WO1999049890A1 - VACCIN PREVENTIF ET THERAPEUTIQUE CONTRE LES MALADIES ASSOCIEES A $i(HELICOBACTER PYLORI) - Google Patents

VACCIN PREVENTIF ET THERAPEUTIQUE CONTRE LES MALADIES ASSOCIEES A $i(HELICOBACTER PYLORI) Download PDF

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WO1999049890A1
WO1999049890A1 PCT/KR1998/000072 KR9800072W WO9949890A1 WO 1999049890 A1 WO1999049890 A1 WO 1999049890A1 KR 9800072 W KR9800072 W KR 9800072W WO 9949890 A1 WO9949890 A1 WO 9949890A1
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glu
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PCT/KR1998/000072
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Byung-O Kim
Byoung-Kwang Lee
Suk-Won Yoon
Seung-Kook Park
Young-Hyo Yu
Suhkneung Pyo
Deok-Joon Choi
Sung-Seup Shin
Hyung-Jin Jung
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Daewoong Pharmaceutical Co., Ltd.
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Priority to PCT/KR1998/000072 priority Critical patent/WO1999049890A1/fr
Priority to AU65247/98A priority patent/AU6524798A/en
Publication of WO1999049890A1 publication Critical patent/WO1999049890A1/fr

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    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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 a preventive and therapeutic vaccine for Helicobacter pylori -associated diseases, more specifically, to a preventive and therapeutic vaccine for H. pylori-associated diseases which comprises an active ingredient of a chimeric protein consisting of adhesin, an antigenic protein of H. pylori and A2 and B subunits of Vibrio cholerae toxin as an adjuvant.
  • 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 in 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: Leymg, 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 cytotoxm gene may have toxicity of cytotoxm itself, though it provides excellent immunogenicity
  • a vaccine employing a non-toxic varient gene of the vacuolating cytotoxm 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 functional 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 3 ac ⁇ d(pH 1-2) to provide poor activities.
  • the present inventors first, prepared a chimeric protein expressed from a recombmant DNA which contains adhesin gene coding for an antigenic determinant of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin. Also, they discovered that the chimeric protein successfully solves the problems of the conventional vaccines and can be used as an effective vaccine for H. pylori . based on its excellent immunogenicity for H. pylori . stability under stomach environment, and penetrating property through intestinal mucous membrane to stimulate slgA production.
  • a primary object of the invention is, therefore, to provide a preventive and therapeutic vaccine for H. pylori-associated diseases employing a chimeric protein which consists of adhesin of H. pylori and A2 and B subunits of Vibrio cholerae toxin as an adjuvant.
  • Figure 1 shows a DNA sequence (SEQ ID NO:l) of a fusion gene prepared by ligat g adhesin gene of H. pylori and A2 and B subunit genes of Vibrio cholerae toxin. 4 and an amino acid sequence (SEQ ID NO: 2) translated therefrom.
  • Figure 2 is a schematic diagram showing construction strategy of an expression vector for adhesin/CTXa2B of H. pylori . pHA022.
  • Figure 3 is a photograph showing 1% agarose gel electrophoresis pattern of pTE105 plasmid, a fusion gene and pHA022 expression vector, after Dsal/PstI digestion.
  • Figure 4 is a photograph showing 15% SDS-PAGE pattern of whole cell lysate of E. coli transformed with pHA022 expression vector.
  • Figure 5 is a chromatogram showing comparison of serum IgG production of mice immunized with adhesin/CTXA2B chimeric protein and adhesin, respectively.
  • Figure 6 is a chromatogram showing comparison of slgA production of mice immunized with adhesin/CTXA2B chimeric protein and adhesin, respectively.
