of babB gene and A2/B subunit gene of Vibrio cholerae toxin was prepared in an analogous manner as in Example 4-3, after sequence determination of the fused gene of babB gene and A2/B subunit gene of Vibrio cholerae toxin of 1.4kb, with an exception of employing babB gene of EL_ pylori amplified in Example 3-21.
Example 5 : Preparation of transformants expressing chimeric proteins
Example 5-1 : Preparation of a transformant containing pHU044
In order to transform host cell with the pHU044 expression vector, E. coli JMlOl was first inoculated in liquid LB medium, cultured at 37°C until absorbance at 600nm reached to a level of 0.25 to 0.5, and harvested, which was subsequently washed with 0.1M MgCl2, and centrifuged.- - To the precipitate thus obtained were added solution containing 0.1M CaCl2 and 0.05M MgCl2, and the pHU044 expression vector prepared in Example 4-1 , and incubated on ice . The cells were centrifuged again, and dispersed uniformly in the same solution (see : DNA Cloning Vol . I, A Practical Approach, IRL
Press, 1985) . In this conncetion, all solutions and tubes were used after cooling at 0°C.
And then, 0.2ml of the cell suspension thus obtained was added to petri dishes coated with liquid LB media containing 12.5μg/ml of tetracycline, and cultured at 37°C overnight to obtain transformant of E. coli JMlOl harboring pHU044. The transformant thus prepared was designated as Escherichia coli DW/HU-044, and deposited with the Korean Culture Center of Microorganisms (KCCM) , an international depositary authority located at College of Eng., Yonsei University, Sodaemun-gu, Seoul, Korea, under an accession No. KCCM- 10124 on March 12, 1997.
Example 5-2 : Preparation of a transformant containing pHC033
In order to transform host cell with the pHC033 expression vector, E. coli JMlOl was first inoculated in liquid LB medium, cultured at 37°C until absorbance at 600nm reached to a level of 0.25 to 0.5, and harvested, which was subsequently washed with 0.1M MgCl2, and centrifuged. To the precipitate thus obtained were added solution containing 0.1M CaCl2 and 0.05M MgCl2, and the pHC033 expression vector prepared in Example 4-2 , and incubated on ice . The cellswere centrifuged again, and dispersed uniformly in the same solution (see : DNA Cloning Vol . I, A Practical Approach, IRL press, 1985) . In this conncetion, all solutions and tubes were used after cooling at 0°C. And then, 0.2ml of the cell suspension thus obtained was added to petri dishes coated with liquid LB media containing 12.5μg/ml of tetracycline, and cultured at 37°C overnight to obtain transformant of E. coli JMlOl harboring pHC033. The transformant thus prepared was designated as Escherichia coli DW/HC-033, and deposited with the Korean Culture Center of Microorganisms (KCCM) , an international depositary authority located at College of Eng., Yonsei University, Korea, under an accession No . KCCM- 10123 on March
12, 1997.
Example 5-3 : Preparation of a transformant expressing alpA-fused gene
Transformant was prepared in an analogous manner as in
Example 5-1, with an exception of employing the expression vector containing a fused gene of alpA gene and A2/B subunit gene of Vibrio cholerae toxin which was prepared in Example
4-3.
Example 5-4 : Preparation of a transformant expressing alpB-fused gene
Transformant was prepared in an analogous manner as in
Example 5-1, with an exception of employing the expression vector containing a fused gene of alpB gene and A2/B subunit gene of Vibrio cholerae toxin which was prepared in Example 4-4.
Example 5-5: Preparation of a transformant expressing fliQ- fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of fliQ gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-5.
Example 5-6: Preparation of a transformant expressing babAl-fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of babAl gene and A2/B- subunit gene of Vibrio cholerae toxin prepared in Example 4-6.
Example 5-7: Preparation of a transformant expressing babA2- fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of babA2 gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-7.
Example 5-8 : Preparation of a transformant expressing ureC- fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of ureC gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-8.
Example 5-9: Preparation of a transformant expressing ureD- fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of ureD gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-9.
Example 5-10: Preparation of a transformant expressing ureA- fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of ureA gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-10.
Example 5-11: Preparation of a transformant expressing sodB- fused gene Transformant was prepared in an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of sodB gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-11.
Example 5-12 : Preparation of a transformant expressing urel -fused gene
Transformant was prepared m an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of urel gene and A2/B subunit gene of Vibrio cholerae toxin prepared m Example 4-12.
Example 5-13 : Preparation of a transformant expressing ureE- fused gene
Transformant was prepared in an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of ureE gene and A2/B subunit gene of Vibrio cholerae toxin prepared m Example 4-13.
Example 5-14 : Preparation of a transformant expressing ureF- fused gene
Transformant was prepared in an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of ureF gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-14.
Example 5-15: Preparation of a transformant expressing ureG- fused gene
Transformant was prepared in an analogous manner as m Example 5-1, with an exception of employing the expression vector containing a fused gene of ureG gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-15. Example 5-16: Preparation of a transformant expressing ureH-fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of ureH gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-16.
Example 5-17: Preparation of a transformant expressing flaA- fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of flaA gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-17.
Example 5-18: Preparation of a transformant expressing flaB- fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of flaB gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-18.
Example 5-19: Preparation of a transformant expressing catA- fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of catA gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-19.
Example 5-20: Preparation of a transformant expressing vacA- fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of vacA gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-20.
Example 5-21: Preparation of a transformant expressing babB-fused gene
Transformant was prepared in an analogous manner as in Example 5-1, with an exception of employing the expression vector containing a fused gene of babB gene and A2/B subunit gene of Vibrio cholerae toxin prepared in Example 4-21.
Example 6 : Expression of chimeric proteins
Example 6-1 : Expression of a chimeric protein in transformant
E. coli DW/HU-044
A transformant E. coli DW/HU-044 was inoculated in about 3ml of a medium which is disclosed in Table 1 below, and overnight cultured at 37°C and 250rpm, and 0.5ml of the culture was inoculated in about 50 ml of the same medium and cultured at 37°C while shaking at 250rpm to reach 1.8 to 2.0 of the absorbance at 600nm. Then, to the culture, was added 0.25ml of IPTG (isopropyl β-D-thiogalactoside) and cultured at 37 °C at 250rpm for 24hours to induce recombinant protein, centrifuged to collect cells, suspended in a buffer solution (lOmM Tris-HCl (pH 8.0) containing 0.1% Triton X- 100, 2mM EDTA and ImM PMSF) to lyse cells, and electrophoresed on 15% SDS-PAGE (see: Figure 7). Table 1 : Composition of medium for transformant culture
Figure imgf000039_0001
In Figure 7, lane M shows molecular size-marker, lane 1 shows cell lysate before IPTG induction; lane 2 shows cell lysate of 24hrs cultured cells after IPTG induction; top arrow indicates locus of a chimeric protein containing ureB of H. pylori and A2 subunit of Vibrio cholerae toxin; and, bottom arrow indicates locus of B subunit of Vibrio cholerae toxin.
As shown in Figure 7, it was found that the transformed E . coli DW/HU-044 successfully expresses a chimeric protein, which is designated as ' UreB/CTXA2B' .
Example 6-2 : Expression of a chimeric protein in transformant
E. coli DW/HC-033
A transformant E. coli DW/HC-033 was cultured similarly as m Example 6-1, and harvested after cetrifugation, suspended in a buffer solution (lOmM Tris-HCl (pH 8.0) containing 0.1% Triton X-100, 2mM EDTA and liriM PMSF) to lyse cells, and electrophoresed on 15% SDS-PAGE (see : Figure 8) .
In Figure 8, lane M shows molecular size marker, lane 1 shows cell lysate before IPTG induction; lane 2 shows cell lysate of 24hrs cultured cells after IPTG induction; top arrow indicates locus of a chimeric protein containing cagA of H. pylori and A2 subunit of Vibrio cholerae toxin; and, bottom arrow indicates locus of B subunit of Vibrio cholerae toxin. As shown in Figure 8, it was found that the transformed E. coli DW/HC-033 successfully expresses a chimeric protein, which is designated as ' CagA/CTXA2B' .
