Cloning And Expressing CagT Gene Of Helicobacter Pylori And Influence Of Inflammatory Factor Expression And Cell Proliferation On SGC-7901 Cells

Objective:To approach the gene function of cagT gene in typeâ…£secretion system(TFSS),which was encoded by cag pathogenicity island (cag PAI)of Helicobacter pylori(H.pylori),and detect the influence of the cagT gene on inflammatory factor secretion and proliferation of gastric cancer epithelial cells,which was profitted to analyse the possible effect of cagT on H.pylori infection and laid a foundation for futher illuminating the pathogenic mechanism of H.pylori.Methods:Oligonucleotide primers were designed according the sequence of H.pylori 22695 in GenBank,using Primer Premier 5.0 software.And in order to facilitate cloning and ensure the exactness of insert direction,we designedly joined some inscribed enzyme points into primers.H.pylori cagT gene was amplified from the genome DNA of H. pylori NCTC 11637 by PCR.The purified PCR products were cloned into pGEM-T vector and transformed into the competent cell of E.coli DH5αby electroporation.It was detected by double restriction enzyme digestion. The nucleotide sequence of the insert was determined by Sangon. Nucleotide sequences and amino sequences encoded by cagT gene were then matched to published sequences by use of the BLAST program available from the National Center for Biotechnology Information. Sequence alignment of CagT with VirB7,the structural gene of TFSS in Agrobacterium tumefaciens(A.tumefaciens)and the biological property at the amino acid level was predicted by using bioinformatics technique.Then the cagT gene fragment was inserted directionally into expression vector pQE30.The recombinant plasmid was transformed into E.coli M15.And the positive clone was selected by Ampicillin fastness and double restriction enzyme digestion.Recombinant protein was expressed by Isopropylthio-β-D-Galacgoside(IPTG)induction and confirmed by Western blot.Fusion protein with 6×His tag was purified using Ni²⁺-NTA agarose.Prepared antibody of the protein by immunizing rabbit,and detected the titer by using ELISA assay.The protein was dialyzed and renaturated before it was added into SGC-7901 cells,then IL-8 mRNA expression was determined by reverse transcriptase polymerase chain reaction(RT-PCR),and the effect of cell proliferation in SGC-7901 cells induced by the recombinant protein was observed by MTT assay.Results:1.The cagT gene of H.pylori NCTC 11637 is 843 base pairs long, which encodes a product of 280 amino acids.GenBank accession number is EF114758.The sequence analysis for cagT showed that it shares 96%～99%homology with other strains of H.pylori in Genbank.CagT is the homologs of VirB7.DNAStar software predicted that its molecular weight was 32.33kDa and has high immunogenicity.2.The prokaryotic expression vector pQE30-cagT was efficiently transformed into E.coli M15.The protein was expressed in E.coli M15 at 30℃after 0.8mmol/L IPTG induction for 4 hours.After expressed by Isopropylthio-β-D-Galacgoside(IPTG)induction,the result of Western blot indicates that the recombinant protein could be recognized by the antibody of anti-His.The expressed product reached a purity of 98%after Ni²⁺-NTA column chromatography,with a relative molecular weight of 32kDa.Using recombinant CagT protein to immunize rabbit,polyclonal antibody titer achieved 1:1.6×10⁴.3.After the protein was co-cultured with SGC-7901 cells for 4 hours, we had detected the IL-8 fragment by RT-PCR.4.When SGC-7901 cells were exposed to CagT of different concentrations for 6,24 and 48 hours,the growth of cell was inhibited in a concentration-dependent and time-dependent manner.Conclusion:1.We have cloned the cagT gene successfully,it has homology with virB7 of the TFSS in A.tumefaciens.It was one of the structure genes of TFSS encoded by cag PAI in H.pylori.2.We have constructed the prokaryotic expression vector,obtained CagT recombinant protein and prepared polyclonal antibody.3.The CagT recombinant protein can induce SGC-7901 cells to express cytokine IL-8 mRNA.4.The CagT recombinant protein can inhibit proliferation of SGC7901 cells in a time-and concentration-dependent manner.