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

作　　者：营孙阳[1] 熊加秀 麦洪旭 林佳佳[1] 姜丽娜[1] 程龙[1] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《军事医学》2016年第2期137-141,165,共6页Military Medical Sciences

基　　金：国家自然科学基金资助项目(81330053);北京市科技新星计划资助项目(Z131102000413034)

摘　　要：目的构建人端粒酶RNA组分(h TR)的真核表达载体,并对其生物学功能进行初步检测。方法以人胚肾293T细胞RNA逆转录得到的c DNA为模板,采用PCR技术扩增出h TR编码序列,将其插入到p CDNA 3.0载体中;将重组质粒与空载体分别转染293T细胞,实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)检测其表达情况,在Hep G2细胞中构建此基因的稳定细胞系并用qRT-PCR检测其效果,通过RNA结合免疫共沉淀(RIP)实验验证其和人端粒酶逆转录酶(h TERT)及角化不良蛋白(dyskerin)的相互作用,通过端粒酶重复序列扩增技术(telomerase repeat amplification protocol,TRAP)检测过表达h TR后Hep G2细胞的端粒酶活性。结果双酶切和测序结果表明,p CDNA3.0-h TR的真核表达载体构建成功;转染293T细胞用qRT-PCR证明成功表达;qRT-PCR证实Hep G2 p CDNA3.0-h TR稳定细胞系筛选成功;RIP实验证实了h TR与h TERT和角化不良蛋白之间存在相互作用;TRAP实验发现过表达h TR并不影响Hep G2细胞的端粒酶活性。结论成功构建了p CDNA 3.0-h TR真核表达载体,为进一步研究以针对h TR为靶标的癌症治疗奠定了基础。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 HepG2 ceils 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 pCDNA3.0-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 

分 类 号：Q78[生物学—分子生物学]