WO2013019098A1

WO2013019098A1 - Helicobacter pylori proteins for diagnostic kit and vaccine

Info

Publication number: WO2013019098A1
Application number: PCT/MY2011/000195
Authority: WO
Inventors: Dr. Rahmah NOORDIN; Dr. Amutha SANTHANAM; Akbar KHALIPOUR; Chun Wei LEE; Sabariah OSMAN
Original assignee: Universiti Sains Malaysia
Priority date: 2011-08-03
Filing date: 2011-08-25
Publication date: 2013-02-07
Also published as: MY182584A

Abstract

The present invention relates to an isolated antigenic protein of Helicobacter pylori. More specifically, the present invention provides an isolated antigenic protein of Helicobacter pylori selected from a group comprising polypeptides having a molecular weight of 13 kDa, 25 kDa, 29 kDa, 30 kDa, 31 kDa or 1 18 kDa, or any combination thereof, wherein said antigenic protein is in substantially purified form. The present invention also relates to a use of said isolated antigenic protein of Helicobacter pylori in vaccines and diagnostic kits for treatment of Helicobacter pylori infection in humans.

Description

HELICOBACTER PYLORI PROTEINS FOR DIAGNOSTIC KIT AND VACCINE

FIELD OF INVENTION

The present invention relates to Helicobacter pylori proteins for diagnostic kit and vaccine.

BACKGROUND OF INVENTION

Helicobacter pylori, also known as H.pylori,\s a gram negative, spiral-shaped, microaerophilic, flagellated slow growing bacteria. It colonizes the apical side of human gastric epithelial cells and mucous layer and wasfirst isolated in 1982 by Marshall and Warren. H.pylori infections are associated with various gastric diseases; among them the most common is active gastritis, and is also implicated in more severe gastric diseases including chronic atrophic gastritis (a precursor of gastric carcinomas), peptic ulceration, duodenal ulcer and mucosa-associated lymphoid tissue lymphomas. The severity of the infection depends on the strain virulence, host susceptibility and environmental co-factors.

However, 6-20% of the infected individuals tend to develop duodenal ulceration and a small proportion of them will eventually advance to gastric cancer. H.pylorns also associated with other clinical problems such as asthma, atherosclerotic heart disease. Consequently the early detection and treatment of H.pylori infection can prevent or reduce morbidity and mortality rates of patients.

During the process of H. pylori infection, the antigens secreted from the bacterium elicit strong humoral immune response; these antigens can thus be regarded as prospective candidates that may serve as infection biomarkers. Various H.pylori proteins had been employed as infection markers for diagnosis, such as CagA, VacA, HspB, FlaA, FlaB, UreC. There are also markers that utilize stool antigen and carbon labeled urease breath test. In some commercially available diagnostic kits, a mixture of antigen extract is used as it provides a higher sensitivity and specificity as compared to utilizing a single antigen. Use of H.pylori proteins as infection markers were disclosed in US Pending Application No. US 2003/0180330 and US Pending Application No. US 2007/0243204.

The accuracy of the diagnostic markers varies from test to test, depending greatly on type of sample and type of diagnostic assay. These differences may be due to several reasons, such as the presence of strain heterogeneity in different geographic regions, cross-reactivity to other intestinal pathogens which vary in different parts of the world and varying immunological responses to H. pylori antigens in different patient populations. Thus it is important to identify H. pylori antigens from local isolates that can detect H. pylori infections in the local population with high sensitivity and specificity. These antigens would probably also be suitable for use in populations in other countries in this region of the world. Accordingly, a need still exits to identify H. pylori antigens from isolates with high sensitivity and specificity for development of diagnostic kits and vaccines for treatment and detection of H. pylori infections in humans.

SUMMARY OF INVENTION

Accordingly, the present invention relates to an isolated antigenic protein of Helicobacter pylori. More specifically, the present invention provides an isolated antigenic protein of Helicobacter py/onselected from a group comprises of a polypeptide having a molecular weights of 13 kDa, or 25 kDa.or 29 kDa.or 30 kDa.or 31 kDaorl 18kDa, or any combination thereof.wherein said antigenic protein is in substantially purified form.

The present invention also relates to a use of the isolated antigenic protein of Helicobacter pylori in vaccines and diagnostic kits for treatment of Helicobacter pylori infection in humans.

BRIEF DESCRIPTION OF DRAWINGS

The drawings constitute part of this specification and include an exemplary or preferred embodiment of the invention, which may be embodied in various forms. It should be understood, however, the disclosed preferred embodiments are merely exemplary of the invention. Therefore, the figures disclosed herein are not to be interpreted as limiting, but merely as the basis for the claim and for teaching one skilled in the art of the invention.

In the appended drawings:

FIG. 1 illustrates IgG immunoblot of H. pylori antigen (fraction 3) incubated with human sera. Lane 1 : serum from H.pylori patient; Lane 2: serum of healthy person: M: molecular weight marker.

FIG. 2 illustrates IgG immunoblot of H. pylori antigen (fraction 7) incubated with human sera. Lane 1 : serum from H. py/onpatient; Lane 2: serum of healthy person: M: molecular weight marker.

FIG. 3 illustrates IgA immunoblot of H. pylori antigen (fraction 3) incubated with human sera. Lane 1 : serum from H.pylori patient; Lane 2: serum of healthy person: M: molecular weight marker.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Detailed descriptions of preferred embodiments of the present invention are disclosed herein. It should be understood, however, that the embodiments are merely exemplary of the present invention, which may be embodied in various forms. Therefore, the details disclosed herein are not to be interpreted as limiting, but merely as the basis for the claim and for teaching one skilled in the art of the invention.

A study was conducted using local bacterial isolates to identify diagnostic markers that would be useful for diagnosis of H. pylori infection.

H. pylori antigen was prepared by freeze-thawing technique using liquid nitrogen and 37 Ό water bath. Positive serum samples were obtained from patients with culture-positive H. pylori infection, and negative control serum samples came from three groups of people i.e. healthy, gastrointestinal patients without H. pylori infection and those with other bacterial/parasitic infections.

The H. pylori antigen was subjected to two dimensional electrophoresis, followed by Western blot analysis using the above sera and probed with peroxidase-conjugated anti-human IgG and IgA antibodies. Selected antigenic bands were excised out and sent for mass spectrometry analysis.

The results showed that antigenic bands with approximate molecular weights of 13, 25, 30 and 1 18 kDa were detected in IgG blots. The 13 kDa, 25kDa bands showed sensitivities of 73.3% and 76.6% respectively, whereas combination of the two bands gave a sensitivity rate of 86.6%. The combination of 13 kDa, 25kDa and 30 kDa bands were positive in 93.3% of serum samples. When all four bands were combined, both the sensitivity was 100%. In IgA blots, the antigenic bands with approximate molecular weights of 29 and 31 were detected, and the sensitivity of the combination of this double band was 70%. Specificities in all cases were 95-100%. MALDI TOF-TOF analysis were used to determine the identities of each of the bands.

The following examples further illustrate but by no means limit the scope of present invention: Material and Methods

Bacterial Strain and Growth Conditions The H.pylori used in this study was isolated from human duodenal biopsy sample obtained from a patient with duodenal ulcer at the Sebarang Jaya Hospital, Penang, Malaysia. The bacteria were cultured on Tryptic Soy Blood Agar with 5% defribinated sheep blood under microaerophilic condition (10% C02 , 5% oxygen, 85% nitrogen in air) at 37^SC for 5 to 7 days.

