H Pylori
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 1, 2004; 10(7): 985-990
Published online Apr 1, 2004. doi: 10.3748/wjg.v10.i7.985
Construction of a prokaryotic expression system of vacA gene and detection of vacA gene, VacA protein in Helicobacter pylori isolates and ant-VacA antibody in patients’ sera
Jie Yan, Ya-Fei Mao
Jie Yan, Ya-Fei Mao, Department of Medical Microbiology and Parasitology, College of Medical Science, Zhejiang University, Hangzhou 310031, Zhejiang Province, China
Author contributions: All authors contributed equally to the work.
Supported by the Excellent Young Teacher Fund of Chinese Education Ministry and the General Science and Technology Research Program of Zhejiang Province, No. 001110438
Correspondence to: Jie Yan, Department of Medical Microbiology and Parasitology, College of Medical Science, Zhejiang University, 353 Yan An Road, Hangzhou 310031, Zhejiang Province, China. yanchen@mail.hz.zj.cn
Telephone: +86-571-87217385 Fax: +86-571-87217044
Received: October 15, 2003
Revised: November 3, 2003
Accepted: December 6, 2003
Published online: April 1, 2004

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.

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