Example 6-3 : Expression of AlpA/CTXA2B in transformant
The transformant E. coli prepared in Example 5-3 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'AlpA/CTXA2B' .
Example 6-4 : Expression of AlpB/CTXA2B in transformant
The transformant E. coli prepared in Example 5-4 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'AlpB/CTXA2B' .
Example 6-5: Expression of FliQ/CTXA2B in transformant
The transformant E. coli prepared in Example 5-5 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'FliQ/CTXA2B' .
Example 6-6 : Expression of BabAl/CTXA2B in transformant
The transformant E. coli prepared in Example -5-6 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as ' BabAl/CTXA2B' . Example 6-7: Expression of BabA2/CTXA2B in transformant
The transformant E. coli prepared in Example 5-7 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'BabA2/CTXA2B' .
Example 6-8: Expression of UreC/CTXA2B in transformant
The transformant E. coli prepared in Example 5-8 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreC/CTXA2B' .
Example 6-9: Expression of in UreD/CTXA2B transformant
The transformant E. coli prepared in Example 5-9 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreD/CTXA2B' .
Example 6-10: Expression of UreA/CTXA2B in transformant
The transformant E. coli prepared in Example 5-10 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreA/CTXA2B' .
Example 6-11: Expression of SodB/CTXA2B in transformant
The transformant E. coli prepared in Example 5-11 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'SodB/CTXA2B' .
Example 6-12 : Expression of Urel/CTXA2B in transformant The transformant E. coli prepared in Example 5-12 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'Urel/CTXA2B' .
Example 6-13 : Expression of UreE/CTXA2B in transformant
The transformant E. coli prepared in Example 5-13 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreE/CTXA2B' .
Example 6-14: Expression of UreF/CTXA2B in transformant
The transformant E. coli prepared in Example 5-14 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreF/CTXA2B' .
Example 6-15: Expression of UreG/CTXA2B in transformant
The transformant E. coli prepared in Example 5-15 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreG/CTXA2B' .
Example 6-16: Expression of UreH/CTXA2B in transformant
The transformant E. coli prepared in Example 5-16 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'UreH/CTXA2B' .
Example 6-17: Expression of FlaA/CTXA2B in transformant
The transformant E. coli prepared in Example 5-17 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as
'FlaA/CTXA2B' .
Example 6-18: Expression of FlaB/CTXA2B in transformant
The transformant E. coli prepared in Example 5-18 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'FlaB/CTXA2B' .
Example 6-19: Expression of CatA/CTXA2B in transformant
The transformant E. coli prepared in Example 5-19 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'CatA/CTXA2B' .
Example 6-20: Expression of VacA/CTXA2B in transformant
The transformant E. coli prepared in Example 5-20 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'VacA/CTXA2B' .
Example 6-21: Expression of BabB/CTXA2B in transformant
The transformant E. coli prepared in Example 5-21 was cultured in an analogous manner as in Example 6-1, to express desired recombinant protein, which is designated as 'BabB/CTXA2B' .
Example 7 : Purification of chimeric proteins from the culture
Example 7-1: Purification of UreB/CTXA2B chimeric protein
The E. coli DW/HU-044 (KCCM-10124) was cultured in a LB medium and further cultured for 4 hours after IPTG induction. The cultured cells were harvested by centrifugation and lysed with lysozyme. The lysed cells were washed several times with 0.5% Triton X-100, and washed with 8M urea to remove contaminated proteins. Then, inclusion bodies were dissolved in 8M urea and 0. IM DTT, diluted with glutathione redox buffer to refold the UreB/CTXA2B protein. Centrifugation was carried out to obtain the refolded chimeric protein, and size-exclusion chromatography was performed to obtain the UreB/CTXA2B chimeric protein only. SDS-PAGE, Western-blot and GMi-ganglioside analysis confirmed that the obtained protein is UreB/CTXA2B chimeric protein.
Example 7-2: Purification of CagA/CTXA2B chimeric protein
The E. coli DW/HC-033 (KCCM-10123) was cultured in a LB medium and further cultured for 4 hours after IPTG induction. The cultured cells were harvested by centrifugation and lysed with lysozyme. The lysed cells were washed several times with 0.5% Triton X-100, and washed with 8M urea to remove contaminated proteins. Then, inclusion bodies were dissolved in 8M urea and 0. IM DTT, diluted with glutathione redox buffer to refold the CagA/CTXA2B protein Centrifugation was carried out to obtain the refolded chimeric protein, and size-exclusion chromatography was performed to obtain the CagA/CTXA2B chimeric protein only. SDS-PAGE, Western-blot and GMi-ganglioside analysis confirmed that the obtained protein is CagA/CTXA2B chimeric protein.
Example 7-3 : Purification of AlpA/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-3, which was prepared and identified as AlpA/CTXA2B in accordance with the method described in Example 7-1.
Example 7-4: Purification of AlpB/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-4, which was prepared and identified as AlpB/CTXA2B in accordance with the method described in Example 7-1.
Example 7-5: Purification of FliQ/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-5, which was prepared and identified as FHQ/CTXA2B in accordance with the method described in Example 7-1.
Example 7-6 : Purification of BabAl/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-6, which was prepared and identified as BAbAl/CTXA2B in accordance with the method described in Example 7-1.
Example 7-7 : Purification of BabA2/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-7, which was prepared and identified as BabA2/CTXA2B in accordance with the method described in Example 7-1.
Example 7-8: Purification of UreC/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-8, which was prepared and identified as UreC/CTXA2B in accordance with the method described in Example 7-1.
Example 7-9: Purification of UreD/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-9, which was prepared and identified as UreD/CTXA2B in accordance with the method described in Example 7-1.
Example 7-10 : Purification of UreA/CTXA2B chimeric -protein
Chimeric protein was expressed in Example 6-10, which was prepared and identified as UreA/CTXA2B in accordance with the method described in Example 7-1. Example 7-11 : Purification of SodB/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-11, which was prepared and identified as SodB/CTXA2B in accordance with the method described in Example 7-1.
Example 7-12 : Purification of Urel/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-12, which was prepared and identified as Urel/CTXA2B in accordance with the method described in Example 7-1.
Example 7-13 : Purification of UreE/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-13, which was prepared and identified as UreE/CTXA2B in accordance with the method described in Example 7-1.
Example 7-14 : Purification of UreF/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-14, which was prepared and identified as UreF/CTXA2B in accordance with the method described in Example 7-1.
Example 7-15 : Purification of UreG/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-15, which was prepared and identified as UreG/CTXA2B in accordance with the method described in Example 7-1.
Example 7-16 : Purification of UreH/CTXA2B chimeric protein
Chimeric protein was expressed in Example 6-16., ..which was prepared and identified as UreH/CTXA2B in accordance with the method described in Example 7-1.
Example 7-17 : Purification of FlaA/CTXA2B chimeric protein Chimeric protein was expressed in Example 6-17, which was prepared and identified as FlaA/CTXA2B m accordance with the method described in Example 7-1.
Example 7-18 : Purification of FlaB/CTXA2B chimeric protein
Chimeric protein was expressed m Example 6-18, which was prepared and identified as FlaB/CTXA2B m accordance with the method described in Example 7-1.
Example 7-19 : Purification of CatA/CTXA2B chimeric protein
Chimeric protein was expressed m Example 6-19, which was prepared and identified as CatA/CTXA2B m accordance with the method described m Example 7-1.
Example 7-20 : Purification of VacA/CTXA2B chimeric protein
Chimeric protein was expressed m Example 6-20, which was prepared and identified as VacA/CTXA2B m accordance with the method described m Example 7-1.
Example 7-21 : Purification of BabB/CTXA2B chimeric protein
Chimeric protein was expressed m Example 6-21, which was prepared and identified as BabB/CTXA2B m accordance with the method described in Example 7-1.
