军事医学2016，Vol.40Issue(2)：137-141,165,6.DOI:10.7644/j.issn.1674-9960.2016.02.015
人TR基因真核表达载体的构建
Construction of eukaryotic expression vector of human telomerase RNA component and its function
摘要
Abstract
Objective To construct the eukaryotic expression vector of human telomerase RNA component ( hTR) and study its biological function tentatively .Methods hTR Gene was obtained by PCR from cDNA template , which was reverse transcribed from 293T mRNA and cloned into pCDNA3.0 vector.The recombinant plasmid and empty vector were trans-fected into 293T cells, and hTR expression was identified by qRT-PCR.HepG2 cells that stably transfected with pCDNA3.0-hTR were constructed and identified by qRT-PCR.These cells were used to assess the interaction of hTR with human telomerase revese transcriptase ( hTERT ) and dyskerin .Telomerase activity was also detected in HepG 2 cells transfected with pCDNA3.0-hTR.Results pCDNA3.0-hTR eukaryotic expression vector was successfully constructed by double digestion identification .The inserted fragment was confirmed by sequencing .The expression of hTR in human 293T cells and HepG2 pCDNA3.0-hTR stable cell line was identified.In addition, qRT-PCR and Western blotting results showed that hTR could interact with hTERT and dyskerin , while hTR overexpression could not regulate the telomerase activity in HepG2 cells.Conclusion The eukaryotic expression vector of pCDNA 3.0-hTR is successfully constructed and expressed.This study will contribute to the further study of cancer therapy targeting hTR .关键词
端粒酶/hTR/基因克隆/真核表达Key words
telomerase/hTR/gene cloning/eukaryotic expression分类
生物科学引用本文复制引用
营孙阳,熊加秀,麦洪旭,林佳佳,姜丽娜,程龙,叶棋浓..人TR基因真核表达载体的构建[J].军事医学,2016,40(2):137-141,165,6.基金项目
国家自然科学基金资助项目( 81330053 ) （ 81330053 ）
北京市科技新星计划资助项目(Z131102000413034) （Z131102000413034）