Preparation of Antigens

The bacterial cells were aseptically harvested by adding 3m L phosphate-buffered saline to each plate; the bacterial suspension was then placed in 10ml centrifuge tube. After centrifugation at 3,000 X g, 4°C for 15 minutes, one washing step was performed, using 1 ml of phosphate buffered saline containing 40 μΙ protease inhibitor (Roche Applied Science). The resulting pellet of H. pylori was res-uspended with 40m M Tris (pH 7.8) containing protease inhibitor at a ratio of 1 :2 and vortexed vigorously for 5 min. Lysis of the bacterial suspension was then performed by three cycles of freeze-thawing using liquid nitrogen and 37"C water bath. After centrifugation for 15 min at 10, 000 X g, the bacterial pellet was re- suspended in a sample buffer containing urea, thiourea, DTT, glycerol, then, vortexed well and centrifuged for 15 minutes at 10,000 X g. The protein content of the supernatant was determined by BioRad RCDC method. 2-D electrophoresis-based protein separation

For the first dimension of the 2-D gel electrophoresis, the OFF-GEL apparatus (Agilent Technologies) and 12-well IPG gel strip of pH 3-10 was used according to the manufacturer's instructions. The IPG gel strip was rehydrated in the assembled device with 40μΙ of rehydration buffer per well for 15 minutes prior to sample loading.

An amount of 2mg of the H. pylori bacterial preparation was mixed with OFFGEL sample buffer to a final volume of 2ml, and 150μΙ sample volume was loaded into each well. The sample was focused with a maximum current of 50μΑ, with a maximum power of 200mW and typical voltages ranging from 400 to 4000V until 64kVh was reached for approximately 17 hrs. For the second dimension, samples from OFFGEL fractions were mixed with sample buffer (4:1 ratio) containing 28.6% SDS and 4.76% 2-mercaptoethanol, then electrophoresed using a 12% Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) in running buffer containing 0.3% tris, 1.44% glycine and 0.1 % SDS with a pH of 8.3. A sample volume of 23ul per well was evenly loaded, and electrophoresis was ran at room temperature with a constant current of 100V for 100 minutes. Western blotting

Protein bands on the SDS-PAGE was electrophoretically transferred onto a nitrocellulose membrane (NCP), 0.45μπι by using a using semi dry transblot (Bio Rad -USA) at a constant current of 12A for 30 minutes. The membranes were blocked for 1 hr at room temperature with blocking solution (Roche Diagnostic), then, washed three times with 0.05% Tween20 in TBS for 10 minutes. Then the membrane was cut into strips and incubated with various categories of human sera at 1 :100 dilutions. The following serum samples were used: 30 from H.pylori infected patients with duodenal ulcer whose biopsy samples were culture- positive. They were also seropositive with an in-house IgG-ELISA which uses H.pylori excretory secretory antigen (ESA), this in-house assay has previously been shown to produce results comparable with a commercial kit (Adaltis, Italy). Three kinds of control sera were used i.e. 20 from healthy people (without history of gastric disorders), 30 patients who attended endoscopy clinic but were H.pylori culture negative and 10 people with other disease such as typhoid, leptospirosis, Escherichia coli septicaemia, shigellosis, Staphylococcus aureus septicaemia and amoebic liver abscess. All control sera tested negative for anti-H. pylori antibodies.

Immunodetection was performed with monoclonal mouse anti-human IgG and IgA conjugated with horseradish peroxides (HRP), each at a dilution of 1 :2000 for 1 hour. Substrate development was performed using enhanced chemiluminescence blotting reagent (Roche diagnostics, Germany). The molecular weight of proteins specific to H.pylori was determined by reference to commercially available standards. The antigenic bands which have reacted with most of the H.pylori patients' sera and not cross-reactive with sera from the control groups were selected for further analysis.

In-Gel digestion of proteins and sample clean up

Selected proteins bands were manually excised from the silver-stained gels and transferred into microfuge tubes. An in-gel digestion was carried out; first, the silver-stained protein was de-stained by adding 100mM of sodium thiosulphate and 30mM of potassium ferricyanide in a ratio of 1 :1 for 20 minutes. Then, the supernatant was removed and replaced by 100μΙ of 200mM ammonium bicarbonate for 20 minutes at room temperature. De-stained protein band was then incubated for 15 minutes in 50μΙ of acetonitrile and the supernatant was removed followed by rehydration in 25μΙ of 25mM ammonium bicarbonate for 10 minutes. The supernatant was removed and air dried for 1 hour. It was then enzymatically digested by re-suspending in 0ng/μΙ trypsin on ice for 5 minutes, the gel pieces were then immersed in 25μΙ of 25mM ammonium bicarbonate and incubated overnight at 37°C. Then, acetonitrile was added and the mixture was left for 20 minutes at room temperature. The supernatant which contained the digested protein samples was transferred to a fresh microfuge tube and dried using a SpeedVac concentrator. The resulting sample was cleaned up by ZipTipC18 pipette tips (Milipore) and dissolved in 0.1 % trifluoroacetic acid. Mass spectrometry analysis & protein Identification

The above processed samples were then sent to the Proteomic Laboratory at the National University of Singapore for mass-spectrometry analysis. Bioinformatic analysis was used to elucidate the amino acid sequences of the proteins. The corresponding nucleotide sequences were then deduced.

Results

The sensitivity and specificities of the antigenic band is calculated as follows:

Sensitivity : number of H. p or/^'-patient sera reactive with the band

total number of H. py/o/7-patient sera tested (n=30)

Specificity : number of control sera not reactive with the band

total number control sera tested (n=60)

In IgG blots, four bands of approximate molecular weights (MWs) of 13, 25, 30 and 1 18 kDa were identified to be of potential diagnostic value , as shown is FIGS. 1 -2. The sensitivities of each antigenic band to detect anti- H.pylori antibodies in patients' sera samples and the specificities when tested with the control sera groups are depicted in TABLE 1 .

The 13 kDa, 25kDa bands showed sensitivities of 73.3% and 76.6% respectively, whereas combination of the two bands gave a positive rate of 86.6%. The combination of 13 kDa, 25kDa and 30 kDa bands were positive with 93.3% of the H. pylori patients' serum samples. When all four bands were combined, both the sensitivity and specificity were 100%.

TABLE 1 : Results of IgG & IgA blots of H.pylori antigen probed with human sera

In IgA blots, antigenic bands were detected at 29kDa and 3 kDa as shown in FIG. 3. Combination of these two bands was observed with 70% of H.pylori serum samples, with a specificity of 100%. Table 2 showed the results of the identifications of the six selected bands when analysed by MALDI-TOF/TOF.

Amino acid and DNA nucleotide sequences of each of the identified antigenic proteins are shown in sequence listing. TABLE 2: Mass spectrometric identification of antigenic proteins of H.pylori

Discussion

Six antigenic proteins that are sensitive and specific in detecting H. pylori infection were identified. The band with ~MWs of 1 18kDa was found to have the highest sensitivity (100%). In addition, combinations of 13, 25, 30kDa with or without 100kDa also gave high sensitivity.

In summary, the six H. pylori infection markers identified in this study have good potential diagnostic value, and can be used for future development of diagnostic kits. In addition, they are also potentially useful as vaccine candidates against H. pylori infection.