Example 8 : Immunological reaction of the chimeric proteins
Example 8-1: Immunological reaction of the chimeric protein (UreB/CTXA2B)
In order to determine an antibody production rate of the UreB/CTXA2B chimeric protein obtained m Example 7-1, an animal experiment was carried out, m accordance with a protocol of the National Institutes of Health (NIH) : That is, taking 4 Balb/C mice of 11 to 12 -week as one experimental group, lOOμg of the UreB/CTXA2B chimeric protein dissolved in 0.5ml of 350mM NaHC03, lOOμg of UreB dissolved in 0.5ml of 350mM NaHC03, and only 0.5ml of 350mM NaHC03 as a control were administered orally into stomach three times at 10- day intervals for immunization, respectively. The test animals were starved for 2 hours before the oral administration and for 1 hour after the oral administration. Sera were obtained by tail bleeding at 1 day before immunization (0 -day) and every week after immunization (8 , 18, 28-day) . Antibodies of extract of gastric juice were prepared by administering 0.5ml of a lavage solution (containing of 25mM NaCl, 40mMNa2SO , lOm KCl, 20mM NaHC03 and 48.5mM polyethyleneglycol) four times at 15-minute intervals into mice, injecting 0.2ml of pilocarpine (0.5mg/ml) peritoneally at 30 minutes after the last administration and obtaining extracts of gastric juice from mice at 30 minutes after injection.
Quantitation of the antibody produced by UreB/CTXA2B was carried out using ELISA as followings: That is, after sera and extract of gastric juice were treated into a 96-well plate treated with goat anti -mouse IgG and IgA antibodies, goat peroxidase-conjugated antibodies against each isotype of mouse antibody as secondary antibodies were treated. Absorbance at 405nm was measured using p-nitrophenyl phosphate as substrates of peroxidase to determine an antibody production rate. As a result, it was found that: when the UreB/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days and increased 3 -fold or more compared with mice administered with only UreB (see : Figure 9) . Also, it was revealed that amount of IgA in extract of gastric juice increased 3 -fold or more compared with mice administered with only UreB (see : Figure 10) .
Example 8-2 : Immunological reaction of the chimeric - protein (CatA/CTXA2B)
In order to determine an antibody production rate of the CatA/CTXA2B chimeric protein obtained in Example 7-2, an animal experiment was carried out, in accordance with a protocol of the National Institutes of Health (NIH) : That is, taking 4 Balb/C mice of 11 to 12 -week as one experimental group, lOOμg of the CatA/CTXA2B chimeric protein dissolved in 0.5ml of 350mM NaHC03, lOOμg of CatA dissolved in 0.5ml of 350mM NaHC03, and only 0.5ml of 350mM NaHC03 as a control were administered orally into stomach three times at 10- day intervals for immunization, respectively. The test animals were starved for 2 hours before the oral administration and for 1 hour after the oral administration.
Sera were obtained by tail bleeding at 1 day before immunization (0 -day) and every week after immunization (8 , 18,
28-day) . Antibodies of extract of gastric juice were prepared by administering 0.5ml of a lavage solution (containing of 25mM NaCl, 40mMNa2SO , lOmMKCl, 20mM NaHCO< and 48.5mM polyethyleneglycol ) four times at 15 -minute intervals into mice, injecting 0.2ml of pilocarpine (0.5mg/ml) peritoneally at 30 minutes after the last administration and obtaining extracts of gastric juice from mice at 30 minutes after injection.
Quantitation of the antibody produced by CatA/CTXA2B was carried out using ELISA as followings: That is, after sera and extract of gastric juice were treated into a 96-well plate treated with goat anti -mouse IgG and IgA antibodies, goat peroxidase-conjugated antibodies against each isotype of mouse antibody as secondary antibodies were treated. Absorbance at 405nm was measured using p-nitrophenyl phosphate as substrates of peroxidase to determine an antibody production rate. As a result, it was found that: when the CatA/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days and increased 0.3 -fold or more compared with mice administered with CatA only (see : Figure 11) . Also, it was revealed that amount of IgA in extract of gastric juice increased 0.3- fold or more compared with mice administered with only CatA (see: Figure 12) .
Example 8-3 : Immunological reaction of the chimeric protein (AlpA/CTXA2B) The animal experiment and antibody quantitation were carried out m an analogous manner as in Example 8-1, with an exception that AlpA/CTXA2B chimeric protein was employed for the determination of antibody productivity of AlpA/CTXA2B prepared m Example 7-3. As a result , it was found that : when the AlpA/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only AlpA. Also, it was revealed that amount of IgA m extract of gastric juice increased compared with mice administered with only AlpA.
Example 8-4 : Immunological reaction of the chimeric protein (AlpB/CTXA2B)
The animal experiment and antibody quantitation erp carried out m an analogous manner as m Example 8-1, with an exception that AlpB/CTXA2B chimeric protein was employed for the determination of antibody productivity of AlpB/CTXA2B prepared m Example 7-4. As a result , it was found that : when the AlpB/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only AlpB . Also, it was revealed that amount of IgA m extract of gastric juice increased compared with mice administered with only AlpB.
Example 8-5: Immunological reaction of the chimeric protein (FlιQ/CTXA2B)
The animal experiment and antibody quantitation were carried out m an analogous manner as in Example 8-1, with an exception that FlιQ/CTXA2B chimeric protein was employed for the determination of antibody productivity of FlιQ/CTXA2B prepared m Example 7-5. As a result , it was found that : when the FlιQ/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only AlpA. Also, it was revealed that amount of IgA m extract of gastric juice increased compared with mice administered with only FliQ. Example 8-6: Immunological reaction of the chimeric protein (BabAl/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that BabAl/CTXA2B chimeric protein was employed for the determination of antibody productivity of BabAl/CTXA2B prepared in Example 7-6. As a result, it was found that: when the BabAl/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only BabAl .
Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only BabAl .
Example 8-7: Immunological reaction of the chimeric protein (BabA2/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that BabA2/CTXA2B chimeric protein was employed for the determination of antibody productivity of BabA2/CTXA2B prepared in Example 7-7. As a result, it was found that: when the BabA2/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only BabA2.
Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only BabA2.
Example 8-8: Immunological reaction of the chimeric protein (UreC/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreC/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreC/CTXA2B prepared in Example 7-8. As a result, it was found that : when the UreC/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreC. Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only UreC.
Example 8-9: Immunological reaction of the chimeric protein (UreD/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreD/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreD/CTXA2B prepared in Example 7-9. As a result, it was found that : when the UreD/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreD . Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only UreD.
Example 8-10: Immunological reaction of the chimeric protein (UreA/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreA/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreA/CTXA2B prepared in Example 7-10. As a result, it was found that: when the UreA/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreA.
Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only UreA.
Example 8-11: Immunological reaction of the chimeric protein (SodB/CTXA2B) The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that SodB/CTXA2B chimeric protein was employed for the determination of antibody productivity of SodB/CTXA2B prepared in Example 7-11. As a result, it was found that : when the SodB/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only SodB. Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only SodB.
Example 8-12 : Immunological reaction of the chimeric protein (Urel/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that Urel/CTXA2B chimeric protein was employed for the determination of antibody productivity of Urel/CTXA2B prepared in Example 7-12. As a result, it was found that: when the Urel/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only Urel . Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only Urel.
Example 8-13 : Immunological reaction of the chimeric protein (UreE/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreE/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreE/CTXA2B prepared in Example 7-13. As a result, it was found that: when the UreE/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreE .
Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only UreE .
Example 8-15 : Immunological reaction of the chimeric protein (UreG/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreG/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreG/CTXA2B prepared in Example 7-15. As a result, it was found that: when the UreG/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreG.
Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only UreG .
Example 8-16: Immunological reaction of the chimeric protein (UreH/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that UreH/CTXA2B chimeric protein was employed for the determination of antibody productivity of UreH/CTXA2B prepared in Example 7-16. As a result, it was found that: when the UreH/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only UreH.
Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only UreH.
Example 8-17: Immunological reaction of the chimeric protein (FlaA/CTXA2B) The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that FlaA/CTXA2B chimeric protein was employed for the determination of antibody productivity of FlaA/CTXA2B prepared in Example 7-17. As a result, it was found that : when the FlaA/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only FlaA. Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only FlaA.
Example 8-18 : Immunological reaction of the chimeric protein (FlaB/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that FlaB/CTXA2B chimeric protein was employed for the determination of antibody productivity of FlaB/CTXA2B prepared in Example 7-18. As a result, it was found that : when the FlaB/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only FlaB. Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only FlaB.
