中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (11): 1977-1980.doi: 10.3969/j.issn.1673-8225.2012.11.018

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

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★

卢钧雄，江慧贤，曹  莹，庞建新   

  1. 南方医科大学药学院，广东省广州市  510515
  • 收稿日期:2011-10-24 修回日期:2011-12-01 出版日期:2012-03-11 发布日期:2012-03-11
  • 通讯作者: 庞建新，教授，南方医科大学药学院，广东省广州市 510515
  • 作者简介:卢钧雄★，男，1986年生，广东省中山市人，汉族，南方医科大学在读硕士，主要从事抗肿瘤药理和新药评价方面研究。 furture5@65w163.com

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

Lu Jun-xiong, Jiang Hui-xian, Cao Ying, Pang Jian-xin   

  1. School of Pharmaceutical Sciences, Southern Medical University, Guangzhou  510515, Guangdong Province, China
  • Received:2011-10-24 Revised:2011-12-01 Online:2012-03-11 Published:2012-03-11
  • Contact: author: Pang Jian-xin, Professor, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, Guangdong Province, China
  • About author:Lu Jun-xiong★, Studying for master’s degree, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, Guangdong Province, China furture5@163.com

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488

被引次数

2

摘要：

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。
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
doi:10.3969/j.issn.1673-8225.2012.11.018

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

Abstract:

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.

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