【摘要】 目的:构建含MT1GcDNA的重组真核表达载体,并观察在人食管癌细胞EC9706中表达情况。方法:用PCR法从含MT1G基因cDNA质粒pACT2-MT1G中扩增获得目的基因片段,正向克隆入C端带Myc和6×His标签的真核表达质粒pcD-NA3.1/Myc-His(-)中。经酶切及测序鉴定后,以脂质体法转染EC9706细胞,经G418加压筛选获得阳性单克隆细胞,用RT-PCR和蛋白质印迹法技术检测MT1G mRNA和蛋白的表达。结果:成功构建了真核表达载体pcDNA3.1/Myc-His(-)-MT1G,转染pcDNA3.1/Myc-His(-)-MT1G的EC9706细胞内有带His标签MT1G融合蛋白表达。结论:获得了人MT1G重组载体和稳定表达带His标签MT1G融合蛋白的EC9706细胞株,为进一步研究MT1G的功能奠定了基础。更多还原

【Abstract】 OBJECTIVE:To construct a recombinant eukaryotic expression vector MT1G cDAN and explore its expression in EC9706 cells. METHODS:The target sequence was amplified by PCR from pACT2-MT1G plasmid containing human MT1G cDNA and cloned into eukaryotic expression vector pcDNA3.1/Myc-His(-) with C terminal of Myc epitope and 6×His-tag. After restriction endonuclease digestion and DNA sequencing confirmation,the recombinant plasmid was tranfected into EC9706 cells by lipofectamine 2000. The positive molyclone was screened by G418. RT-PCR and Western blot was used to detect the expressions of mRNA and protein of MT1G gene respectively. RESULTS:The eukaryotic expression vector pcDNA3.1/Myc-His(-)-MT1G was successfully constructed and MT1G fused protein with His-tag was expressed in tranfected EC9706 cells. CONCLUSION:The human MT1G recombinant plasmid and the cell strains expressing MT1G fused protein with His-tag are obtained,which provide the basis for further study on biology functions of MT1G.更多还原