Example 8-19: Immunological reaction of the chimeric protein (CatA/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that CatA/CTXA2B chimeric protein was employed for the determination of antibody productivity of CatA/CTXA2B prepared in Example 7-19. As a result, it was found that : when the CatA/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only CatA.
Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only CatA.
Example 8-20: Immunological reaction of the chimeric protein (VacA/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that VacA/CTXA2B chimeric protein was employed for the determination of antibody productivity of VacA/CTXA2B prepared in Example 7-20. As a result, it was found that : when the VacA/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only VacA.
Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only VacA.
Example 8-21: Immunological reaction of the chimeric protein (BabB/CTXA2B)
The animal experiment and antibody quantitation were carried out in an analogous manner as in Example 8-1, with an exception that BabB/CTXA2B chimeric protein was employed for the determination of antibody productivity of BabB/CTXA2B prepared in Example 7-8. As a result, it was found that: when the BabB/CTXA2B chimeric protein was administered, the amount of serum IgG increased remarkably after 18 days compared with mice administered with only BabB . Also, it was revealed that amount of IgA in extract of gastric juice increased compared with mice administered with only BabB.
Example 9 : Effect of the chimeric proteins as a vaccine for H. pylori-associated disease Example 9-1: Effect of the UreB/CTXA2B chimeric protein as a vaccine
Effect of the UreB/CTXA2B chimeric protein as an active ingredient for a potential vaccine against H. pylori was determined by investigating infection of mice immunized with the UreB/CTXA2B chimeric protein and UreB, respectively, with H. pylori .
After 11 to 12 C57BL/6 mice were taken as one experimental group, lOOug of the UreB/CTXA2B chimeric protein dissolved in 0.5ml of a physiological saline, lOOug of UreB dissolved in 0.5ml of a physiological saline, and only 0.5ml of a physiological saline as a control were administered orally into stomach three times at 1-week intervals using polyethylene catecher, respectively. At 7 days after the last administration, H. pylori Q- 35 (obtainable from the College of Medicine, Kyungsang National University, Korea) strain was suspended in 0.1ml of a physiological saline in a concentration of 107CFU and administered into mice three times at 2 -day intervals using polyethylene catecher.
After 2 weeks, pylori of stomachs of all mice were cut in a size of 0.5cm x 0.5cm and soaked in 1ml of a sterilized Brain Heart Infusion broth (Difco, U.S.A.). After each sample was diluted with a sterilized physiological saline in a serial dilution of 10-fold, lOOμl of the sample was inoculated in a medium (Blood Agar Base No .2 containing 5. horse serum, lOmg/ml vancomycin, 5mg/ml trimethoprim and 4mg/ml amphotericin B) and cultured at 37°C for 5 days in a C02 incubator (10% C02, humidity of 90% or more). After cultivation, number of colonies showing appearance of H. pylori was measured and the corresponding colonies were transferred onto a fresh medium and cultured for 3 days .
The cultured strains were suspended in 500ml of a physiological saline and catalase, oxidase and" Urease reactions were carried out as followings: First, lOOul of each sample was added to 1ml of an urease-detecting reagent (2 Og/1 urea, 0.05% (w/v) phenolred, 0.044g/l NaH2P0H20, 1.02g/l Na2HP04, 0.2g/l NaN3) , vortexed well , and incubated at room temperature for 4 hours, and its absorbance at 550nm was measured. In this connection, a sample having a value of 0.1 or more higher than a control without a sample was considered as a sample showing a positive reaction. On the other hand, in order to detect catalase, one drop of a sample was added onto a slide glass and one drop of 3% H202 was dropped onto it. In this connection, a reaction showing generation of gas and bubbles was considered as a positive reaction. Also, in order to detect oxidase, one drop of a sample was added onto a filter paper and one drop of 1% N,N'- tetramethyl-p-phenylenediamine dissolved in isoamylalcohol was dropped onto it. In this connection, a reaction showing a purple color within several minutes was considered as a positive reaction. A sample showing positive reactions in all three experiments mentioned as above was regarded as a sample infected with H. pylori . As can be seen in Table 2 below, the experiments revealed that the experimenatl groups administered with UreB/CTXA2B and UreB showed prevention rate of 75% and 27%, respectively. On the other hand, it was found that all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Table 2: Infection of mice immunized with the UreB/CTXA2B chimeric protein and UreB, respectively, with H. pylori
Figure imgf000058_0001
Example 9-2 : Effect of the CagA/CTXA2B chimeric protein as a vaccine
Effect of the CagA/CTXA2B chimeric protein as an active ingredient for a potential vaccine against H. pylori was determined by investigating infection of mice immunized with the CagA/CTXA2B chimeric protein and CagA, respectively, with H. pylori . After 8 to 11 C57BL/6 mice were taken as one experimental group, lOOug of the CagA/CTXA2B chimeric protein dissolved m 0.5ml of a physiological saline, lOOug of CagA dissolved m 0.5ml of a physiological saline, and only 0.5ml of a physiological saline as a control were administered orally into stomach three times at 1-week intervals using polyethylene catecher, respectively. At 7 days after the last administration, H. pylori (Q-35 , ATCC 11637) strain was suspended m 0.1ml of a physiological saline m a concentration of 107CFU and administered into mice three times at 2 -day intervals using polyethylene catecher.
After 2 weeks, H. pylori of stomachs of all mice were cut m a size of 0.5cm x 0.5cm and soaked in 1ml of a sterilized Brain Heart Infusion broth(Dιfco, U.S.A.). After each sample was diluted with a sterilized physiological saline m a serial dilution of 10-fold, lOOμl of the sample was inoculated m a medium (Blood Agar Base No .2 containing 5% horse serum, lOmg/ml vancomycm, 5mg/ml trimethopπm and 4mg/ml amphotericm B) and cultured at 37°C for 5 days m a C02 incubator (10% C02, humidity of 90% or more). After cultivation, number of colonies showing appearance of EL pylori was measured and the corresponding colonies were transferred onto a fresh medium and cultured for 3 days .
The cultured strains were suspended m 500ml of a physiological saline and catalase, oxidase and urease reactions were carried out as followings : First, lOOul of each sample was added to 1ml of an urease-detectmg reagent (20g/l urea, 0.05% (w/v) phenolred, 0.044g/l NaH2P0 H O, 1.02g/l Na2HP04, 0.2g/l NaN3) , vortexed well, and incubated at room temperature for 4 hours, and its absorbance at 550nm was measured. In this connection, a sample having a value of 0.1 or more higher than a control without a sample was considered as a sample showing a positive reaction. On the other hand, in order to detect catalase, one drop of a sample was added onto a slide glass and one drop of 3% H202 was dropped onto it. In this connection, a reaction showing generation of gas and bubbles was considered as a positive reaction. Also, in order to detect oxidase, one drop of a sample was added onto a filter paper and one drop of 1% N,N'- tetramethyl-p-phenylenediamine dissolved in isoamylalcohol was dropped onto it. In this connection, a reaction showing a purple color within several minutes was considered as a positive reaction.
A sample showing positive reactions in all three experiments mentioned as above was regarded as a sample infected with H. pylori . As can be seen in Table 4 below, the experiments revealed that the experimenatl groups administered with CagA/CTXA2B and CagA showed prevention rate of 80% and 55%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Table 3: Infection of mice immunized with the CagA/CTXA2B chimeric protein and CagA, respectively, with H. pylori
Figure imgf000060_0001
44130 .
59
Example 9-3: Effect of the AlpA/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with AlpA/CTXA2B chimeric protein and AlpA against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of AlpA/CTXA2B as a potential vaccine for H. pylori, with an exception that AlpA/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with AlpA/CTXA2B and AlpA showed prevention rate of 75% and 50%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-4: Effect of the AlpB/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with AlpB/CTXA2B chimeric protein and AlpB against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of AlpB/CTXA2B as a potential vaccine for H. pylori, with an exception that AlpB/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with AlpB/CTXA2B and AlpA showed prevention rate of 65% and 53%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-5: Effect of the FUQ/CTXA2B chimeric protein as a vaccine . .