While embodiments and examples of the present invention have been illustrated and described, it is not intended that these embodiments and examples illustrate and describe all possible forms of the present invention. Rather, words used in the specification are words of description rather than limitation and various changes may be made without departing from the scope of the invention. SEQUENCE LISTING

13 kDa antigenic protein

Description: ribosomal protein L7/L12 [Helicobacter pylori G27] Protein Sequence: SEQ ID No .1

MAISKEEVLE YIGSLSVLEL AELVKMFEEK FGVSATPTW AGAAVAGGAA AESEEKTEFN VILADSGAEK IKVIKWREI TGLGLKEAKD ATEKTPHVLK EGVNKEEAET IKKKLEEVGA KVEVK

DNA Sequence: SEQ ID No .2

ATGGCAATTTCAAAAGAAGAAGTGTTAGAGTATATTGGTTCATTGAGCGTTTTAGAGCTT GCTGAATTGGTTAAAATGTTTGAGGAAAAATTTGGCGTGAGCGCGACTCCAACGGTCGTA GCGGGTGCGGCTGTAGCTGGTGGTGCAGCGGCTGAAAGCGAAGAAAAAACCGAATTTAAT GTGATTTTGGCCGATAGCGGTGCTGAAAAAATTAAGGTGATTAAAGTGGTTCGTGAAATC ACTGGACTTGGCCTGAAAGAAGCTAAAGACGCTACTGAAAAAACCCCTCATGTGCTTAAA GAGGGCGTGAATAAAGAAGAAGCTGAAACCATCAAGAAGAAACTTGAAGAAGTAGGCGCT AAGGTTGAAGTCAAGTAA

Description: 50S ribosomal protein L7/L12 [Helicobacter pylori J99] Protein Sequence: SEQ ID No.3

MAISKEEVLE YIGSLSVLEL SELVKMFEEK FGVSATPTW AGAAVAGGAA AESEEKTEFN VILADSGAEK IKVIKWREI TGLGLKEAKD ATEKTPHVLK EGVNKEEAET IKKKLEEVGA KVEVK

DNA Sequence: SEQ ID No .

ATGGCAATTTCAAAAGAAGAAGTGTTAGAGTATATTGGTTCATTGAGCGTTTTGGAGCTT TCTGAATTGGTTAAAATGTTTGAGGAAAAATTTGGCGTGAGTGCGACTCCAACGGTCGTA GCGGGTGCGGCTGTAGCTGGCGGTGCAGCGGCTGAGAGCGAAGAAAAAACCGAATTTAAT GTGATTTTGGCCGATAGCGGCGCTGAAAAAATCAAGGTGATTAAAGTGGTTCGTGAAATC ACCGGGCTTGGCTTGAAAGAAGCTAAAGACGCTACCGAAAAAACCCCTCATGTGCTTAAA GAGGGCGTGAATAAAGAAGAAGCTGAAACCATCAAGAAGAAACTTGAAGAAGTAGGTGCT AAGGTTGAAGTCAAGTAA

25 kDa antigenic protein

Description: cag island protein [Helicobacter pylori J99]

Protein Sequence: SEQ ID No .5

MELGFNEAER QKILDSNSSL IRNANEVRDK FIQNYAISLK DSNDPQDFLR RVQELRINMQ KNFISFDTYY NYLNNLVLAS YNRCKQEKTF AESTIKNELT LGEFVAEISD NFNNFMCDEV ARISDLVASY LPREYLPPFI DGNMMGVAFQ ILGIDDFGRK LNEIVQDIGT KYI ILSKNKT YLTSLERAKL ITQLKLN

DNA Sequence: SEQ ID No .6

ATGGAACTAGGTTTCAATGAAGCAGAAAGACAAAAGATCTTAGACAGCAACAGCTCTCTT ATAAGAAATGCGAATGAAGTAAGGGATAAGTTTATTCAAAATTACGCCATTTCTTTAAAA GATAGCAACGATCCGCAAGATTTTTTGAGAAGAGTTCAAGAGTTAAGAATCAATATGCAA AAGAATTTTATTAGTTTTGATACTTATTACAACTATTTAAACAACCTTGTGTTAGCCAGT TACAATCGTTGCAAACAAGAAAAGACTTTTGCAGAAAGCACGATCAAAAATGAACTAACG CTTGGAGAGTTTGTTGCAGAAATTTCTGACAACTTCAATAATTTCATGTGTGATGAAGTG GCAAGAATTTCAGACCTAGTGGCTTCTTATCTGCCAAGAGAGTATTTACCGCCATTCATA GATGGCAATATGATGGGCGTGGCGTTTCAGATTCTAGGGATAGATGATTTTGGGAGAAAG CTCAATGAGATTGTCCAAGATATAGGGACTAAATATATTATTTTGAGCAAAAATAAGACT TATCTCACTTCTTTAGAAAGGGCTAAATTGATAACCCAGTTAAAATTAAATTTGGAATAA Description: cag pathogenicity island protein [Helicobacter pylori] Protein Sequence: SEQ ID No.7

MELGFNEAER QKILDSNRSL MGNANEVRDK FIQNYAISLK DSNDPQDFLR RVQELRINMQ KNFISFDAYY NYLNNLVLAS YNRCKQEKTF AESTIKNELT LGEFVVEISD NFNNFMCDEV ARISDLVASY LPREYLPPFI DGNMMGVAFQ ILGIDDFGRK LNEIVQDIGT KYIILSKNKT YLTSLERAKL ITQLKLNLE

DNA Sequence: SEQ ID No .8

ATGGAACTCGGTTTCAATGAAGCAGAAAGACAAAAGATCTTAGATAGCAACAGATCTCTT ATGGGAAATGCAAATGAAGTAAGGGATAAGTTTATTCAAAATTACGCCATTTCTTTAAAA GATAGCAACGATCCGCAAGATTTTTTGAGAAGAGTTCAAGAGTTAAGAATCAATATGCAA AAGAATTTTATTAGTTTTGATGCTTATTACAACTATTTGAACAACCTTGTGTTAGCTAGC TACAATCGTTGCAAACAAGAAAAGACTTTTGCAGAAAGCACGATCAAAAATGAACTAACG CTTGGGGAGTTTGTTGTAGAAATTTCTGACAACTTCAATAATTTTATGTGTGATGAAGTG GCAAGAATTTCAGACCTAGTGGCTTCTTATCTGCCAAGAGAGTATTTACCGCCATTCATA GATGGCAATATGATGGGCGTGGCGTTTCAGATTCTAGGGATAGATGATTTTGGGAGAAAG CTCAATGAGATTGTCCAAGATATAGGGACTAAATATATTATTTTGAGCAAAAATAAGACT TATCTCACTTCTTTAGAAAGAGCTAAATTGATAACCCAATTAAAATTAAATTTGGAATAA

30 kDa antigenic protein

Description: acyl-carrier-protein S-malonyltransferase

[Helicobacter pylori HPAG1]

