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Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori.

ABSTRACT: UNLABELLED:Objective(s) : Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. MATERIALS AND METHODS:The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. RESULTS:Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient's sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E . co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .

SUBMITTER: Hasanzadeh L

PROVIDER: S-EPMC3758054 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori.

Hasanzadeh Leila L Ghaznavi-Rad Ehsanollah E Soufian Safieh S Farjadi Vahideh V Abtahi Hamid H

Iranian journal of basic medical sciences 20130701 7

<h4>Unlabelled</h4>Objective(s) : Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity.<h4>Materials and methods</h4>The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction ...[more]

PMID: 23997913

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Project description:AIM:To construct a recombinant prokaryotic expression vector inserted with Helicobacter pylori vacA gene and identify the immunity of the expressed recombinant protein, and to determine prevalence of vacA-carrying/VacA expressing H pylori isolates and seroprevalence of specific ant-VacA antibody in H pylori infected patients. METHODS:Polymerase chain reaction technique was used to amplify complete vacA gene of H pylori strain NCTC11637 and to detect vacA gene in 109 H pylori isolates. The amplification product of the complete vacA gene was sequenced after T-A cloning. A recombinant expression vector inserted with a complete vacA gene fragment, named as pET32a-vacA, was constructed. Expression of the target recombinant protein VacA (rVacA) was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and an immunodiffusion assay using self-prepared rabbit anti-rVacA antibody were applied to determine immunoreaction and antigenicity of rVacA. Two ELISA methods were established to detect VacA expression in H pylori isolates and the specific anti-VacA antibody in sera from 125 patients infected with H pylori. RESULTS:In comparison with the reported corresponding sequences, homologies of nucleotide and putative amino acid sequences of the cloned vacA gene were 99.82% and 100%, respectively. The constructed recombinant prokaryotic expression system efficiently produced rVacA. rVacA was able to combine with the commercial antibodies against whole cell of H pylori and to induce the immunized rabbit to produce specific antibody with an immunodiffusion titer of 1:4. All tested H pylori isolates carried vacA gene, but only 66.1% expressed VacA protein. Of the serum samples tested, 42.4% were positive for specific anti-VacA antibody. CONCLUSION:A prokaryotic expression system of H pylori vacA gene was successfully constructed. The expressed rVacA can be used to detect specific anti-VacA antibody in human and to prepare antiserum in animals. The high frequency of vacA gene in H pylori isolates, but with a low frequency of VacA expression and specific anti-VacA antibody in H pylori infected patients implies that VacA is not an ideal antigen for H pylori vaccine.

Dataset Information

Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori.

Publications

Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori.

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