1.Prokaryotic Expression Of NapA Gene Of Helicobacter Pylori And Its Preliminary Analysis 2.Study On Prokaryotic Expression Of Fusion Gene Of Vacuolating Segment Of VacA And Caga From Helicobacter Pylor

Objective: To obtain the recombinant neutrophil activating protein in Helicobacter pylori by gene-engineering, which 　would lay a foundation for the development of diagnostic reagent and vaccine for H.pylori infection. Methods: PCR was used to amplify the napA gene encoding the neutrophil activating protein in Helicobacter pylori from chromosomal DNA. The prokaryotic expression vectors pQE30-napA was constructed and sequenced. Then the engineered bacteria E.coli DH5a was induced by IPTG to express the corresponding protein and optimal conditions of induction were studied.The fusion expression protein was analyzed by SDS-PAGE and its antigenicity was confirmed by Western blot.The expression 　product inclusions were denatured, and then were purified and renatured with Ni2+-NTA chromatography and mltistep dialysis. After purification,　the protein was used to immunize rabbits to prepare antiserum to assess its immunogenicity. The indirect ELISA assay with the recombinant protein was preliminarily applied to detect the IgG in sera of patients infected with H.pylori.Results: 1.The prokaryotic expression vectors pQE30-napA was successfully constructed. Restriction enzyme cleavage analysis, electrophoresis analysis of PCR product of recombiannt plasmid and sequencing showed that target gene has been successfully inserted into expression vector; 2. When engineered bacteria was induced by IPTG and optimal conditions of induction were achieved ,the anticipated Mr 17×103protein band appeared on SDS-PAGE gel and amount to 38.8% of total Bacteria protein; 3. Western blot showed that the recombinant protein could be recognized by sera from patients infected with Helicobacter pylori; 4. SDS-PAGE showed that the recombinant protein mainly expressed as inclusion bodies. After purification, the purity of target protein was 92.5%; 5. The good immunogenicity of recombinant protein was confirmed by detecting the antiserum of rabbit immunized with the recombinant protein; 6. ELISA assay showed the recombinant protein renatured through multistep dialysis can detected sera from patients infected with H.pylori ,　which pave the way for further study of Nap as a diagnostic reagent for H.pylori infection. Conclusion: The napA gene was successfully cloned. The recombinant protein was efficiently expressed in E.coli and highly purified, which provided antigen basis for the development of the prophylactic and therapeutic vaccine and the diagnostic reagent for H.pylori infection.