Protein Sequence: SEQ ID No .9

1 MQYALLFPGQ GSQCIGMGKS FYESHTLAKE LFERASNALK VD KKTLFEE

51 NELLKESTYT QPAIYLVSYI AYQLLNKQVN GGLKPVFALG HSLGEVSAVS

101 LSGALDFEKA LKLTHQRGKM MQEACANKDA S MWLGVSE ESLLSLCQRT

151 KNVWCANFNG GMQVVLAGIK DDLKALEPTL KEMGAKRVVF LEMSVASHCP

201 FLEPMIFKFQ ELLEKSLKDK FHFEIISNAT NEAYHNKAKA VELLSLQLTQ

251 PVRYQDCVKS NNDRVDVFFE LGCGSVLKGL NKRLSNKPTI SVGDNKGLDE

301 AIEFLEEYV

DNA Sequence: SEQ ID No.10

ATGCAATACGCGCTATTATTTCCAGGGCAAGGCTCGCAATGTATAGGAATGGGAAAGTCA TTCTATGAGAGCCACACCCTAGCTAAAGAATTGTTTGAAAGGGCTTCTAACGCGCTTAAA GTGGATATGAAAAAAACGCTTTTTGAAGAAAACGAGCTTTTAAAAGAGAGCGCTTACACC CAGCCTGCCATTTATTTAGTGAGCTATATCGCTTACCAATTGCTCAACAAGCAAGTGAAT GGAGGGTTAAAACCCGTTTTTGCTTTAGGGCATTCGCTCGGCGAAGTGAGCGCGGTGTCT TTGAGCGGGGCGTTAGATTTTGAAAAAGCCCTTAAACTCACGCACCAAAGAGGCAAAATG ATGCAAGAAGCGTGCGCGAATAAAGACGCTTCCATGATGGTCGTTTTGGGCGTTTCTGAA GAGAGTCTTTTGAGTTTGTGTCAAAGAACCAAAAATGTGTGGTGCGCGAATTTCAATGGC GGCTTGCAAGTGGTTTTAGCCGGGATTAAAGACGATTTGAAAGCCCTAGAGCCGACTTTA AAGGAAATGGGGGCTAAAAGAGTGGTTTTTTTAGAAATGAGCGTGGCGAGCCATTGCCCT TTTTTAGAGCCTATGATTTTTAAATTCCAGGAATTGCTAGAAAAAAGCCTGAAAGATAAA TTCCATTTTGAAATCATCTCCAATGCGACTAACGAAGCGTATCACAACAAAGCAAAAGCC GTTGAACTATTGAGCTTGCAACTCACTCAGCCGGTGCGTTATCAAGACTGCGTGAAATCC AACAATGACCGAGTGGATGTCTTTTTTGAATTAGGCTGTGGGAGTGTGTTAAAAGGGCTT AACAAGCGATTGAGCAACAAGCCTACTATAAGCGTGGGGGATAATAAAGGGCTTGATGAA GCGATTGAGTTTTTAGAAGAATACGTGTGA

Description: malonyl coenzyme A-acyl carrier protein

transacylase [Helicobacter pylori Shi470]

Protein Sequence: SEQ ID No.11

1 MQYALLFPGQ GSQCIGMGKS FYESHTLAKE LFERASNALK VDMKKTLFEE

51 NELLKESTYT QPAIYLVSYI AYQLLNKQVN GGLKPVFALG HSLGEVSAVS

101 LSGALDFEKA LKLTHQRGKM MQEACANKDA SMMWLGVSE ESLLSLCQRT

151 KNVWCANFNG GMQVVLAGIK DDLKALEPTL KEMGAKRVVF LEMSVASHCP 201 FLEPMIFKFQ ELLEKSLKDK FHFEIISNAT NEAYHNKAKA VELLSLQLTQ 251 PVRYQDCVKS NNDRVDVFFE LGCGSVLKGL NKRLSNKPTI SVGDNKGLDE 301 AIEFLEEYV

DNA Sequence: SEQ ID No.12

ATGCAATACGCGCTATTATTTCCAGGGCAAGGCTCGCAATGTATAGGAATGGGGAAATCA TTCTATGAGAGCCACACCCTAGCTAAAGAATTGTTTGAAAGGGCTTCTAACGCGCTTAAA GTGGATATGAAAAAAACGCTTTTTGAAGAAAATGAGCTTTTAAAAGAGAGCACTTACACC CAGCCTGCCATTTATTTAGTGAGCTATATCGCTTACCAATTGCTCAACAAACAAGTAAAT GGGGGGTTAAAACCCGTTTTTGCTTTGGGGCATTCGCTCGGCGAAGTGAGCGCGGTGTCT TTGAGCGGGGCGCTAGATTTTGAAAAAGCCCTTAAACTCACGCACCAAAGAGGCAAAATG ATGCAAGAAGCGTGCGCGAATAAAGACGCTTCCATGATGGTCGTTTTGGGCGTTTCTGAA GAGAGCCTTTTGAGTTTGTGTCAAAGAACCAAAAATGTGTGGTGCGCGAATTTCAATGGC GGCATGCAAGTGGTTTTAGCCGGGATTAAAGACGATTTGAAAGCCCTAGAGCCGACTTTA AAGGAAATGGGGGCTAAAAGGGTGGTTTTTTTAGAAATGAGCGTGGCGAGCCATTGCCCT TTTTTAGAGCCTATGATTTTTAAATTCCAGGAATTGCTAGAAAAAAGCCTGAAAGATAAA TTCCATTTTGAAATCATCTCCAATGCGACTAACGAAGCGTATCATAACAAAGCAAAAGCC GTTGAATTATTGAGCTTGCAACTCACTCAGCCGGTGCGTTATCAAGACTGCGTGAAATCC AATAATGACCGAGTGGATGTCTTTTTTGAATTAGGCTGTGGGAGTGTGTTAAAGGGGCTT AACAAGCGATTGAGCAACAAGCCCACTATAAGCGTGGGGGATAATAAAGGGCTTGATGAA GCCATTGAGTTTTTAGAAGAATACGTGTGA

118 kDa antigenic protein

Description: proline/pyrroline-5-carboxylate dehydrogenase [Helicobacter pylori J99]

Protein Sequence: SEQ ID No.13

1 MQKI IEDSLE LAKKLQDS IS NHLSEQEKAF HFKMQKLLNN PENKVMLIEL

51 MDRSFRCLDN KARFEMIEHV LDKYKSREIF SSFEKWLLMG FLSFGKMLPD 101 MSVPFFVNKI RSDTKAMVLD QEESQLRERI LKRKNEKIIL NVNFIGEEVL 151 GEEEASARFE KYSQALKSNY IQYISIKITT IFSQINILDF EYSKKEIVKR 201 LDALYALALE EEKKQGMPKF INLDMEEFRD LELTVESFME SIAKFDLNAG 251 IVLQAYIPDS YEYLKKLHAF SKERVLKGLK PIKIRFVKGA NMESEETIAS 301 MKDWALPTFS SKQDTDSNYN KMLDFVLEGD NYKYIHIGAA SHNIFEIAYV 351 YTRIHALNDP WLEHFSFEM LEGMSLQASQ ELKEMHKLIL YAPVCDEAHF 401 NNAIAYLVRR LDENTSSDNF MKAFFNLKVG TSEWKDQEQR FLNSLKGIAT 451 LDNTTHRTQD RNAKQTGHTT YPNHSFKNES DTDFILKANR EWAKKVRDKM 501 HNAPILELYP EIDGRFEDPN LTPLEVFDRI HHKKIASVHL ADKEAILKAL 551 EVAKSDKSRF SQKSFTEIHA LLSQTAQLFR ERRGDLIGIS ALEVGKTFAE 601 TDAEVSEAID FLEFYPYSLR VLQEQNKKTK FTPKGVGWI APWNFPVGIS 651 VGTIAAPLAA GNRVIYKPSS LSSVTGYKLC ECFWDAGVPR DALIYLPSKG 701 SDISEHLLKD ESIKFAILTG GEDTAYKMLK ANPTLALSAE TGGKNATIVS 751 KMADRDQAIK NVIHSAFSNS GQKCSATSLL VLEKEVYEDE NFKKTLIDAT 801 LSLSVGDPFD FKNKIGALAD KPNEKVIKAI DEL SYENYE IPASFVDDNP 851 YLMKPS IKYG TKKGDFTHQT ELFTPILSVM KAKDLDEAIE IVNSTGYGLT 901 SALESLDERE WEYYLERIEA GNIYINKPTT GAIVLRQPFG GVKKSAVGFG 951 RKVGIFNYIT QFVNIHQEEE DENALKNPLS EALEGLTQKG YDEHTHELKR 1001 AIFMAKSYAY HYKHEFSQAK DYVKIRGEDN LFSYTKVTSV GYRITEKDTL 1051 SDMLGVALAC LISQIPLTIS IENERANKDL TFFLECLKAL QANAPIVYES 1101 LQKFSEKLNA FNRVRYLKSD LDLLHEQASR LGMVLATAKP CLNGRFELLY 1151 YHLERSVSIS YHRYGNLGSR VLRQPTCHKS CCAEK

