Helicobacter Pylori Hpaa,-ctxb Fusion Protein Development And Immunogenicity Study

Objective: To obtain the recombinant adhesion gene hpaA in H.pylori by gene-engineering, which would lay a foundation for the development of diagnostic reagent and vaccine for H.pylori infection. To establish an indirect ELISA with the recombinant protein,which would provide a solid basis for obtaining diagnostic reagent kit detecting H.pylori infection.Methods: The high homogeneity gene fragment was found in comparing adhesin gene hpaA of domestic typical H.pylori strains. By using a pair of primers designed according to the hpaA sequence from Gene Bank, the hpaA gene was amplified by PCR and cloned into prokaryotic expression vectors pQE30 to construct pQE30-hpaA. The pQE30-hpaA was transformed into DH5α E.coli and expressed in the presence of IPTG. The expression product was analyzed by SDS-PAGE and its antigenicity was confirmed by Western blot. The recombinant protein was purified by Ni2+-NTA agarose. The feasibility of its clinical applications with comparative evaluation was done by H.pylori diagnostic standard of reseach. Results: 1.The prokaryotic expression vectors pQE30-hpaA was constructed successfully. The total length of the gene cloned was 783 bp with a 97.3% sequence homology with adhesion genes in Gene Bank; 2.SDS-PAGE showed that the recombinant protein that Mr was about 30 000 represented 31.67% of total cell protein; 3. Western blot showed that the recombinant protein could be recognized by H.pylori antiserum of patients; 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 90% after purified with Ni2+-NTA agarose of supernatant; 5. The specific antibody titer was 1:16 in rabbit immunized with HpaA protein detected by double immunodiffusion; 6. The indirect ELISA based on HpaA protein was successfully established to assay the anti-HpaA in serum, and its sensitivity and specificity was 100.0% and 90.1% respectively.Conclusion: The successful expression and purification of the HpaA of H.pylori , provided antigen basis for the development of the vaccine and the diagnostic reagent for H.pylori infection. The indirect ELISA was created to assay anti-HpaA in serum provided a basis for the preparation of diagnostic reagent kit detecting H.pylori infection.