Construction of eukaryotic expression vector of human telomerase RNA component and its function
10.7644/j.issn.1674-9960.2016.02.015
- VernacularTitle:人TR基因真核表达载体的构建
- Author:
Sunyang YING
;
Jiaxiu XIONG
;
Hongxu MAI
;
Jiajia LIN
;
Lina JIANG
;
Long CHENG
;
Qinong YE
- Publication Type:Journal Article
- Keywords:
telomerase;
hTR;
gene cloning;
eukaryotic expression
- From:
Military Medical Sciences
2016;40(2):137-141,165
- CountryChina
- Language:Chinese
-
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 .
