Helicobacter Pylori Vaca Gene Toxic Fragment Hpaa Of Genes In Prokaryotic Expression, And Preliminary Applications

Objective: To obtain the recombinant vacuolating segment of VacA in Helicobacter pylori by gene-engineering, which would lay a foundation for the development of diagnostic reagent and vaccine for Hp infection. To establish an indirect ELISA with the recombinant protein,which would provide a solid basis for obtaining diagnostic reagent kit detecting Hp toxicity strain infection.Methods: The high homogeneity gene fragment was found in comparing VacA toxic subunit gene of domestic typical Hp strains. By using a pair of primers designed according to the vacA sequence of AF050320, the v segment of vacA gene was amplified by PCR and cloned into prokaryotic expression vectors pQE30 to construct pQE30-V. The pQE30-V was transformed into DH5α E.coli andexpressed in the presence of IPTG. The expression product was analyzed by SDS-PAGE and its antigenicity was confirmed by Western blot. The recombinant protein purified with Ni2+-NTA agarose was used to immunize rabbits to prepare antiserum, in which the anti-VacA was detected by helico-blot kit. The indirect ELISA was established with the recombinant protein to detect the IgG in sera of patients infected with Hp, which was compared with helico-blot kit, and then its sensitivity and specifity was evaluated.Results: 1.The prokaryotic expression vectors pQE30-V was constructed successfully. The v segment of vacA gene confirmed by DNA sequence was integrated. When compared with the sequence of standard strain AF 361700, 1.5% cloned gene was mutated, and 1% amino acid radices was changed; 2.SDS-PAGE showed that the recombinant protein that Mr was about 27000 represented 30% total protein of E.coli; 3. Western blot showed that the recombinant protein could be recognized by antiserum against Hp; 4.SDS-PAGE showed that the recombinant protein existed in the supernatant and precipitation of the lysed bacteria broken by ultrasonic wave, and its purity was over 93% after purified with Ni2+-NTA agarose; 5. The good immunogenicity of recombinant protein was confirmedby detecting the anti-VacA in the serum of rabbit immunized with the recombinant protein with helico-blot kit; 6.The indirect ELISA based on recombinant protein was successfully established to assay the anti-VacA in serum, and its sensitivity and specificity was 93.1% and 92.5% respectively. Conclusion: The successful expression and purification of the V segment of VacA in Helicobacter pylori, provided antigen basis for the development of the vaccine and the diagnostic reagent for Hp infection. The indirect ELISA was created to assay anti-VacA in serum provided a basis for the preparation of diagnostic reagent kit detecting Hp toxicity strain infection.