Infectivity of the mice immunized with FliQ/CTXA2B chimeric protein and FliQ against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of FliQ/CTXA2B as a potential vaccine for H. pylori, with an exception that FUQ/CTXA2B chimeric protein was employed instead of FliQ/CTXA2B. As a result, it was determined that the experimenatl groups administered with FHQ/CTXA2B and FliQ showed prevention rate of 75% and 57%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-6: Effect of the BabAl/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with BabAl/CTXA2B chimeric protein and BabAl against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of BabAl/CTXA2B as a potential vaccine for H. pylori, with an exception that BabAl/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with BabAl/CTXA2B and BabAl showed prevention rate of 80% and 57%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-7: Effect of the BabA2/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with BabA2/CTXA2B chimeric protein and BabA2 against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of BabA2/CTXA2B as a potential vaccine for H. pylori, with an exception that BabA2/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, . it was determined that the experimenatl groups administered with BabA2/CTXA2B and BabA2 showed prevention rate of 75% and 55%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-8: Effect of the UreC/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with UreC/CTXA2B chimeric protein and UreC against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of UreC/CTXA2B as a potential vaccine for H. pylori, with an exception that UreC/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with UreC/CTXA2B and UreC showed prevention rate of 78% and 60%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-9: Effect of the UreD/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with UreD/CTXA2B chimeric protein and UreD against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of UreD/CTXA2B as a potential vaccine for H. pylori, with an exception that UreD/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with UreD/CTXA2B and UreD showed prevention rate of 70% and 52%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-10 : Effect of the UreA/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with UreA/CTXA2B chimeric protein and UreA against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of UreA/CTXA2B as a potential vaccine for H. pylori, with an exception that UreA/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with UreA/CTXA2B and UreA showed prevention rate of 70% and 50%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-11 : Effect of the SodB/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with SodB/CTXA2B chimeric protein and SodB against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of SodB/CTXA2B as a potential vaccine for H. pylori, with an exception that SodB/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with SodB/CTXA2B and SodB showed prevention rate of 70% and 55%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-12: Effect of the Urel/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with Urel/CTXA2B chimeric protein and Urel against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of UreI/CTXA2B as a potential vaccine for H. pylori, with an exception that Urel/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, .It was determined that the experimenatl groups administered with UreI/CTXA2B and Urel showed prevention rate of 65% and 53%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-13 : Effect of the UreE/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with UreE/CTXA2B chimeric protein and UreE against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of UreE/CTXA2B as a potential vaccine for H. pylori,' with an exception that UreE/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with UreE/CTXA2B and Urel showed prevention rate of 70% and 55%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-14 : Effect of the UreF/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with UreF/CTXA2B chimeric protein and UreF against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of UreF/CTXA2B as a potential vaccine for H. pylori, with an exception that UreF/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with UreF/CTXA2B and UreF showed prevention rate of 75% and 55%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-15: Effect of the UreG/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with UreG/CTXA2B chimeric protein and UreG against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of UreG/CTXA2B as a potential vaccine for H. pylori, with an exception that UreG/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with UreG/CTXA2B and UreG showed prevention rate of 78% and 53%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-16: Effect of the UreH/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with UreH/CTXA2B chimeric protein and UreH against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of UreH/CTXA2B as a potential vaccine for H. pylori, with an exception that UreH/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with
UreH/CTXA2B and UreH showed prevention rate of 65% and 45%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-17: Effect of the FlaA/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with FlaA/CTXA2B chimeric protein and FlaA against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of FlaA/CTXA2B as a potential vaccine for H. pylori, with an exception that FlaA/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with FlaA/CTXA2B and FlaA showed prevention rate of 70% and 52%, respectively. On the other hand, all mice of the control /44130
65 group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-18: Effect of the FlaB/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with FlaB/CTXA2B chimeric protein and FlaB against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the' effect of FlaB/CTXA2B as a potential vaccine for H. pylori, with an exception that FlaB/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with FlaB/CTXA2B and FlaB showed prevention rate of 78% and 50%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-19: Effect of the CatA/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with CatA/CTXA2B chimeric protein and CatA against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of CatA/CTXA2B as a potential vaccine for H. pylori, with an exception that CatA/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with CatA/CTXA2B and CatA showed prevention rate of 75% and 50%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-20 : Effect of the VacA/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with VacA/CTXA2B chimeric protein and VacA against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of VacA/CTXA2B as a potential vaccine for H. pylori, with an exception that VacA/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with VacA/CTXA2B and VacA showed prevention rate of 68% and 53%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
Example 9-21: Effect of the BabB/CTXA2B chimeric protein as a vaccine
Infectivity of the mice immunized with BabB/CTXA2B chimeric protein and BabB against H. pylori was investigated in an analogous manner as in Example 9-1, to determine the effect of BabB/CTXA2B as a potential vaccine for H. pylori, with an exception that BabB/CTXA2B chimeric protein was employed instead of UreB/CTXA2B. As a result, it was determined that the experimenatl groups administered with BabB/CTXA2B and BabB showed prevention rate of 72% and 53%, respectively. On the other hand, all mice of the control group administered with only a physiological saline were infected with H. pylori , which showed no preventive effect.
As shown in Examples 9-1 to 9-21, it was clearly demonstrated that: the chimeric proteins of antigenic proteins of H. pylori and A2 and B subunits of Vibrio cholerae , can induce specific antibodies neutralizing H. pylori and be used as a preventive or therapeutic vaccine for H. pylori -associated diseases.
Preparative Example 1: Solution containing UreB/CTXA2B chimeric protein
UreB/CTXA2B chimeric protein lOOμg 0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing UreB/CTXA2B chimeric protein was prepared as described above.
Preparative Example 2 : Solution containing CagA/CTXA2B chimeric protein
CagA/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing CagA/CTXA2B chimeric protein was prepared as described above.
Preparative Example 3 : Solution containing AlpA/CTXA2B chimeric protein
AlpA/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing AlpA/CTXA2B chimeric protein was prepared as described above. Preparative Example 4 : Solution containing AlpB/CTXA2B chimeric protein
AlpB/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing AlpB/CTXA2B chimeric protein was prepared as described above.
Preparative Example 5: Solution containing FliQ/CTXA2B chimeric protein
FUQ/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing FliQ/CTXA2B chimeric protein was prepared as described above.
Preparative Example 6: Solution containing BabAl/CTXA2B chimeric protein
BabAl/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl Total 500μl
A solution containing BabAl/CTXA2B chimeric protein was prepared as described above.
Preparative Example 7 : Solution containing BabA2/CTXA2B chimeric protein
BabA2/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing BabA2/CTXA2B chimeric protein was prepared as described above.
Preparative Example 8: Solution containing UreC/CTXA2B chimeric protein
UreC/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing UreC/CTXA2B chimeric protein was prepared as described above.
Preparative Example 9: Solution containing UreD/CTXA2B chimeric protein UreD/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing UreD/CTXA2B chimeric protein was prepared as described above.
Preparative Example 10: Solution containing UreA/CTXA2B chimeric protein
UreA/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing UreA/CTXA2B chimeric protein was prepared as described above.
Preparative Example 11: Solution containing SodB/CTXA2B chimeric protein
SodB/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl . .
Total 500μl
A solution containing SodB/CTXA2B chimeric protein was prepared as described above .
Preparative Example 12 : Solution containing Urel/CTXA2B chimeric protein
Urel/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing UreI/CTXA2B chimeric protein was prepared as described above.
Preparative Example 13 : Solution containing UreE/CTXA2B chimeric protein
UreE/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing UreE/CTXA2B chimeric protein was prepared as described above.
Preparative Example 14: Solution containing UreF/CTXA2B chimeric protein
UreF/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl 4130
72 Distilled water 250μl
Total 500μl
A solution containing UreF/CTXA2B chimeric protein was prepared as described above .
Preparative Example 15: Solution containing UreG/CTXA2B chimeric protein
UreG/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing UreG/CTXA2B chimeric protein was prepared as described above.
Preparative Example 16: Solution containing UreH/CTXA2B chimeric protein
UreH/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing UreH/CTXA2B chimeric protein was prepared as described above.
