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C端带His标签重组MT1G真核表达载体构建及其在EC9706中的表达 被引量:4

Expression of recombinant eukaryotic expression vector of MT1G with C terminal of His-tag in EC9706 cells
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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。 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 eukaryotie 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 lipofeetamine 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 tranfeeted EC9706 cells. CONCLUSION: The human MT1G recombinant plasmid and the cell strains expressing MTIG fused protein with His-tag are obtained, which provide the basis for further study on biology functions of MTIG.
出处 《中华肿瘤防治杂志》 CAS 2009年第8期596-599,共4页 Chinese Journal of Cancer Prevention and Treatment
关键词 食管肿瘤 基因 MT1G DNA 重组 基因表达 遗传载体 esophageal neoplasms genes, MT1G DNA, recombinant gene expression genetic vectors
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