人端粒酶反转录酶936-1132氨基真核表达载体的构建及在293T细胞中表达  

Construction of an eukaryotic expression vector carrying amino acids 936-1132 of human telomerase reverse transcriptase and its expression in 293T cells

作　　者：卢钧雄[1] 江慧贤[1] 曹莹[1] 庞建新[1] 

机构地区：[1]南方医科大学药学院,广东省广州市510515

出　　处：《中国组织工程研究》2012年第11期1977-1980,共4页Chinese Journal of Tissue Engineering Research

摘　　要：背景:人端粒酶反转录酶是端粒酶的重要组成之一,可使端粒酶表现活性从而拮抗端粒缩短或细胞衰老。目的:构建人端粒酶反转录酶C端936-1132氨基真核表达载体,并在人胚肾细胞293T表达,为其在细胞内定位研究奠定基础。方法:以人端粒酶反转录酶cDNA为模板,PCR扩增出带有酶切位点其C端936-1132氨基序列片段,并与真核表达载体pcDNA3.1-HisA连接,构建出重组质粒pcDNA3.1-HisA-hTERT aa936-1132,将重组质粒转染入人胚肾细胞293T中,使蛋白表达,并对蛋白进行免疫印迹鉴定。结果与结论:经双酶切和基因测序等方法证实,人端粒酶反转录酶C端936-1132氨基重组质粒pcDNA3.1-HisA-hTERT aa936-1132构建成功,经免疫印迹鉴定可检出融合蛋白。BACKGROUND： Human telomerase reverse transcriptase is an important component of telomerase. The display on telomerase activity results in anti-telomere shortening or cell senescence. OBJECTIVE： To construct an eukaryotic expressing vector containing the C-terminal amino acid 936-1132 of human telomerase reverse transcriptase; to express the constructed vector in human embryonic kidney 293T cells; to lay the foundation for the research of intracellular localization. METHODS： The C-terminal fragment of human telomerase reverse transcriptase containing amino acids 936-1132 and enzyme sites was amplified by PCR using human telomerase reverse transcriptase as templates. The amplified fragments were inserted into the eukaryotic expressing vector pcDNA3.1-HisA to construct recombinant plasmid pcDNA3.1-HisA-hTERT aa936-1132. This recombinant plasmid was transfected into the human embryonic kidney 293T cells, and then the protein expression was examined by western blot. RESULTS AND CONCLUSION： Double enzyme digestion and gene sequencing results showed that the recombinant plasmid pcDNA3.1-HisA-hTERT aa936-1132 containing the C-terminal amino acids 936-1132 of human telomerase reverse transcriptase was constructed successfully. The fusion protein was identified using western blot.

关 键 词：人端粒酶反转录酶C 端 重组质粒 人胚肾细胞293T 蛋白表达 免疫印迹 

分 类 号：R318[医药卫生—生物医学工程]