Distribution Of Porins Gene Of Differwnt Strains Of Helicobacter Pylori And Study On Clone And Prokaryotic Expression Of Its Genes

Objective to investigate the distribution of hopw, hopx and opiA genes of 67 H.pylori clinical strains and NCTC11637 in Zhenjiang city; And to assess the relationship between hopw gene and hopx gene and the relationship between the two porins genes status of isolates and occurrence of gastroduodenal diseases. To make a homologous comparison of nucleotide between NCTC11637,43504,26695 and J99; to clone and express the hopw of NCTC11637 and identify immunity, make a homologous comparison of amino acid encoded by HopW; to investigate the chemical and immunological characteristics of the peptides encoded by the genes for development of HP vaccine.Methods The three genes (hopw, hopx, opiA ) were detected with PCR in NCTC11637 and 67 H.pylori clinical strains isolated from clinical gastric biopsies after culture and urease test. Then PCR products were sent out for nucleotide sequence analysis, compared with 43504,26695 and J99. Cloned into the vector PET32a and PQE30; To express HopW through SDS-PAGE, induced by IPTG at different time . Western blot was applied to determine immunity.Results (1) Of the 67 clinical isolates, the frequency of hopw,hopx and opiA gene was 92.5%,95.2%,39.9%, respectively. There was no significant difference in the presence of the two genes of the H.pylori isolates from chronic gastritis, duodenal ulcer, peptic ulcer, gastric cancer (P>0.05); the frequency of opiA gene was significant high in peptic ulcer(p<0.05). (2)The full length of the two porins genes was obtained by PCR and ligated into PGEM-T vector by T-A clone. The hopw gene was confabulated into hopw-PET32a; and the hopx gene into hopx-PQE30. (3) The fusion protein about 36kDa was inducted under the induction environment with IPTG concentration as 1mmol/L, 30°C for 4h and purified with Ni~+-NTA column. There is relatively higher reactivity and specificity was detected by Western-blot.Conclusion (1) H.pylori clinical strains in Zhenjiang city, Jiangsu province were predominantly hopx, hopw positive. The relationship between hopx gene and hopw gene of H.pylori and occurrence of gastroduodenal disease can not be identified. There was no correlation between the two genes. (2) The recombinant plasmid hopw-PET32a and hopx-PQE30 were constructed successfully which were confirmed by sequencing. The homogenicity of nucledtide and that of amino acid was high. The GenBank accession number was EF59968, EF208122. (3) The HopW protein was expressed successfully in vitro which facilitated further research in the function of porins and vaccine of H.pylori.