DNA Sequence: SEQ ID No.14

ATGCAAAAAATCATTGAGGATTCATTAGAATTAGCTAAAAAACTGCAAGATAGTATCAGT AACCATTTGAGCGAGCAAGAAAAAGCGTTCCATTTTAAAATGCAAAAGCTTTTAAACAAC CCCGAAAACAAAGTCATGCTTATAGAGCTTATGGATCGGAGTTTTAGGTGTTTGGACAAT AAAGCCCGCTTTGAAATGATTGAGCATGTTTTAGACAAATACAAAAGCCGTGAGATTTTT TCTTCGTTTGAAAAATGGCTTTTAATGGGGTTTTTAAGCTTTGGGAAAATGCTGCCTGAT ATGAGCGTGCCTTTCTTTGTCAATAAAATCAGAAGCGACACGAAAGCAATGGTCTTGGAT CAAGAAGAGAGCCAATTAAGAGAGCGGATTTTAAAAAGAAAAAATGAGAAAATCATTTTG AACGTGAATTTTATTGGCGAGGAAGTTTTAGGCGAAGAAGAAGCGTCTGCGCGTTTTGAA AAATACTCTCAAGCCCTAAAATCCAACTACATCCAATACATTTCCATTAAAATCACAACG ATTTTTTCTCAAATCAATATCCTTGATTTTGAATACTCTAAAAAAGAGATTGTCAAACGC

TTAGACGCTCTTTATGCTTTAGCTTTAGAAGAAGAAAAAAAGCAAGGCATGCCTAAATTC ATCAACTTGGATATGGAGGAATTTAGGGATTTAGAGCTTACGGTGGAATCGTTTATGGAG TCTATCGCCAAATTTGATTTGAACGCCGGTATTGTGTTGCAAGCTTATATCCCTGATTCT TATGAATATTTGAAAAAACTGCACGCTTTTTCTAAAGAAAGGGTTTTAAAAGGGTTAAAG CCTATTAAAATCCGCTTTGTTAAGGGAGCGAACATGGAGAGCGAAGAGACGATCGCTTCT ATGAAAGACTGGGCGTTACCCACATTTTCTAGTAAGCAAGACACCGATTCTAATTACAAC AAAATGCTGGATTTTGTTTTAGAGGGCGACAATTATAAATACATTCATATTGGTGCAGCG AGCCATAATATTTTTGAAATCGCCTATGTCTATACGCGCATCCATGCCCTTAATGACCCT GTTGTGTTAGAGCATTTTAGCTTTGAAATGCTAGAGGGCATGAGCTTGCAAGCGAGCCAG GAATTAAAAGAGATGCACAAACTCATTCTTTATGCGCCGGTGTGCGATGAAGCGCATTTC AACAATGCAATCGCTTACTTGGTGAGGAGGCTAGATGAAAACACTTCAAGCGATAATTTC ATGAAAGCTTTCTTTAACCTCAAAGTAGGCACGAGCGAATGGAAAGACCAAGAGCAACGC TTTTTAAACAGCCTTAAAGGGATCGCCACTTTAGACAATACCACCCACAGGACCCAAGAC AGAAACGCCAAACAAACCGGGCATACCACCTACCCAAACCACTCCTTTAAAAACGAAAGC GATACCGATTTTATTTTAAAAGCCAACCGAGAATGGGCTAAAAAAGTGCGCGATAAAATG CATAACGCCCCTATTTTAGAGCTTTACCCAGAAATAGATGGGAGGTTTGAAGATCCTAAT TTAACCCCTTTAGAAGTCTTTGATAGAATCCATCATAAAAAAATCGCCAGCGTGCATTTA GCGGATAAAGAAGCGATTTTAAAAGCCCTAGAAGTGGCTAAAAGCGATAAGAGCCGTTTC AGTCAAAAAAGCTTCACAGAAATCCATGCTTTATTGAGTCAAACCGCTCAGCTTTTTAGA GAAAGAAGAGGCGATTTAATAGGGATTTCCGCTTTAGAAGTGGGTAAGACTTTCGCTGAA ACGGACGCTGAAGTGAGCGAAGCCATTGACTTTTTAGAGTTTTACCCTTACAGCTTAAGG GTGTTACAAGAGCAAAATAAAAAAACGAAATTCACCCCCAAAGGCGTGGGTGTGGTCATC GCTCCATGGAATTTCCCTGTGGGCATTTCTGTAGGCACTATCGCTGCCCCCCTAGCCGCT GGCAATCGGGTGATTTACAAGCCCTCAAGTTTGTCTAGCGTAACGGGTTATAAGCTTTGC GAGTGCTTTTGGGATGCGGGCGTGCCTAGAGATGCGCTCATTTACTTGCCCTCTAAAGGG AGCGATATTAGCGAGCATCTTTTAAAAGATGAAAGCATCAAGTTTGCCATTTTAACAGGG GGTGAAGACACCGCTTATAAAATGCTCAAGGCTAACCCCACTTTAGCCTTAAGCGCTGAA ACAGGCGGTAAAAACGCCACTATTGTGAGCAAAATGGCAGACAGAGACCAGGCGATTAAG AATGTTATCCATTCAGCTTTTAGCAATTCGGGGCAAAAATGCTCCGCCACTTCGCTTTTA GTGTTAGAAAAAGAAGTCTATGAAGATGAGAATTTCAAAAAGACTCTAATAGATGCGACT CTAAGCCTTAGCGTGGGCGATCCTTTTGATTTCAAAAACAAAATCGGTGCTCTAGCGGAC AAGCCTAATGAAAAGGTCATCAAAGCCATAGATGAATTGAAAAGCTATGAAAATTACGAA ATCCCAGCAAGCTTTGTTGATGATAACCCCTATTTGATGAAGCCAAGCATCAAATACGGC ACTAAAAAAGGCGATTTCACGCACCAAACTGAGCTTTTTACGCCCATTTTATCCGTGATG AAAGCAAAAGATTTAGACGAGGCGATAGAAATAGTCAATTCTACCGGTTACGGGCTGACT AGCGCGTTAGAGTCTTTGGACGAAAGGGAGTGGGAATATTATTTAGAACGCATTGAAGCC GGTAATATCTATATCAACAAACCCACCACAGGAGCGATTGTCTTGCGCCAGCCTTTTGGG GGGGTTAAAAAATCCGCTGTGGGGTTTGGGAGGAAAGTAGGCATTTTCAACTATATCACG CAATTTGTGAATATCCATCAAGAAGAAGAAGATGAAAACGCCTTAAAAAACCCCTTAAGC GAAGCTTTAGAGGGCTTGACTCAAAAAGGCTATGATGAACACACGCATGAGTTGAAACGC GCGATTTTTATGGCAAAAAGCTACGCTTATCATTATAAGCATGAATTCAGCCAAGCTAAA GACTATGTCAAAATCAGAGGCGAAGACAACCTTTTTTCCTACACTAAAGTTACAAGCGTG GGCTATCGCATCACCGAAAAGGACACTTTAAGCGACATGTTAGGCGTTGCTTTAGCATGC TTAATTTCTCAAATCCCTTTAACGATCAGCATAGAAAACGAACGAGCGAACAAAGATTTA ACCTTTTTTTTAGAATGCTTAAAAGCACTCCAAGCAAACGCCCCTATCGTTTATGAAAGC TTGCAAAAATTTAGCGAGAAGTTGAACGCTTTCAATCGTGTCCGTTATCTCAAAAGCGAT TTGGATTTATTGCACGAACAAGCAAGCAGATTAGGGATGGTTTTAGCCACGGCTAAACCC TGCTTGAATGGGCGTTTTGAATTGCTGTATTACCACTTAGAGCGATCGGTTAGCATTTCG TATCATCGTTATGGGAATTTAGGCTCAAGGGTTTTAAGGCAACCCACTTGCCACAAATCA TGCTGTGCTGAAAAATAA