  • 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 its 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 a chimeric protein containing A2 and B subunits of cholera toxin and adhesin of H. pylori which has an excellent immunogenicity, in order to stimulate production of antibody to H. pylori :
  • adhesin gene 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 gene thus prepared was cleaved with Dsal and PstI, and inserted into pTE105 plasmid(see: Korean patent No.102993, KCCM-10027) to prepare an expression vector, pHA022. Then, E. coli was transformed with the expression vector to prepare a recombinant Escherichia coli DW/HP- 222 (KCCM-10078) (see: Korean patent laid-open publication No. 97-59278) . And then, the recombinant E. coli was cultured, and a chimeric protein of adhesin of H. pylori and A2 and B subunits of Vibrio cholerae toxin was obtained, which was designated as ' adhesin/CTXA2B chimeric protein 1 .
  • mice immunized with the said chimeric protein showed prevention rate against infection with H.
  • chimeric protein may be used as an active ingredient of a vaccine for prevention and treatment of H. pylori- 6 associated diseases, a diagnostic kit for H. pylori infection, and used in the production of anti-H. pylori antibody.
  • the adhesin/CTXA2B chimeric protein of the invention induces mucosal immune response to bring about infiltration of anti -adhesin IgA antibodies and/or lymphocytes into gastric mucosa .
  • prevention of H. pylori infection or removal of H. pylori already infected can be accomplished.
  • the adhesin/CTXA2B chimeric protein 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 adhesin/CTXA2B chimeric protein 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 it with pharmaceutically acceptable buffering agents such as sodium bicarbonate, potassium bicarbonate and sodium phosphate to protect the adhesin/CTXA2B chimeric protein stably by increasing pH of gastric juice or neutralizing the gastric juice, and manufactured by formulating it 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 adhesin/CTXA2B chimeric protein in case of an adult of 60kg body weight, may be administered preferably in one dose of lO ⁇ g to l,000mg 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 protein 7 has LD 5 o of 4g/kg or more, which shows that the adhesin/CTXA2B chimeric protein is sufficiently safe in the range of effective dose.
  • the present invention further provides a preventive and therapeutic vaccine for H. pyroli -associated diseases which comprises the adhesin/CTXA2B chemeric protein's 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.
  • 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 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. & 9
  • lO ⁇ l of lOx Taq polymerase buffer (lOmM T ⁇ s-HCl (pH 8.3) containing 500mMKCl, 15mM MgCl- and 0.1% (v/v) gelatin), 10 / -1 of dNTP's mixture (containing an equimolar concentration of 1.25mM dGTP, dATP, dTTP and dCTP) , 2 ⁇ g of each primer (oligonucleotides synthesized m Example 2) and 0.5 ⁇ l of Ampli Taq DNA polymerase (Perkm-Elmer Cetus , USA), was added distilled water to be a final volume of lOO ⁇ l.
  • lOmM T ⁇ s-HCl (pH 8.3) containing 500mMKCl, 15mM MgCl- and 0.1% (v/v) gelatin
  • 10 / -1 of dNTP's mixture containing an equimolar concentration of 1.25mM dGTP, dATP,
  • nucleic acid was dissolved 20 ⁇ l of TE buffer for later use.
  • nucleotide sequence of base position 1 to 54 corresponds to a signal peptide sequence of adhesin
  • nucleotide sequence of base position 1010 to 1070 corresponds to a signal peptide sequence of B subunit of Vibrio cholerae toxin.
  • FIG. 2 is a schematic diagram showing the construction strategy of pHA022.
  • FIG. 3 is a photograph showing that the fusion gene was correctly inserted into pTE105 plasmid.
  • lane 1 shows lkb ladder of DNA size marker
  • lane 2 shows pTE105 plasmid after Dsal/PstI digestion
