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Construction of hpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein.

ABSTRACT: AIM:To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein. METHODS:The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E.coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE. Western blot with commercial antibody against whole cell of H.pylori as well as immunodiffusion assay with self-prepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein. ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H.pylori and to examine HpaA expression of 109 clinical isolates of H.pylori. RESULTS:In comparison with the reported corresponding sequences, the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25-97.32 % and 95.38-98.46 %, respectively. The output of HpaA fusion protein in its expression system of pET32a-hpaA-BL21DE3 was approximately 40 % of the total bacterial proteins. HpaA fusion protein was able to combine with the commercial antibody against whole cell of H.pylori and to induce rabbit producing specific antiserum with 1:4 immunodiffusion titer after the animal was immunized with the fusion protein. 81.6 % of the serum samples from 125 patients infected with H.pylori (102/125) were positive for HpaA antibody and all of the tested isolates of H.pylori (109/109) were detectable for HpaA. CONCLUSION:A prokaryotic expression system with high efficiency of H.pylori hpaA gene was successfully established. The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity. High frequencies of HpaA expression in different H.pylori clinical strains and specific antibody production in H.pylori infected patients indicate that HpaA is an excellent and ideal antigen for developing H.pylori vaccine.

SUBMITTER: Mao YF

PROVIDER: S-EPMC4615498 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Construction of hpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein.

Mao Ya-Fei YF Yan Jie J Li Li-Wei LW Li Shu-Ping SP

World journal of gastroenterology 20030701 7

<h4>Aim</h4>To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein.<h4>Methods</h4>The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E.col ...[more]

PMID: 12854157

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Project description:AIM:To clone ureB gene from a clinical isolate of Helicobacter pylori and construct a prokaryotic expression system of the gene and identify immunity of the expressed recombinant protein. METHODS:ureB gene from a clinical H pylori strain Y06 was amplified by the high fidelity polymerase chain reaction technique. The target DNA fragment amplified from ureB gene was sequenced after T-A cloning. Prokaryotic recombinant expression vector pET32a inserted with ureB gene (pET32a-ureB) was constructed. The expression of recombinant UreB protein (rUreB) in E. coli BL21DE3 induced by isopropylthio-beta-D-galactoside (IPTG) at different concentrations was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and an immunodiffusion assay using a self-prepared rabbit anti-rUreB antibody were applied to determine immunity of the target recombinant protein. ELISA was used to detect the antibody against rUreB in sera of 125 H pylori infected patients and to examine rUreB expression in 109 H pylori isolates. RESULTS:In comparison with the reported corresponding sequences, the nucleotide sequence homology of the cloned ureB gene was from 96.88-97.82% while the homology of its putative amino acid sequence was as high as 99.65-99.82%. The rUreB output expressed by pET32a-ureB-BL21DE3 was approximate 30% of the total bacterial proteins. rUreB specifically combined with the commercial antibodies against whole cell of H pylori and strongly induced rabbits to produce antibody with a 1:8 immunodiffusion titer after the animals were immunized with the recombinant protein. Serum samples from all H pylori infected patients were positive for UreB antibody and UreB expression were detectable in all tested H pylori isolates. CONCLUSION:A prokaryotic expression system with high expression efficiency of H pylori ureB gene was successfully established. The expressed rUreB showed qualified immunoreactivity and antigenicity. High frequencies of UreB expression in different H pylori isolates and specific antibody against UreB in sera of H pylori infected patients indicate that UreB is an excellent antigen candidate for developing H pylori vaccine.

Project description:AIM:To assess the variability of adhesin gene hpaA between different Helicobacter pylori (H pylori) strains with PCR-restriction fragment length polymorphism (RFLP). METHODS:Twelve different H pylori strains were chosen to amplify the 710-bp segments of gene hpaA. These strains were NCTC11637, SS1; Chongqing clinical isolates CCS9801, CCS9802, CCS9803, CCS9806, CCS9809, CCS9810, CCS9813, which were gained from patients of gastritis; Mongolia gerbil adapted H pylori strains (abbreviation MG), which were gained from the following steps: gastric mucosal specimens of Mongolia gerbils infected by clinical isolate CCS9803 were cultured and detected, the positive H pylori strains were named as the first generation of Mongolia gerbil adapted H pylori strains (abbreviation MG1) and then were subcultured with healthy Mongolia gerbil to generate MG2, in turn to gain the ninth generation (abbreviation MG9). All hpaA segments, obtained from 12 different H pylori strains, were digested by HhaI and HaeIII individually and analyzed by agarose gel electrophoresis. RESULTS:In all 12 strains, the 710-bp PCR products were successfully amplified and products were cloned to pMD18-T vector respectively, then the recombinant plasmids were digested simultaneously with NcoI and XhoI to recover the small fragments. The objective fragments from 12 different H pylori strains digested with Hae III could be seen as 4 types of bands and 5 types with Hha I. According to the hpaA RFLP patterns, the 12 H pylori strains could be divided into 5 groups: group I, NCTC11637 and SS1; group II, CCS9809, which RFLP type digested with HaeIII was the same as strains of group I, but HhaI RFLP showed difference compared with the other groups; group III, CCS9810; group IV, CCS9803; group V: CCS9801, CCS9802, CCS9806, CCS9813, MG1, MG3 and MG9. The sequence data of 12 hpaA segments were analyzed by DNAsis software and it was observed that: (1) The homologies of base pair and amino acid sequence between strains NCTC11637, SS1, CCS9809 were 99.6% and 98.9%, respectively; (2) The homology of base pair and amino acid sequence between CCS9803 and CCS9810 was 97.7% and 99.1%; (3) That of the rest strains, CCS9801, CCS9802, CCS9806, CCS9813, MG1, MG3, MG9 reached 99.4% and 98.4%; (4) The base pair homologies between all hpaA fragments of different sources were higher than 94.6%, therefore the correspondence of deduced amino acid sequence was higher than 96.8% between each other. CONCLUSION:The gene hpaA from different H pylori strains revealed variation, and this might provide an effective method for molecular epidemiological survey of H pylori.