Preparative Example 17: Solution containing FlaA/CTXA2B chimeric protein 4130
73
FlaA/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing FlaA/CTXA2B chimeric protein was prepared as described above .
Preparative Example 18: Solution containing FlaB/CTXA2B chimeric protein
FlaB/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing FlaB/CTXA2B chimeric protein was prepared as described above .
Preparative Example 19: Solution containing CatA/CTXA2B chimeric protein
CatA/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl A solution containing CatA/CTXA2B chimeric protein was prepared as described above.
Preparative Example 20: Solution containing VacA/CTXA2B chimeric protein
VacA/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing VacA/CTXA2B chimeric protein was prepared as described above .
Preparative Example 21: Solution containing BabB/CTXA2B chimeric protein
BabB/CTXA2B chimeric protein lOOμg
0.6M sodium bicarbonate 250μl
Distilled water 250μl
Total 500μl
A solution containing BabB/CTXA2B chimeric protein was prepared as described above .
As clearly illustrated and demonstrated as aboves, the present invention provides a series of recombinant DNAs which are prepared by ligating antigenic determinant coding genes of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin, and a process for preparing the chimeric proteins of antigenic proteins of H. pylori and A2 and B subunits of Vibrio cholerae toxin, employing recombinant microorganisms transformed with the recombinant expression vectors comprising the recombinant DNAs. The recombinant DNAs which are designed for convenient expression and gene manipulation, can express chimeric proteins having excellent immunogenicity to H. pylori , which are stable in stomach, and penetrate mucous membrane of intestines easily, finally to stimulate production of slgA. Accordingly, the chimeric proteins expressed from the recombinant DNAs may be used as an active ingredient of the diagnostic kit for H. pylori infection and preventive or therapeutic vaccine for H. pylori -associated diseases , and may be used in the production of anti-H. pylori antibody.

Claims

WHAT IS CLAIMED IS:
1. A recombinant DNA comprising a fusion gene which is prepared by ligating antigenic determinant coding gene of Helicobacter pylori and A2 and B subunit genes of Vibrio cholerae toxin.
2. The recombinant DNA of claim 1, wherein the antigenic determinant coding gene of Helicobacter pylori is selected from the group consisting of ureB, cagA, alpA, alpB, fliQ, babAl, babA2 , ureC, ureD, ureA, sodB, urel, ureE, ureF, ureG, ureH, flaA, flaB, catA, vacA and babB.
3. The recombinant DNA of claim 1, wherein the fusion gene which is prepared by ligating ureB gene of Helicobacter pylori and A2 and B subunit genes of Vibrio cholerae toxin,
5 has a nucleotide sequence represented as following, or its functional equivalents:
1 atgaaaaaga ttagcagaaa agaatatgct tctatgtatg gccctactac aggcgataaa
10 61 gtgagattgg gcgatacaga cttgatcgct gaagtagaac atgactacac catttatggt 121 gaagagctta aattcggcgg cggtaaaacc ctaagagaag gcatgagcca atctaacaac 181 cctagcaaag aagaactgga tctaatcatc actaacgctt taatcgtgga ttacaccggt 241 atttataaag cggatattgg tattaaagat ggcaaaatcg ctggcattgg taaaggcggt 301 aacaaagaca cgcaagatgg cgttaaaaac aatcttagcg tgggtcctgc tactgaagcc 361 ttagccggtg aaggtttgat tgtaactgct ggtggtattg acacacacat ccacttcatc
1 421 tccccccaac aaatccctac agcttttgca agcggtgtaa caaccatgat tggtggcgga 481 actggccctg ctgatggcac taacgcaacc actatcactc caggtagaag aaatttaaaa 541 ttcatgctca gagcggctga agaatattct atgaactttg gtttcttggc taaaggtaac 601 gcttctaacg atgcaagctt agccgatcaa attgaagctg gtgcgattgg ccttaaaatc 661 cacgaagact ggggcaccac tccttctgca atcaatcatg cgttagatgt tgcggacaaa 721 tacgatgtgc aagtcgctat ccacacagac actttgaatg aagccggttg cgtggaagac
20 781 actatggcag ctattgccgg acgcactatg cacacttacc acactgaagg cgctggcggc 841 ggacacgctc ctgatattat taaagtggcc ggtgaacaca acatcctacc cgcttccact 901 aaccccacta tccctttcac cgtgaataca gaagccgaac acatggacat gcttatggtg 961 tgccaccact tggataaaag cattaaagaa gatgtccagt tcgctgattc aaggattcgc 1021 cctcaaacca ttgcggctga agacactttg catgacatgg ggattttctc aatcactagt 1081 tctgactctc aagcgatggg ccgtgtgggt gaagttatca ctagaacttg gcaaacagct
25 1141 gacaaaaata aaaaagaatt tggccgcttg aaagaagaaa aaggcgataa cgacaacttc 1201 aggatcaaac gctacttgtc taaatacacc attaacccag cgatcgctca tgggattagc 1261 gagtatgtcg gttctgtaga agtgggcaaa gtggctgact tggtattgtg gagtcccgca 1321 ttctttggtg tgaaacccaa catgatcatc aaaggcgggt tcatcgcatt gagtcaaatg 1381 ggtgatgcga acgcttctat ccctacccca caaccagttt attacagaga aatgttcgct 1441 catcatggta aagctaaata cgatgcaaac atcacttttg tgtctcaagc ggcttatgac
30 1501 aaaggcatta aagaagaatt agggcttgaa agacaagtgt tgccggtaaa aaattgcaga 1561 aatatcacta aaaaagacat gcaattcaac gacactaccg ctcacattga agtcaattct 1621 gaaacttacc atgtgttcgt ggatggcaaa gaagtaactc taaaccagcc aataaagtga 1681 gaattcgaag agccgtggat tcatcatgca ccgccgggtt gtgggaatgc tccaagatca 1741 tcgatcagta atacttgcga tgaaaaaacc caaagtctag gtgtaaaatt ccttgacgaa 1801 taccaatcta aagttaaaag acaaatattt tcaggctatc aatctgatat tgatacacat
35 1861 aatagaatta aggatgaatt aatgattaaa ttaaaatttg gtgttttttt tacagtttta 1921 ctatcttcag catatgcaca tggaacacct caaaatatta ctgatttgtg tgcagaatca 1981 cacaacacac aaatatatac gctaaatgat aagatatttt cgtatacaga atctctagct 2041 ggaaaaagag agatggctat cattactttt aagaatggtg caatttttca agtagaagta
2101 ccaagtagtc aacatataga ttcacaaaaa aaagcgattg aaaggatgaa ggataccctg
2161 aggattgcat atcttactga agctaaagtc gaaaagttat gtgtatggaa taataaaacg
2221 cctcatgcga ttgccgcaat tagtatggca aattaagata taaaaagccc acctcagtgg
2281 gcttttttgt ggttcgatga tgagaagcaa ccgttttgcc caaacatgta ttactgcaag 2385 tatgatgttt ttattccaca tccttagtgc gtattatgtg ctgca
