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Isolation of recombinant protective Helicobacter pylori antigens.

ABSTRACT: A total of seven clones producing both new and previously described Helicobacter pylori proteins were isolated from a library of H. pylori genomic DNA. The screening approach by which these proteins were detected relied on the use of antisera raised in mice vaccinated with Helicobacter felis sonicate plus cholera toxin, a regimen which protects mice from H. pylori challenge. This strategy was designed to maximize the possibility of obtaining antigens which might be capable of conferring protection from H. pylori infection. Two of the clones were shown to encode the urease enzyme and the heat shock protein HspB, which have already been identified as protective antigens. The other five clones were sequenced, protein coding regions were deduced, and these sequences were amplified by PCR for incorporation into Escherichia coli expression vectors. The proteins produced from these expression systems were purified to allow testing for protective efficacy in an H. pylori mouse model. All five proteins were able to facilitate the clearance of a challenge with H. pylori, as judged by an assay of gastric urease activity and light microscopy on stomach sections. These results clearly indicate that the screening strategy has successfully identified candidate vaccine antigens.

SUBMITTER: Hocking D

PROVIDER: S-EPMC96799 | biostudies-literature | 1999 Sep

REPOSITORIES: biostudies-literature

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Isolation of recombinant protective Helicobacter pylori antigens.

Hocking D D Webb E E Radcliff F F Rothel L L Taylor S S Pinczower G G Kapouleas C C Braley H H Lee A A Doidge C C

Infection and immunity 19990901 9

A total of seven clones producing both new and previously described Helicobacter pylori proteins were isolated from a library of H. pylori genomic DNA. The screening approach by which these proteins were detected relied on the use of antisera raised in mice vaccinated with Helicobacter felis sonicate plus cholera toxin, a regimen which protects mice from H. pylori challenge. This strategy was designed to maximize the possibility of obtaining antigens which might be capable of conferring protecti ...[more]

PMID: 10456921

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Project description:BACKGROUND: Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities. METHODS: The coding sequence of H. pylori UreG was cloned and the recombinant protein expressed and purified by affinity chromatography using nickel nitrilotriacetic acid (Ni-NTA) resin. The antigenicity of rUreG to detect H. pylori specific antibodies was determined by western blot, using HRP-conjugated anti-human IgG and IgA antibodies as probes. A total of 70 sera, comprising 30 positive and 40 control serum samples, were used. The positive sera were from culture-positive H. pylori-infected patients with duodenal ulcers, gastric ulcers, or gastritis. The control sera comprised three types of samples without detectable H. pylori antibodies, i.e. healthy individuals (with no history of gastric disorders) (n = 10); patients who attended an endoscopy clinic (because of gastrointestinal complaints) but were H. pylori culture negative (n = 20); and people with other diseases (n = 10). Additionally, hyperimmune mice serum against rUreG was raised and tested with the native and recombinant UreG protein. RESULTS: The ureG gene fragment was successfully cloned and expressed in both soluble and insoluble forms. Western blots on rUreG protein showed 70% (21/30) and 60% (18/30) reactivity with patients' sera when probed with HRP-conjugated anti-human IgG and IgA antibodies, respectively; and the combination of the IgG and IgA western blots showed reactivity of 83.3% (25/30). By comparison, 97.5% and 92.5% of the control sera showed no reactivity when probed with HRP-conjugated anti-human IgG and IgA antibodies, respectively. Both the H. pylori lysate antigen and rUreG protein displayed a distinctive band at the expected molecular weight when probed with the hyperimmune mice serum. CONCLUSION: The rUreG protein was successfully cloned and expressed and showed good reactivity with H. pylori culture-positive patients' sera and no reactivity with most control sera. Thus, the diagnostic potential of this recombinant protein merits further investigation.

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Isolation of recombinant protective Helicobacter pylori antigens.

Publications

Isolation of recombinant protective Helicobacter pylori antigens.

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