Description: NAD-dependent aldehyde dehydrogenase [Helicobacter pylori 52] Protein Sequence: SEQ ID No.15

MQKI IDDSLE LAKKLQDSIS NHLSEQEKAF HSKMQKLLNN PENKVMLIEL MDRSFRCLDN KARFEMIEHV LDKYKSREIF SSFEKLLLMG FLSFGK LPD MSVPFFVNKI RSDTKAMVLD QEESRLKERI LKRKNEKIIL NVNF IGEEVL GEEEASARFE KYSQALKSNY IQYISIKITT IFSQINILDF EYSKKEIVKR LDALYALALE EEKKQGMPKF INLDMEEFRD LELTVESFME SIAKFDLNAG IVLQAYIPDS YEYLKKLHAF SKERVLKGLK PIKIRFVKGA NMESEETIAS MKDWALPTFS NKQDTDSNYN KMLDFVLEGD NYKYIHIGAA SHNIFEIAYV YTRIHALNDP WLEHFSFEM LEGMSLQASQ ELKE HKLIL YAPVCDEAHF NNAIAYLVRR LDENTSSDNF MKAFFNLKVG TSEWKDQEQR FLNSLKGIAT LDNATHRTQD RNAKQSGHTT YPNHSFKNES DTDFILKANR EWAKKVREKM HNAPILELYP EMDGRFEDPN LTPLEVFDKI HHRKIASVHL ADKGAILKAL EVAKSDKSRF SQKSFTEIHA LMSQTAQLFR ERRGDLIGIS ALEVGKTFAE TDAEVSEAID FLEFYPYSLR VLQEQNKKTK FTPKGVGWI AP NFPVGIS VGTIAAPLAA GNRVIYKPSS LSSVTGYKLC ECFWDAGVPR DALIYLPSKG SDISEHLLKD ESIKFAILTG GEDTAYKMLE ANPTLALSAE TGGKNATIVS KMADRDQAIK NVIHSAFSNS GQKCSATSLL VLEKEVYEDE NF KTLIDAT LSLSVGDPFD FKNKIGTLAD KPNEKVIKAI GELKSYENYE IPVSFVNDNP YLMKPSI YG TKKGDFTHQT ELFTPILSVM EAQDLDEAID IVNSTGYGLT SALESLDERE WEYYLERIEA GNIYINKPTT GAIVLRQPFG GVKKSAVGFG RKVGIFNYIT QFVNIHQEEE DENALKNPLS EALEGLIQKG YDEHTHELKR AIFMAKSYAY HYKHEFSQAK DYVKIRGEDN LFSYTKVKSV GYRITEKDTL SDMLGVALAC LISQIPLTLS TENERANKDL TFFLECLKAL QANAPI YES LQKFSEKLNA FNRVRYLKSD LDLLHEQASR LGIVLATAKP CLNGRFELLY YHLERSVSIS YHRYGNLGSR VLRQPTCHKS CCAEK

DNA Sequence: SEQ ID No.16

ATGCAAAAAATCATTGACGATTCATTAGAATTAGCCAAAAAACTGCAAGATAGCATCAGT AACCATTTGAGCGAGCAAGAAAAAGCGTTCCATTCTAAAATGCAAAAGCTTTTAAATAAC CCTGAAAACAAAGTCATGCTCATAGAGCTTATGGATCGGAGTTTTAGATGCTTGGACAAT AAAGCCCGCTTTGAAATGATTGAGCATGTTTTAGACAAATACAAAAGCCGTGAGATTTTT TCTTCGTTTGAAAAATTGCTTTTAATGGGGTTTTTGAGCTTTGGGAAAATGCTCCCTGAT ATGAGCGTGCCTTTCTTTGTCAATAAAATTAGAAGTGACACAAAAGCGATGGTCTTGGAT CAAGAAGAGAGCCGATTAAAAGAGCGGATTTTAAAAAGAAAAAATGAAAAAATCATTTTG AACGTGAATTTTATTGGCGAAGAGGTTTTAGGCGAAGAAGAAGCTAGTGCGCGTTTTGAA AAATACTCTCAAGCCCTAAAATCCAACTACATTCAATACATTTCTATTAAAATCACGACG ATTTTTTCTCAAATCAATATCCTTGATTTTGAATACTCTAAAAAAGAGATTGTCAAACGC TTAGACGCTCTTTATGCCCTGGCTTTAGAAGAAGAAAAAAAGCAAGGCATGCCGAAATTT ATTAATTTGGATATGGAGGAATTTAGGGATTTAGAGCTCACGGTGGAGTCTTTTATGGAG TCTATCGCTAAATTTGATTTGAACGCTGGTATTGTGTTGCAAGCCTATATCCCGGATTCT TATGAATATTTGAAGAAACTGCACGCTTTTTCTAAAGAAAGGGTTTTAAAAGGGTTAAAG CCCATTAAAATCCGCTTTGTTAAGGGAGCGAACATGGAGAGCGAAGAGACTATCGCTTCT ATGAAAGACTGGGCGTTACCCACATTTTCTAATAAGCAAGACACCGATTCTAATTACAAT AAAATGCTGGATTTTGTTTTAGAGGGCGATAATTATAAATACATTCATATTGGCGCAGCG AGTCATAATATTTTTGAAATCGCTTATGTTTATACGCGCATCCATGCCCTTAATGACCCT GTTGTGTTAGAGCATTTTAGCTTTGAAATGCTAGAGGGCATGAGCTTGCAAGCGAGCCAG GAACTAAAAGAGATGCACAAACTCATTCTTTATGCGCCGGTGTGCGATGAAGCACATTTT AACAATGCGATCGCTTACTTGGTGAGAAGACTAGATGAAAACACCTCAAGCGATAATTTC ATGAAAGCTTTCTTTAATCTCAAAGTAGGCACGAGCGAATGGAAAGACCAAGAACAACGC TTTTTAAACAGCCTTAAAGGAATCGCTACTTTAGACAATGCCACCCACAGGACTCAAGAC AGGAACGCCAAACAAAGCGGGCATACCACCTACCCAAACCATTCTTTTAAAAACGAAAGC GATACCGATTTTATTTTAAAAGCCAACCGAGAATGGGCTAAAAAAGTGCGCGAAAAAATG CATAACGCCCCTATTTTAGAGCTTTACCCAGAAATGGATGGGAGGTTTGAAGATCCTAAT TTAACCCCTTTAGAAGTCTTTGATAAAATCCATCATAGAAAAATCGCTAGCGTGCATTTA GCGGATAAGGGAGCGATTTTAAAAGCCCTAGAAGTGGCTAAAAGCGATAAGAGCCGTTTC AGCCAAAAAAGCTTTACAGAAATCCATGCCTTAATGAGTCAAACCGCCCAGCTTTTTAGA GAAAGGCGAGGCGACTTAATAGGGATTTCGGCTTTAGAAGTGGGTAAGACTTTCGCTGAA ACGGACGCTGAAGTGAGCGAAGCCATTGACTTTTTAGAGTTTTACCCTTACAGCTTAAGG GTGTTACAAGAGCAGAATAAAAAAACGAAATTCACCCCCAAAGGCGTGGGTGTGGTCATT GCCCCATGGAATTTCCCTGTGGGCATTTCTGTAGGCACTATCGCTGCCCCCCTAGCCGCT GGCAATCGGGTGATTTACAAGCCCTCAAGTTTGTCTAGCGTAACGGGTTATAAGCTTTGT GAGTGCTTTTGGGATGCGGGCGTGCCTAGAGATGCGCTCATTTACTTGCCCTCTAAAGGG AGCGATATTAGCGAGCATCTTTTAAAAGATGAAAGCATCAAGTTTGCCATTTTAACAGGG GGCGAAGACACCGCTTATAAAATGCTAGAGGCTAACCCCACTTTAGCCTTGAGCGCTGAA ACAGGCGGTAAAAACGCCACGATTGTGAGCAAAATGGCAGACAGAGATCAAGCGATTAAG AATGTTATCCATTCAGCTTTTAGCAATTCGGGGCAAAAATGCTCCGCCACTTCGCTTTTA GTGTTAGAAAAAGAAGTTTATGAAGATGAGAACTTTAAAAAGACTCTAATAGATGCGACT CTAAGCCTTAGCGTGGGCGATCCTTTTGATTTCAAAAACAAAATCGGCACTCTAGCAGAC AAGCCTAATGAAAAGGTCATTAAAGCCATAGGCGAATTGAAAAGCTATGAAAATTACGAA ATCCCGGTAAGCTTTGTCAATGATAACCCCTATTTGATGAAGCCAAGCATCAAATACGGC ACTAAAAAAGGCGATTTCACGCACCAAACTGAGCTTTTTACGCCCATTTTATCCGTGATG GAAGCGCAAGATTTAGACGAAGCGATAGACATAGTCAATTCTACCGGTTACGGGCTGACG AGCGCGTTAGAGTCTTTGGACGAAAGGGAGTGGGAATATTATTTAGAGCGCATTGAAGCC GGTAATATCTATATCAACAAGCCCACCACAGGAGCGATTGTCTTGCGCCAGCCTTTTGGG GGGGTTAAAAAATCCGCTGTGGGGTTTGGGAGGAAGGTAGGCATTTTTAACTATATCACG CAATTTGTGAATATCCACCAAGAAGAAGAAGATGAAAACGCCCTAAAAAACCCTTTAAGC GAAGCTTTAGAGGGCTTGATCCAAAAAGGCTATGATGAGCATACGCATGAGTTGAAGCGC GCGATTTTTATGGCAAAGAGCTACGCTTATCATTACAAGCATGAATTCAGCCAAGCTAAA GACTATGTCAAAATCAGAGGCGAAGACAACCTTTTTTCCTACACTAAAGTTAAAAGCGTG GGCTATCGCATCACCGAAAAGGACACTTTAAGCGATATGTTAGGCGTTGCTTTAGCATGT TTAATTTCTCAAATCCCTTTAACGCTCAGCACAGAAAACGAGCGAGCGAACAAAGATTTA ACATTTTTCTTAGAATGCTTAAAAGCGCTCCAAGCAAACGCCCCTATTGTTTATGAAAGC