  • lane 3 shows adhesin gene
  • lane 4 shows A2 , B subunit gene of cholera toxin
  • lane 5 shows a fusion gene digested with Dsal/PstI
  • lane 6 shows pHA022 digested with Dsal/PstI .
  • 1% agarose gel electrophoresis revealed that the pHA022 expression vector has unique restriction site for each restriction enzyme.
  • E. coli JM101 was first inoculated m 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 11 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 pHA022 expression vector prepared in Example 4, 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.
  • 0.2ml of the cell suspension thus obtained was added to petri dishes coated with liquid LB media containing 12.5 ⁇ g/ml of tetracycline, and cultured at 37°C overnight to obtain a transformant of E. coli JM101 harboring pHA022.
  • the transformant thus prepared was designated as Escherichia coli DW/HP-222, and deposited with the Korean Culture Center of Microorganisms (KCCM) , an international depositary authority under an accession No. KCCM- 10078 on December 11, 1995.
  • KCCM Korean Culture Center of Microorganisms
  • the transformed E. coli DW/HP-222 (KCCM-10078) was cultured in liquid LB medium at 37°C for 18 hours, centrifuged to harvest cells, resuspended in a buffer (lOmM Tris-HCl (pH 8.0) containing 0.1% Triton X-100, 2mM EDTA and ImM PMSF) , and lysed to prepare E. coli extract . Then, 15% SDS-PAGE was carried out with lO ⁇ l of E. coli extract, in accordance with Laemmli's method (see: Laemmli, et al . , Nature, 277:680 (1970) ) (see: Figure 4(A)).
  • lane 1 shows protein size-marker
  • lane 2 shows extract of E. coli harboring a pTE105 plasmid as a control
  • lane 3 to lane 7 show time-dependent extracts of DW/HP-222 , E . coli harboring the pHA022 after IPTG induction
  • top arrow indicates locus of chimeric protein of adhesin of H. pylori and A2 subunit of a Vibrio cholerae toxin
  • bottom arrow indicates locus of B subunit of Vibrio cholerae toxin.
  • the E. coli DW/PHA022 (KCCM-10078) 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 adhesin/CTXA2B protein.
  • 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 4 , lOmMKCl, 20mM NaHC0 3 and 48.5mM polyethyleneglycol) four times at 15-minute intervals into mice, injecting 0.2ml of 13 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 4 , lOmMKCl, 20mM NaHC0 3 and 48.5mM polyethyleneglycol
  • Quantitation of the antibody produced by adhesin/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 9 Effect of adhesin/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 10CFU and administered into mice three 14 times at 2 -day intervals using polyethylene catecher.
  • the cultured strains were suspended in 500ml of a physiological saline and catalase, oxidase and urease reactions were carried out as followings: First, lOO ⁇ l of each sample was added to 1ml of an urease-detecting reagent (2Og/1 urea, 0.05%(w/v) phenolred, 0.044g/l NaH 2 P0 4 H 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 2Og/1 urea, 0.05%(w/v) phenolred, 0.044g/l NaH 2 P0 4 H 2 0, 1.02g/l Na 2 HP0 4 , 0.2g/
  • a solution containing adhesin/CTXA2B chimeric protein was prepared as described above .
  • the present invention provides a preventive and therapeutic vaccine for H. pylori -associated diseases employing a chimeric protein which consists of adhesin of H. pylori and A2 and B subunits of Vibrio cholerae toxin. Since the adhesin/CTXA2B chimeric protein can induce specific antibodies neutralizing H. pylori , it can be used as an active ingredient of diagnostic kit for H. pylori infection and preventive or therapeutic vaccine for H. pylori-associated diseases as well.
  • ORGANISM Helicobacter pylori and Vibrio cholerae
  • AAATCAGAAC CCGGGTTATT ATTCTCCACC GGTTTGGACA AAATGGAAGG 500
  • ORGANISM Helicobacter pylori
  • ORGANISM Helicobacter pylori
  • ORGANISM Vibrio cholerae
  • ORGANISM Vibrio cholerae

