The Role Of Hp0540 In H.Pylori Pathogenesis

Objective:Construct the expression system and gene mutant of hp0540 from strain NCTC11637 of H.pylori.Moreover,we explored the role of hp0540 in the process of CagA's translocation.All of these results will shed light on the role of hp0540 in H. pylori pathogenesis.Methods:1.The hp0540 gene was amplified by PCR with the genomic DNA of H.pylori NCTC 11637 as template.The plasmid pMD18-2T-hp0540 was constructed via T-A clone method,and sequenced by Sangong(Shanghai,China).The sequence of hp0540 was analyzed through bioinformatics approach.2.The expression vector pET-28a-hp0540 was constructed and transformed into E.coli BL21DE3 by molecular cloning method.The protein was expressed and characterized via SDS-PAGE method after induced by IPTG.The target protein CagI was purified and collected by Ni²⁺-NTA agarose.The puried protein was used to immunize rabbit to prepare polyclonal antibody serum.3.Basing on the allelic exchange,we designed and amplified the upstream homologous fragment and downstream homologous fragment of hp0540 gene via PCR method,and then constructed the suicide plasmid pBlueKM402Δhp0540.We introduced the suicide plasmid pBlueKM402Δhp0540 into Helicobacter pylori 11637 by electroporation and screened the mutant based on antibiotic selection.The mutant was checked by PCR and gene sequencing.4.The hp0540 gene mutant and wild type were co-cultured with human gastric epithelial cell GES-1.The ability of CagA's translocation was dertermined by Western blot method.Results:1.We cloned hp0540 gene of H.pylori NCTC 11637 genomic DNA.It is 1086 base pairs long,which encodes a product of 361 amino acids.GenBank accession number is HM126476.The sequence analysis for hp0540 showed that it shares 98% homology with other strains of H.pylori in Genbank.DNAStar software predict that the CagI's molecular weight is 39.37kDa and has high immunogenicity.2.We constructed the expression vector pET-28a-hp0540 and transformed into E.coli BL21DE3.The result of SDS-PAGE sugGES-1ted that CagI protein expressed after induced by IPTG.The target protein CagI was purified and collected by Ni²⁺-NTA agarose.The potency of the polyclonal antibody serum is 1:1.6×10⁵.3.We amplified the upstream homologous fragment and downstream homologous fragment of hp0540 gene,and constructed the suicide plasmid pBlueKM402Δhp0540.The PCR result sugGES-1ted that the hp0540 deletion mutant was selected from antibiotic medium successfully.4.The co-cultured assay indicated that hp0540 could attenuate the CagA's translocation.Conclusion:In this study,we constructed the prokaryotic expression system and the deletion mutant of hp0540 successfully.The co-cultured assay indicated that hp0540 could attenuate the CagA's translocation,which sugGES-1ted that the hp0540 gene might be a component of Cag Typeâ…£like secretion apparatus that involoved in CagA's translocation.