Project description:The ability of certain strains of Helicobacter pylori to cause sialic acid-sensitive agglutination of erythrocytes has been attributed to the HpaA protein (D.G. Evans, T.K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C.H. Lee, J. Bacteriol. 175:674-683, 1993), the gene for which has been cloned and sequenced. On the basis of the hydropathy plot of HpaA and the presence of a potential lipoprotein signal sequence and modification site, and because of the similarities of these features with those of the cell envelope lipoprotein Lpp20 of H. pylori, we examined the possibility that HpaA was also a lipoprotein. Posttranslational processing of the HpaA protein expressed by the cloned gene was sensitive to globomycin, an inhibitor of the lipoprotein-specific signal peptidase II. Antibodies raised to the putative sialic acid-binding region of HpaA failed to bind to the surface of H. pylori cells in immunoelectron microscopy but instead were observed to have labeled the cytoplasm when thin sections were examined. This antibody recognized a 29,000-M(r) protein in Western blots (immunoblots) of cell extracts of H. pylori and Escherichia coli cells expressing the cloned hpaA gene. Determination of the sequence of hpaA from strain CCUG 17874 indicated significant differences from that determined by Evans and coworkers in the above-mentioned study, including extension of the gene into the open reading frame 3 downstream of hpaA to produce a protein with an M(r) of 26,414. Localization of HpaA indicated that it was predominantly located in the cytoplasmic fraction of the cell in both E. coli and H. pylori. HpaA was not observed in the sarkosyl-insoluble outer membrane fraction. An isogenic mutant generated by insertional inactivation of hpaA was unaffected in its ability to bind four different human cell lines as well as fixed sections of gastric tissue and had hemagglutination properties identical to those of the wild type. The data collectively suggest that HpaA is a nonessential lipoprotein internal to the H. pylori cell and that it is not involved in adhesion.

Project description:The localization of lipoprotein (Lol) system is used by Gram-negative bacteria to export lipoproteins to the outer membrane. Lol proteins and models of how Lol transfers lipoproteins from the inner to the outer membrane have been extensively characterized in the model organism Escherichia coli, but in numerous bacterial species, lipoprotein synthesis and export pathways deviate from the E. coli paradigm. For example, in the human gastric bacterium Helicobacter pylori, a homolog of the E. coli outer membrane component LolB is not found, E. coli LolC and LolE correspond to a single inner membrane component (LolF), and a homolog of the E. coli cytoplasmic ATPase LolD has not been identified. In the present study, we sought to identify a LolD-like protein in H. pylori. We used affinity-purification mass spectrometry to identify interaction partners of the H. pylori ATP-binding cassette (ABC) family permease LolF and identified the ABC family ATP-binding protein HP0179 as its interaction partner. We engineered H. pylori to conditionally express HP0179 and showed that HP0179 and its conserved ATP binding and ATP hydrolysis motifs are essential for H. pylori growth. We then performed affinity purification-mass spectrometry using HP0179 as the bait and identified LolF as its interaction partner. These results indicate that H. pylori HP0179 is a LolD-like protein and provide a more complete understanding of lipoprotein localization processes in H. pylori, a bacterium in which the Lol system deviates from the E. coli paradigm. IMPORTANCE Lipoproteins are critical in Gram-negative-bacteria for cell surface assembly of LPS, insertion of outer membrane proteins, and sensing envelope stress. Lipoproteins also contribute to bacterial pathogenesis. For many of these functions, lipoproteins must localize to the Gram-negative outer membrane. Transporting lipoproteins to the outer membrane involves the Lol sorting pathway. Detailed analyses of the Lol pathway have been performed in the model organism Escherichia coli, but many bacteria utilize altered components or are missing essential components of the E. coli Lol pathway. Identifying a LolD-like protein in Helicobacter pylori is important to better understand the Lol pathway in diverse bacterial classes. This becomes particularly relevant as lipoprotein localization is targeted for antimicrobial development.

Dataset Information

Construction of hpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein.

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Construction of hpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein.

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