【摘要】 【目的】初步研究幽门螺杆菌(Helicobacter pylori,H.pylori)NCTC11637cagT(HP0532)基因对SGC-7901细胞增殖和白细胞介素-8(IL-8)分泌的影响。【方法】应用PCR技术从H.pylori基因组DNA中扩增cagT编码基因片段,将其定向插入pQE30载体中,双酶切鉴定筛选阳性克隆。测序分析确认后,IPTG诱导表达,表达蛋白以Ni2+-NTA柱进行纯化,并经Westernblot鉴定,纯化的蛋白作用于SGC-7901细胞,提取细胞总RNA,RT-PCR扩增IL-8,并用MTT法检测蛋白对细胞增殖的影响。【结果】成功克隆了cagT基因,全长843bp(GenBank登录号为EF114758),编码280个氨基酸,与GenBank公布的其他H.pylori菌株基因序列的核苷酸同源性为97%~99%。工程菌诱导后SDS-PAGE显示新生表达蛋白带,相对分子质量约为32kDa,经Ni2+-NTA柱纯化后可获得纯度为98%重组蛋白。经透析复性后的蛋白(终浓度10μg/mL)作用于SGC-7901细胞后,可诱导细胞因子IL-8在mRNA水平上的表达。不同浓度CagT蛋白与SGC-7901细胞作用,细胞增殖的程度随着蛋白浓度的增加逐渐降低,细胞的增殖受到抑制。【结论】CagT蛋白可刺激SGC-7901细胞IL-8的表达,并抑制细胞增殖,为进一步研究其生物学功能奠定了基础。更多还原

【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 trans-formed into E.coli M15. 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. 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.更多还原