Study On The Biological Functions Of VirB2 And VirB11 Genes In The Plastic Region Of Helicobacter Pylori

ObjectiveHelicobacter pylori(H.pylori)is an important bacterium associated with human gastric and duodenal diseases.virB2 and virB11 are structural genes of tfs3 a gene cluster of Helicobacter pylori IV secretory system.Their biological characteristics are rarely reported in the literature.Therefore,the virB2 and virB11 genes and their proteins in the plasticity region of H.pylori WH-21,a clinical isolate of H.pylori,were studied in this paper.virB2 and virB11 genes and their coding products were further studied by molecular biology,immunology and bioinformatics,which laid a foundation for further research on the pathogenesis and diagnosis and treatment of H.pylori.Methods1.H.pylori WH-21 strain was cultured in Karmali medium under microaerobic environment.2.Prokaryotic expression vector of virB11 gene was constructed.IPTG induced host expression.The fusion protein was purified by Ni2+-NTA affinity chromatography column.Urea gradient dialysis refolded protein was used to immunize rabbits to prepare polyclonal antiserum against recombinant VirB11 protein.3.The subcellular localization of VirB11 protein was detected by Western blotting,and the interaction protein of VirB11 protein was analyzed by immunoprecipitation and mass spectrometry.4.Purified VirB11 protein was co-cultured with human normal gastric epithelial cell GES-1 at different concentrations and the content of IL-8 in supernatant was determined.5.VirB2 protein was synthesized and co-cultured with normal gastric epithelial cell GES-1 for MTT assay.Polyclonal antibodies were prepared from mice and their titers were determined.6.The amino acid sequences of virB2 and virB11 encoding proteins were deduced.Bioinformatics software was used to analyze the physical and chemical properties,cell localization,signal peptide and spatial structure of VirB2 and VirB11 encoding proteins.Results1.Using H.pylori WH-21 strain as template,the full-length fragment of virB11 gene with 942 bp length was successfully obtained.2.The prokaryotic expression vector pET28a-virB11 of virB11 gene was constructed.VirB11 protein was induced and purified.The rabbit anti-VirB11 polyclonal antibody with titer of 1:16 000 was obtained by immunizing male rabbits.3.After co-culture of VirB11 protein with GES-1,the content of IL-8 in supernatant was analyzed.The results showed that VirB11 protein had a concentration-dependent cytotoxicity,indicating that VirB11 protein could significantly stimulate the secretion of IL-8,which was related to host inflammation.4.Using rabbit anti-VirB11 polyclonal antibody as an antibody,the subcellular localization and interaction proteins of VirB11 protein were determined by immunoblotting and co-precipitation combined with mass spectrometry,respectively.The results showed that VirB11 was located in the cell membrane and interacted with ATP syntheses subunit beta,isocitrate dehydrogenase,cytosol aminopeptidase,F0F1 ATP syntheses subunit alpha and malate: none oxidoreductase.5.Mouse anti-VirB2 polyclonal antibody was obtained by synthesizing VirB2 protein,which indicated that VirB2 was a concentration-dependent cytotoxic protein.6.Biological software analysis showed that virB11 gene could encode a protein with molecular weight of 36 kDa,which was composed of 313 amino groups.The isoelectric point of the protein is 8.46,which belongs to the hydrophilic stable protein.The spatial structure of the protein is hexamer channel,which further confirms the function of VirB11 protein in ATP metabolism.virB2 gene can encode a protein with molecular weight of 11 kDa,which is composed of 100 amino groups.The isoelectric point of the protein is 9.56,which belongs to hydrophobic stable protein unlike VirB11.Conclusion:1.Biological analysis show that VirB2 is a hydrophobic stable protein and yet VirB11 is a hydrophilic stable protein.2.VirB11 is localized in the cell membrane and interacted with ATP synthesessubunit beta,isocitrate dehydrogenase,cytosol aminopeptidase,F0F1 ATP syntheses subunit alpha and malate: none oxidoreductase.The ATP metabolic correlation of VirB11 protein is further confirmed.3.It is clear that VirB11 can stimulate the secretion of IL-8,which is related to the occurrence of tissue inflammation.4.The antigenicity and concentration-dependent cytotoxicity of VirB2 are identified.