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.

Abstract

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. 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. 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. 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.