Cloning and expressing cagT gene of type Ⅳ secretion system in Helicobacter pylori and influence of cytokine secretion and cell proliferation on SGC-7901 cell

Abstract:

[Objective] To detect the influence of the CagT protein on interleukin-8 secretion and proliferation in SGC-7901 cells. [Methods] Helicobacter pylori cagT gene was amplified by PCR with the genomic DNA of H. pylori NCTC 11637 as template, then it was inserted into an expression vector pQE30. The recombinant plasmid was transformed into E.coli M15. Recombinant protein was expressed by Isopropylthio-b-D-Galacgoside (IPTG) induction and confirmed by Western blot. Fusion protein with 6×His tag was purified using Ni2+-NTA agarose. Interleukin-8 mRNA expression of SGC7901 cells was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Cell viability was determined by methyl thiazolyl tetrazolium assay (MTT). [Results] The GenBank accession number of the amplified sequence is EF114758. The sequence analysis for cagT showed that it shares 97%-99% homology with other strains of H. pylori in Gene bank. The molecular mass of the product is 32kDa, and its purity is 98% analyzed by SDS-PAGE. After the protein was dialyzed, it can stimulate SGC7901 cells to express interleukin-8 and the growth of cells was inhibited in a dose- and time-dependent manner. [Conclusion] It is indicated that we have obtained the correct cagT gene and expressed in E.coli M15. The protein can stimulate the cells to express cytokine interleukin-8 and inhibit proliferation of cells, which posed a basis for further research on its biological function.