TTGCAAAAATTTAGCGAGAAATTGAACGCTTTCAATCGTGTCCGTTATTTAAAAAGCGAT TTGGATTTATTGCACGAACAAGCAAGCAGATTAGGGATTGTTTTAGCCACGGCCAAACCC TGCTTAAATGGGCGTTTTGAATTGCTGTATTACCACTTAGAGCGATCGGTTAGCATCTCT TATCATCGTTATGGGAATTTAGGTTCAAGGGTTTTAAGGCAACCAACTTGCCACAAATCA TGCTGTGCTGAAAAATAA

29 kDa antigenic protein

Description: urease accessory protein [Helicobacter pylori J99] Protein Sequence: SEQ ID No.17

1 MVKIGVCGPV GSGKTALIEA LTRHMSKDYD MAVITNDIYT KEDAEF CKN 51 SV PRDRIIG VETGGCPHTA IREDASMNLE AVEEMHGRFP NLELLLIESG 101 GDNLSATFNP ELADFTIFVI DVAEGDKIPR KGGPGITRSD LLVIN IDLA 151 PYVGADLKVM ERDSKKMRGE KPFIFTNIRA KEGLDDVIAW IKRNALLED

DNA Sequence: SEQ ID No.18

ATGGTAAAAATTGGAGTTTGTGGTCCTGTAGGAAGCGGTAAAACCGCCTTGATTGAAGCT TTAACGCGCCACATGTCAAAAGATTATGACATGGCGGTCATCACTAATGATATTTACACG AAAGAAGACGCAGAGTTTATGTGCAAAAATTCGGTGATGCCACGAGATAGGATCATTGGC GTAGAAACAGGAGGCTGTCCGCACACGGCTATTAGAGAAGACGCTTCTATGAATTTAGAA GCGGTAGAAGAAATGCATGGCCGTTTCCCTAATTTGGAATTGCTTTTGATTGAAAGCGGA GGCGATAACCTTTCAGCGACTTTTAACCCGGAGTTGGCGGATTTTACGATTTTTGTGATT GATGTGGCTGAGGGCGATAAAATCCCCAGAAAAGGCGGGCCAGGAATCACGCGCTCAGAC TTGCTTGTCATCAATAAGATTGATTTAGCCCCCTATGTGGGAGCGGACTTGAAAGTCATG GAAAGGGATTCTAAAAAAATGCGCGGCGAAAAGCCCTTTATTTTTACGAATATCCGCGCT AAAGAAGGTTTAGACGATGTGATCGCTTGGATCAAGCGCAACGCTTTATTGGAAGATTGA

31 kDa antigenic protein

Description: elongation factor Tu [Helicobacter pylori J99] Protein Sequence: SEQ ID No.19

1 MAKEKFNRTN PHVNIGTIGH VYHGKTTLSA AISAVLSLKG LAEMKDYDNI 51 DNAPQEKERG ITIATSHIEY ETETRHYAHV DCPGHADYVK NMITGAAQMD 101 GAILWSAAD GPMPQTREHI LLSRQVGVPH IWFLNKQDM VDDQELLELV 151 EMEVRELLSA YEFPGDDTPI VAGSALRALE EAKAGNVGE GEKVLKLMAE 201 VDSYIPTPER DTEKTFLMPV EDVFSIAGRG TVVTGRIERG VV VGDEVEI 251 VGIRATQKTT VTGVEMFRKE LEKGEAGDNV GVLLRGTKKE EVERG VLCK 301 PGSITPHKKF EEEIYVLSKE EGGRHTPFFT NYRPQFYVRT TDVTGSITLP 351 EGVEMVMPGD NVKITVELIS PVALELGTKF AIREGGRTVG AGVVSNIIE

DNA Sequence: SEQ ID No.20

ATGGCAAAAGAAAAGTTTAATAGAACTAACCCGCATGTTAATATTGGAACCATTGGGCAT GTATACCATGGTAAAACGACTTTGAGCGCAGCGATTTCAGCGGTGCTTTCTTTGAAAGGT CTTGCAGAAATGAAAGACTATGACAATATTGATAACGCCCCTCAAGAAAAAGAAAGAGGG ATCACTATCGCTACTTCTCACATTGAATATGAGACTGAAACCAGACACTATGCGCATGTG GATTGCCCAGGACACGCTGACTATGTAAAAAACATGATCACCGGTGCGGCACAAATGGAT GGAGCGATCTTGGTTGTTTCTGCGGCTGATGGCCCCATGCCTCAAACCAGAGAGCATATC TTATTGTCTCGTCAAGTAGGCGTGCCTCACATTGTTGTTTTCTTAAACAAACAAGACATG GTAGATGACCAAGAATTGTTAGAGCTTGTAGAAATGGAAGTACGCGAGTTGTTGAGCGCG TATGAATTTCCTGGCGATGACACTCCTATCGTAGCGGGTTCAGCTTTAAGAGCTTTAGAA GAAGCAAAGGCTGGCAATGTGGGTGAATGGGGCGAAAAAGTGCTTAAACTCATGGCTGAA GTGGATTCCTATATCCCTACTCCAGAAAGAGACACTGAAAAAACTTTCTTGATGCCGGTT GAAGACGTGTTTTCTATCGCAGGTAGAGGAACTGTGGTTACAGGTAGGATTGAAAGAGGC GTGGTGAAAGTGGGCGATGAAGTGGAAATCGTTGGTATCAGAGCCACACAAAAAACGACC GTAACCGGTGTAGAAATGTTTAGAAAAGAATTAGAAAAAGGTGAAGCCGGCGATAATGTG GGCGTGCTTTTGAGAGGAACTAAAAAAGAAGAAGTAGAGCGCGGTATGGTTCTATGCAAA CCAGGTTCTATCACTCCGCACAAGAAATTTGAGGAAGAAATTTATGTCCTTTCTAAAGAA GAAGGCGGGAGACACACTCCATTCTTTACCAACTACCGCCCGCAATTCTATGTGCGTACG ACTGATGTGACTGGCTCTATCACCCTTCCTGAAGGCGTGGAGATGGTTATGCCTGGCGAT AATGTGAAAATCACCGTGGAGTTGATTAGCCCTGTTGCGTTAGAGTTAGGAACCAAATTT GCGATTCGTGAAGGCGGGAGAACCGTTGGCGCTGGTGTTGTGAGCAATATTATTGAATAA

