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

摘要

BACKGROUND: Human telomerase reverse transcriptase is an important component of telomerase. The display on telomeraseactivity 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 telomerasereverse transcriptase; to express the constructed vector in human embryonic kidney 293T cells; to lay the foundation for theresearch of intracellular localization.METHODS: The C-terminal fragment of human telomerase reverse transcriptase containing amino acids 936-1132 and enzymesites was amplified by PCR using human telomerase reverse transcriptase as templates. The amplified fragments were insertedinto 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 wasexamined by western blot.RESULTS AND CONCLUSION: Double enzyme digestion and gene sequencing results showed that the recombinant plasmidpcDNA3.1-HisA-hTERT aa936-1132 containing the C-terminal amino acids 936-1132 of human telomerase reverse transcriptasewas constructed successfully. The fusion protein was identified using western blot.%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-hTERTaa936-1132 构建成功,经免疫印迹鉴定可检出融合蛋白.