10
4. The recombinant DNA of claim 1, wherein the fusion gene which is prepared by ligating cagA gene of Helicobacter 15 pylori and A2 and B subunit genes of Vibrio cholerae toxin, has a nucleotide sequence represented as following, or its functional equivalents:
20 1 atgactaacg aaaccattga ccaacaacca caaaccgaag cggcttttaa cccgcagcaa
61 tttatcaata atcttcaagt agcttttctt aaagttgata acgctgtcgc ttcatacgat
121 cctgatcaaa aaccaatcgt tgataagaac gatagggata acaggcaagc ttttgaagga
181 atctcgcaat taagggaaga atactccaat aaagcgatca aaaatcctac caaaaagaat
241 cagtattttt cagactttat caataagagc aatgatttaa tcaacaaaga caatctcatt
301 gatgtagaat cttccacaaa gagctttcag aaatttgggg atcagcgtta ccgaattttc
25 361 acaagttggg tgtcccatca aaacgatccg tctaaaatca acacccgatc gatccgaaat
421 tttatggaaa atatcataca accccctatc cttgatgata aagagaaagc ggagtttttg
481 aaatctgcca aacaatcttt tgcaggaatc attataggga atcaaatccg aacggatcaa
541 aagttcatgg gcgtgtttga tgagtccttg aaagaaaggc aagaagcaga aaaaaatgga
601 gagcctactg gtggggattg gttggatatt tttctctcat ttatatttga caaaaaacaa
661 tcttctgatg tcaaagaagc aatcaatcaa gaaccagttc cccatgtcca accagatata
30
721 gccactacca ccaccgacat acaaggctta ccgcctgaag ctagagattt acttgatgaa
781 aggggtaatt tttctaaatt cactcttggc gatatggaaa tgttagatgt tgagggagtc
841 gctgacattg atcccaatta caagttcaat caattattga ttcacaataa cgctctgtct
901 tctgtgttaa tggggagtca taatggcata gaacctgaaa aagtttcatt gttgtatggg
961 ggcaatggtg gtcctggagc taggcatgat tggaacgcca ccgttggtta taaagaccaa
1021 caaggcaaca atgtggctac aataattaat gtgcatatga aaaacggcag tggcttagtc
35 1081 atagcaggtg gtgagaaagg gattaacaac cctagttttt atctctacaa agaagaccaa
1141 ctcacaggct cacaacgagc attaagtcaa gaagagatcc aaaacaaaat agatttcatg 1201 gaatttcttg cacaaaataa tgctaaatta gacaacttga gcgagaaaga gaaggaaaaa
1261 ttccgaactg agattaaaga tttccaaaaa gactctaagg cttatttaga cgccctaggg
1321 aatgatcgta ttgcttttgt ttctaaaaaa gacacaaaac attcagcttt aattactgag
1381 tttggtaatg gggatttgag ctacactctc aaagattatg ggaaaaaagc agataaagct
1441 ttagataggg agaaaaatgt tactcttcaa ggtagcctaa aacatgatgg cgtgatgttt
1501 gttgattatt ctaatttcaa atacaccaac gcctccaaga atcccaataa gggtgtaggc
1561 gttacgaatg gcgtttccca tttagaagta ggctttaaca aggtagctat ctttaatttg
1621 cctgatttaa ataatctcgc tatcactagt ttcgtaaggc ggaatttaga ggataaacta
1681 accactaaag gattgtcccc acaagaagct aataagctta tcaaagattt tttgagcagc
1741 aacaaagaat tggttggaaa aactttaaac ttcaataaag ctgtagctga cgctaaaaac
1801 acaggcaatt atgatgaagt gaaaaaagct cagaaagatc ttgaaaaatc tctaaggaaa
1861 cgagagcatt tagagaaaga agtagagaaa aaattggaga gcaaaagcgg caacaaaaat
1921 aaaatggaag caaaagctca agctaacagc caaaaagatg agatttttgc gttgatcaat
1981 aaagaggcta atagagacgc aagagcaatc gcttacgctc agaatcttaa aggcatcaaa
2041 agggaattgt ctgataaact tgaaaatgtc aacaagaatt tgaaagactt tgataaatct
2101 tttgatgaat tcaaaaatgg caaaaataag gatttcagca aggcagaaga aacactaaaa
2161 gcccttaaag gttcggtgaa agatttaggt atcaatccag aatggatttc aaaagttgaa
2221 aaccttaatg cagctttgaa tgaattcaaa aatggcaaaa ataaggattt cagcaaggta
2281 acgcaagcaa aaagcgacct tgaaaattcc gttaaagatg tgatcatcaa tcaaaaggta
2341 acggataaag ttgataatct caatcaagcg gtatcagtgg ctaaagcaac gggtgatttc
2401 agtagggtag agcaagcgtt agccgatctc aaaaatttct caaaggagca attggcccaa
2461 caagctcaaa aaaatgaaag tctcaatgct agaaaaaaat ctgaaatata tcaatccgtt
2521 aagaatggtg tgaatggaac cctagtcggt aatgggttat ctcaagcaga agccacaact
2581 ctttctaaaa acttttcgga catcaagaaa gagttgaatg caaaacttgg aaatttcaat
2641 aacaataaca ataatggact caaaaacgaa cccatttatg ctaaagttaa taaaaagaaa
2701 gcagggcaag cagctagcct tgaagaaccc atttacgctc aagttgctaa aaaggtaaat
2761 gcaaaaattg accgactcaa tcaaatagca agtggtttgg gtgttgtagg gcaagcagcg
2821 ggcttccctt tgaaaaggca tgataaagtt gatgatctca gtaaggtagg gctttcaagg
2881 aatcaagaat tggctcagaa aattgacaat ctcaatcaag cggtatcaga agctaaagca
2941 ggtttttttg gcaatctaga gcaaacgata gacaagctca aagattctac aaaacacaat
3001 cccatgaatc tatgggttga aagtgcaaaa aaagtacctg ctagtttgtc agcgaaacta
3061 gacaattacg ctactaacag ccacatacgc attaatagca atatcaaaaa tggagcaatc
3121 aatgaaaaag cgaccggcat gctaacgcaa aaaaaccctg agtggctcaa gctcgtgaat
3181 gataagatag ttgcgcataa tgtaggaagc gttcctttgt cagagtatga taaaattggc
3241 ttcaaccaga agaatatgaa agattattct gattcgttca agttttccac caagttgaac
3301 aatgctgtaa aagacactaa ttctggcttt acgcaatttt taaccaatgc attttctaca 3361 gcatcttatt actgcttggc gagagaaaat gcggagcatg gaatcaagaa cgttaataca
3421 aaaggtggtt tccaaaaatc ttaagaattc gaagagccgt ggattcatca tgcaccgccg
3481 ggttgtggga atgctccaag atcatcgatc agtaatactt gcgatgaaaa aacccaaagt
3541 ctaggtgtaa aattccttga cgaataccaa tctaaagtta aaagacaaat attttcaggc
3601 tatcaatctg atattgatac acataataga attaaggatg aattaatgat taaattaaaa
3661 tttggtgttt tttttacagt tttactatct tcagcatatg cacatggaac acctcaaaat
3721 attactgatt tgtgtgcaga atcacacaac acacaaatat atacgctaaa tgataagata
3781 ttttcgtata cagaatctct agctggaaaa agagagatgg ctatcattac ttttaagaat
3841 ggtgcaattt ttcaagtaga agtaccaagt agtcaacata tagattcaca aaaaaaagcg
3901 attgaaagga tgaaggatac cctgaggatt gcatatctta ctgaagctaa agtcgaaaag
3961 ttatgtgtat ggaataataa aacgcctcat gcgattgccg caattagtat ggcaaattaa
4021 gatataaaaa gcccacctca gtgggctttt ttgtggttcg atgatgagaa gcaaccgttt
4081 tgcccaaaca tgtattactg caagtatgat gtttttattc cacatcctta gtgcgtatta
4154 tgtgctgca
5. A chimeric protein consisting of antigenic protein of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin which has an amino acid sequence deducable from the recombinant DNA of claim 1, or its functional equivalents. <
6. The chimeric protein of claim 5 , wherein the antigenic protein of Helicobacter pylori is selected from the group consisting of UreB, CagA, AlpA, AlpB, FliQ, BabAl, BabA2 ,
UreC, UreD, UreA, SodB, Urel, UreE, UreF, UreG, UreH, FlaA,
FlaB, CatA, VacA and BabB.