Description: elongation factor Tu [Helicobacter pylori Shi470] Protein Sequence : SEQ ID No.21

MAKEKFNRTK PHVNIGTIGH VDHGKTTLSA AISAVLSLKG LAEMKDYDNI DNAPEEKERG ITIATSHIEY ETENRHYAHV DCPGHADYVK NMITGAAQMD GAILWSAAD GPMPQTREHI LLSRQVGVPH IWFLNKQDM VDDQELLELV EMEVRELLSA YEFPGDDTPI VAGSALRALE EAKAGNVGEW GEKVLKLMAE VDAYIPTPER DTEKTFLMPV EDVFSIAGRG TVVTGRIERG WKVGDEVEI VGIRATQKTT VTGVEMFR E LEKGEAGDNV GVLLRGTK E EVERGMVLCK PGSITPHKKF EGEIYVLSKE EGGRHTPFFT NYRPQFYVRT TDVTGSITLP EGVEMVMPGD NVKITVELIS PVALELGTKF AIREGGRTVG AGVVSNIIE

DNA Sequence : SEQ ID No.22

ATGGCAAAAGAAAAGTTTAATAGAACTAAGCCGCATGTTAATATTGGAACCATTGGGCAT GTAGACCACGGTAAAACGACTTTGAGTGCAGCGATTTCAGCGGTGCTTTCTTTGAAAGGT CTTGCAGAAATGAAAGACTATGATAATATTGATAACGCCCCTGAAGAAAAAGAAAGAGGG ATCACTATCGCTACTTCTCACATTGAATATGAGACTGAAAACAGACACTATGCGCATGTG GATTGCCCAGGACACGCTGACTATGTAAAAAACATGATCACCGGTGCGGCGCAAATGGAC GGAGCGATTTTGGTTGTTTCTGCAGCTGATGGTCCTATGCCTCAAACTAGGGAGCATATC TTATTGTCTCGTCAAGTAGGCGTGCCTCACATCGTTGTTTTCTTAAACAAACAAGACATG GTAGATGACCAAGAATTGTTAGAGTTGGTAGAAATGGAAGTGCGCGAATTGTTGAGCGCG TATGAATTTCCTGGCGACGACACTCCTATCGTAGCGGGTTCAGCTTTAAGAGCTTTAGAA GAAGCAAAGGCTGGCAATGTGGGTGAATGGGGTGAAAAAGTGCTGAAGCTCATGGCTGAA GTGGATGCCTATATCCCTACTCCAGAAAGGGATACTGAAAAAACTTTCTTGATGCCGGTT GAAGATGTGTTCTCTATTGCGGGTAGAGGAACTGTGGTTACAGGTAGGATTGAAAGAGGC GTGGTGAAAGTGGGCGATGAAGTGGAAATCGTTGGTATCAGAGCTACACAAAAAACGACT GTAACCGGTGTGGAAATGTTTAGAAAAGAGTTAGAAAAAGGTGAGGCCGGCGATAATGTG GGCGTGCTTTTGAGAGGAACTAAAAAAGAAGAAGTAGAACGCGGTATGGTTCTATGCAAA CCAGGTTCTATCACTCCGCACAAGAAATTTGAGGGAGAAATTTATGTCCTTTCCAAAGAA GAAGGTGGGAGACACACTCCATTCTTCACCAACTACCGCCCGCAATTCTATGTGCGCACG ACTGATGTGACTGGCTCTATCACCCTTCCTGAAGGCGTGGAAATGGTTATGCCTGGCGAT AATGTTAAAATCACTGTGGAGTTGATTAGCCCTGTTGCATTGGAATTGGGAACTAAATTT GCGATTCGTGAAGGCGGTAGGACCGTTGGTGCTGGTGTTGTGAGCAATATTATTGAATAA

Claims

1. An isolated antigenic protein of Helicobacter pylori selected from a group comprises of: i) a polypeptide having a molecular weight of 13 kDa; or

ii) a polypeptide having a molecular weight of 25 kDa; or

iii) a polypeptide having a molecular weight of 29 kDa; or

iv) a polypeptide having a molecular weight of 30 kDa; or

v) a polypeptide having a molecular weight of 31 kDa; or

vi) a polypeptide having a molecular weight of 1 18 kDa; or

vii) or any combination thereof

wherein said antigenic protein is in substantially purified form.

2. An isolated antigenic protein of Helicobacter pylori as claimed in Claim 1 , wherein the polypeptide having a molecular weight of 13 kDa has an amino-terminal amino acid sequence of SEQ ID No.1 or SEQ ID No.3 and a nucleotide sequence of SEQ ID No.2 or SEQ ID No.4.

3. An isolated antigenic protein of Helicobacter pylori as claimed in Claim 1 , wherein the polypeptide having a molecular weight of 25 kDa has an amino-terminal amino acid sequence of SEQ ID No.5 or SEQ ID No.7 and a nucleotide sequence of SEQ ID No.6 or SEQ ID No.8.

4. An isolated antigenic protein of Helicobacter pylori as claimed in Claim 1 , wherein the polypeptide having a molecular weight of 29 kDa has an amino-terminal amino acid sequence of SEQ ID No.17 and a nucleotide sequence of SEQ ID No.18.

5. An isolated antigenic protein of Helicobacter pylori as claimed in Claim 1 , wherein the polypeptide having a molecular weight of 30 kDa has an amino-terminal amino acid sequence of SEQ ID No.9 or SEQ ID No.11 and a nucleotide sequence of SEQ ID No.10 or SEQ ID No.12.

6. An isolated antigenic protein of Helicobacter pylori as claimed in Claim 1 , wherein the polypeptide having a molecular weight of 31 kDa has an amino-terminal amino acid sequence of SEQ ID No.19 or SEQ ID No.21 and a nucleotide sequence of SEQ ID No.20or SEQ ID No.22.

7. An isolated antigenic protein of Helicobacter pylori as claimed in Claim 1 , wherein the polypeptide having a molecular weight of 1 18 kDa has an amino-terminal amino acid sequence of SEQ ID No.13 or SEQ ID No.15and a nucleotide sequence of SEQ ID No.14or SEQ ID No.16.

8. A use of antigenic protein according to Claims 1 -7 in vaccine for treatment of Helicobacter pylori infection in humans.

9. A use of antigenic protein according to Claims 1 -7 in diagnostic kit for detecting Helicobacter pylori infection in human samples.

10. A diagnostic kit as claimed in Claim 9, wherein the human samples are stool, saliva, serum, whole blood and urine.