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Abstract

L'invention concerne un vaccin préventif et thérapeutique contre les maladies associées à H. pylori, dont le principe actif est une protéine chimère comprenant de l'adhésine, une protéine antigénique de H. pylori, et, comme adjuvant, les sous-unités A2 et B de la toxine de Vibrio cholerae. Du fait que la protéine chimère adhésine/CTXA2B provoque l'apparition d'anticorps spécifiques neutralisant H. pylori, elle peut être utilisée comme principe actif d'une trousse diagnostique permettant de détecter une infection à H. pylori, ainsi que d'un vaccin préventif ou thérapeutique contre les maladies associées à H. pylori.
PCT/KR1998/000072 1998-03-31 1998-03-31 VACCIN PREVENTIF ET THERAPEUTIQUE CONTRE LES MALADIES ASSOCIEES A $i(HELICOBACTER PYLORI) WO1999049890A1 (fr)

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PCT/KR1998/000072 WO1999049890A1 (fr) 1998-03-31 1998-03-31 VACCIN PREVENTIF ET THERAPEUTIQUE CONTRE LES MALADIES ASSOCIEES A $i(HELICOBACTER PYLORI)
AU65247/98A AU6524798A (en) 1998-03-31 1998-03-31 A preventive and therapeutic vaccine for (helicobacter pylori)-associated diseases

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100378223C (zh) * 2003-04-17 2008-04-02 南方医院 具有粘附功能的幽门螺杆菌膜孔素
US7393529B2 (en) 1998-04-09 2008-07-01 Idexx Laboratories, Inc. Methods and compositions for inhibiting binding of IgE to a high affinity receptor
WO2009145603A1 (fr) 2008-05-26 2009-12-03 Universidad De Concepcion Nécessaire moléculaire de diagnostic de souches virulentes d'helicobacter pylori
EP2426140A1 (fr) 2003-08-15 2012-03-07 University of Florida Research Foundation, Inc. Identification de polynucléotides responsables de la virulence de porphyromonas gingivalis dans le diagnostic, le traitement, et la surveillance de maladies parodontales

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002836A1 (fr) * 1995-07-07 1997-01-30 Oravax, Inc. Toxines de clostridium difficile utilisees comme adjuvants agissant sur les muqueuses
WO1997011182A1 (fr) * 1995-09-22 1997-03-27 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin Nouvelle adhesine obtenue a partir de helicobacter pylori
WO1998006428A1 (fr) * 1996-08-16 1998-02-19 The Uab Research Foundation Immunogenes muqueux pour nouveaux vaccins
WO1998012562A1 (fr) * 1996-09-20 1998-03-26 Cortecs International Limited Adhesines obtenues d'heliobacter pylori et leurs utilisations therapeutiques et diagnostiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002836A1 (fr) * 1995-07-07 1997-01-30 Oravax, Inc. Toxines de clostridium difficile utilisees comme adjuvants agissant sur les muqueuses
WO1997011182A1 (fr) * 1995-09-22 1997-03-27 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin Nouvelle adhesine obtenue a partir de helicobacter pylori
WO1998006428A1 (fr) * 1996-08-16 1998-02-19 The Uab Research Foundation Immunogenes muqueux pour nouveaux vaccins
WO1998012562A1 (fr) * 1996-09-20 1998-03-26 Cortecs International Limited Adhesines obtenues d'heliobacter pylori et leurs utilisations therapeutiques et diagnostiques

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7393529B2 (en) 1998-04-09 2008-07-01 Idexx Laboratories, Inc. Methods and compositions for inhibiting binding of IgE to a high affinity receptor
US7931898B2 (en) 1998-04-09 2011-04-26 Idexx Laboratories, Inc. Methods and compositions for inhibiting binding of IgE to a high affinity receptor
US8252907B2 (en) 1998-04-09 2012-08-28 Idexx Laboratories, Inc. Methods and compositions for inhibiting binding of IgE to a high affinity receptor
CN100378223C (zh) * 2003-04-17 2008-04-02 南方医院 具有粘附功能的幽门螺杆菌膜孔素
EP2426140A1 (fr) 2003-08-15 2012-03-07 University of Florida Research Foundation, Inc. Identification de polynucléotides responsables de la virulence de porphyromonas gingivalis dans le diagnostic, le traitement, et la surveillance de maladies parodontales
WO2009145603A1 (fr) 2008-05-26 2009-12-03 Universidad De Concepcion Nécessaire moléculaire de diagnostic de souches virulentes d'helicobacter pylori

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