Cloning,Expressing and Analysis of Helicobacter pylori NCTC 11637 hp0525 Gene and Study the Influence of Proliferation of SGC-7901 Cell / 中国生物工程杂志

ABSTRACT

Objective:Helicobacter pylori is one of the human pathogens causing gastric ulcers and cancers.A key virulence factor of H.pylori is the Cag pathogenicity island,which encodes a type IV secretion system.HP0525 is an essential component of the Cag system and acts as an inner membrane associated ATPase.To construct recombinant plasmid containing hp0525 gene of Helicobacter pylori(H.pylori)NCTC 11637,and analysis of sequence nucleic acid,express it in E.coli BL21 and study the influence of the HP0525 protein on proliferation of SGC-7901 cells.Methods:H.pylor hp0525 gene was amplified from the genome DNA by PCR,then operated T-A cloning and sequenced.The hp0525gene fragment was inserted directionally into vector pMD18-T to construct recombinant clones of hp0525 and was sequenced.The recombinant plasmid was transformed into E.coli BL21for expressing under induction of IPTG.Purify the expressed protein by Ni2+-NTA column chromatography.Expressed product was analyzed by Western blot and MALDI-TOF.Added the purified protein into SGC-7901 cells,the effect of cell proliferation in SGC-7901 cells induced by the recombinant protein was observed by MTT.Results:A 993 base pairs long hp0525 gene,which encodes a product of 330 amino acid,was obtained using PCR method and was cloned into pMD18-T vector successfully.The sequence analysis for hp0525 showed that it shares 97%~99% homology with other strains in Gene bank.SDS-PAGE showed a protein band with a relative molecular weight of 36 000,which was consistent with the expectation.The expressed product reached a purity of 97% after Ni2+-NTA column chromatography.The protein after dialyzed and annealed,was co-cultured with SGC-7901 cells,The protien of different concentration co-cultured with SGC-7901 cells for different times,found that the protein in low concentration stimulates proliferation of cells,to achieve some concentration,it inhibits proliferation of cells along with multiplication of the concentration of the protein;The protein inhibits proliferation of cells relay on the extension of time and concentration.Conclusion:It is indicated that the correct hp0525 gene was obtained and expressed in E.coli BL21.High purified protein was obtained by Ni2+-NTA column chromatography,the protein can inhibits proliferation of SGC-7901cells,and T-ATPase activity which posed a basis for further researching on its biological function.