S2
7. A chimeric protein which is prepared by ligating UreB of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin, has an amino acid sequence represented as following, or its functional equivalents:
1 MK1SRKEYA SMYGPTTGDK VRLGDTDL1A EVEHDYTIYG EELKFGGGKT LREGMSQSXN 61 PSKEELDL1I TNAL1VDYTG 1YKAD1GIKD GK1AG1GKGG NKDTQDGVKN LSVGPATEA 121 LAGEGLIVTA GGIDTHIHF1 SPQQIPTAFA SGVTTM1GGG TGPADGTNAT TITPGRRXLK 181 FMLRAAEEYS MN'FGFLAKGN ASNDASLADQ lEAGAIGLKI HEDWGTTPSA IN'HALDVADK 41 YDVQVAIHTD TLNEAGCVED TMAAIAGRTM HTYHTEGAGG GHAPDIIKVA GEHNILPAST 301 KPTIPFTVNT E.AEHMDMLMV CHHLDKSIKE DVQFADSRIR PQTIAAEDTL HDMGIFSITS 361 SDSQAMGRVG EV1TRTWQTA DKNKKEFGRL KEEKGDNDN'F R1KRYLSKYT INPA1AHG1S 421 EYVGSVEVGK VADLVLWSPA FFGVKPNM11 KGGF1ALSQM GDANAS1PTP QPVYYREMFA 481 HHGKAKYDAN ITFVSQAAYD KG1KEELGLE RQVLPVKCR NITKKDMQFN DTTAHIEVN'S 541 ETYHVFVDGK EVTLNQPIKE FEEPW1HHAP PGCGNAPRSS 1SNTCDEKTQ SLGVKFLDEY 601 QSKVKRQIFS GYQSD1DTHN RIKDELM1KL KFGVFFTVLL SSAYAHGTPQ NITDLCAESH 661 NTQIYTLNDK IFSYTESLAG KREMA1ITFK NGAIFQVEVP SSQH1DSQKK AIERMKDTLR 7501AYLTEAKVE KLCVV7NNKTP HAIAA1SAN
8. A chimeric protein which is prepared by ligating CagA of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin, has an amino acid sequence represented as 5 following, or its functional equivalents:
1 MTNETIDQQP QTEAAFNPQQ FINNLQVAFL KVDNAVASYD PDQKPIVDKN DRDNRQAFEG
10 61 ISQLREEYS . KAIKNPTKKN QYFSDFINKS NDLINKDNLI DVESSTKSFQ KFGDQRYRIF
121 TSWVSHQNDP SKINTRSIRN FMENIIQPPI LDDKEKAEFL KSAKQSFAGI IIGNQIRTDQ
181 KFMGVFDESL KERQEAEKNG EPTGGDWLDI FLSFIFDKKQ SSDVKEAINQ EPVPHVQPDI
241 ATTTTDIQGL PPEARDLLDE RGNFSKFTLG DMEMLDVEGV ADIDPNYKFN QLLIHN'NALS
301 SVLMGSHN'GI EPEKVSLLYG GNGGPGARHD WNATVGYKDQ QGNNVATIIN VHMKNGSGLV
1 361 IAGGEKGINN PSFYLYKEDQ LTGSQRALSQ EEIQNKIDFM EFLAQNNAKL DNLSEKEKEK
421 FRTEIKDFQK DSKAYLDALG NDRIAFVSKK DTKHSALITE FG GDLSYTL KDYGKKADKA
481 LDREKVTLQ GSLKHDGVMF VDYSNFKYTN ASKNPNKGVG VTNGVSHLEV GFNKVAIFN'L
541 PDLNNLAITS FVRRNLEDKL TTKGLSPQEA NKLIKDFLSS NKELVGKTLN FNKAVADAKN
601 TGNYDEVKKA QKDLEKSLRK REHLEKEVEK KLESKSGNKN KMEAKAQA S QKDEIFALIN
20 661 KEANRDARAI AYAQNLKGIK RELSDKLENV NKNLKDFDKS FDEFKNGKNK DFSKAEETLK
721 ALKGSVKDLG INPEWISKVE NLNAALNEFK NGKNKDFSKV TQAKSDLENS VKDVIINQKV
781 TDKVDNLNQA VSVAKATGDF SRVEQALADL KNFSKEQLAQ QAQKNESLNA RKKSEIYQSV
841 KNGVNGTLVG NGLSQAEATT LSKNFSDIKK ELNAKLGNFN NNNNNGLKNE PIYAKVN'KKK
901 AGQAASLEEP IYAQVA KVN AKIDRLNQIA SGLGVVGQAA GFPLKRHDKV DDLSKVGLSR
25 961 NQELAQKIDN LNQAVSEAKA GFFGNLEQTI DKLKDSTKHN PMNLWVESAK KVPASLSAKL
1021 DNYATNSHIR INSNIKNGAI NEKATGMLTQ KNPEWLKLVN DKIVAHNVGS VPLSEYDKIG
1081 FNQKNMKDYS DSFKFSTKLN NAVKDTNSGF TQFLTNAFST ASYYCLAREN AEHGIKNVNT
1141 KGGFQKSEFE EPWIHHAPPG CGNAPRSSIS NTCDEKTQSL GVKFLDEYQS KVKRQIFSGY
1201 QSDIDTHNRI KDELMIKLKF GVFFTVLLSS AYAHGTPQNI TDLCAESHNT QIYTLNDKIF
30 1261 SYTESLAGKR EMAIITFKNG AIFQVEVPSS QHIDSQKKAI ERMKDTLRIA YLTEAKVEKL
1338 CVWNNKTPHA IAAISMAN
35
9. A recombinant expression vector which comprises the recombinant DNA of claim 1, to express a chimeric protein consisting of antigenic protein of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin.
10. A recombinant expression vector which is capable of expressing a chimeric protein consisting of UreB of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin, represented as following genetic map:
Figure imgf000086_0001
11. A recombinant expression vector which is capable of expressing a chimeric protein consisting of CagA of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin, represented as following genetic map:
Figure imgf000086_0002
12. A recombinant Escherichia coli transformed with the recombinant expression vector of claim 9.
13. Escherichia coli D /HU-044 (KCCM-10124) which is capable of expressing a chimeric protein consisting of UreB of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin.
14. Escherichia coli D /HC-033 (KCCM-10123) which is capable of expressing a chimeric protein consisting of CagA of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin.
15. A process for preparing a chimeric protein which comprises the steps of: culturing a microorganism transformed with the recombinant expression vector of claim 9; and, recovering a chimeric protein consisting of antigenic protein of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin.
16. The process for preparing a chimeric protein of claim 15, wherein the antigenic protein of Helicobacter pylori is selected from the group consisting of UreB, CagA, AlpA, AlpB, FliQ, BabAl, BabA2 , UreC, UreD, UreA, SodB, Urel, UreE, UreF, UreG, UreH, FlaA, FlaB, CatA, VacA and BabB.
17. The process for preparing a chimeric protein of claim 15, wherein the microorganism is Escherichia coli DW/HU- 044 (KCCM-10124) .
18. The process for preparing a chimeric protein of claim 15, wherein the microorganism is Escherichia coli DW/HC- 033 (KCCM-10123) . - -
19. A chimeric protein which is prepared by the process comprising the steps of: culturing a microorganism transformed with the recombinant expression vector of claim recombinant expression vector of claim 9; and, recovering a chimeric protein consisting of antigenic protein of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin .
20. The chimeric protein of claim 19, wherein the antigenic protein of Helicobacter pylori is selected from the group consisting of UreB, CagA, AlpA, AlpB, FliQ, BabAl, BabA2, UreC, UreD, UreA, SodB, Urel, UreE, UreF, UreG, UreH, FlaA, FlaB, CatA, VacA and BabB.
21. A preventive and therapeutic vaccine for Helicobacter pylori -associated diseases which comprises an active ingredient of a chimeric protein consisting of antigenic protein of Helicobacter pylori and A2 and B subunits of Vibrio cholerae toxin, and its pharmaceutically acceptable carrier.
22. The preventive and therapeutic vaccine for Helicobacter pylori -associated diseases of claim 21 , wherein the antigenic protein of Helicobacter pylori is selected from the group consisting of UreB, CagA, AlpA, AlpB, FliQ, BabAl, BabA2, UreC, UreD, UreA, SodB, Urel, UreE, UreF, UreG, UreH, FlaA, FlaB, CatA, VacA and BabB.
23. The preventive and therapeutic vaccine for Helicobacter pylori -associated diseases of claim 21, wherein the Helicobacter pylori -associated diseases comprise gastris, gastric ulcer, duodenal ulcer and gastric cancer.
PCT/KR1998/000073 1997-03-31 1998-03-31 RECOMBINANT MICROORGANISMS EXPRESSING ANTIGENIC PROTEINS OF $i(HELICOBACTER PYLORI) WO1998044130A1 (en)

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