人端粒酶逆转录酶基因启动子区的克隆及其真核表达载体的构建

Cloning of human telomerase reverse transcriptase gene promoter and construction of its eukaryotic expression vector

目的 :构建人端粒酶逆转录酶基因启动子区的真核表达载体。方法 :用核酸内切酶NotⅠ和KpnⅠ酶切报告基因GFP ,插入pcDNA3.1 (+)载体 ,得到pcDNA3.1 (+) -GFP(PG)载体 ;用PCR方法扩增hTERT基因ATG上游的启动子序列 ,测序后用核酸内切酶BglⅡ和KpnⅠ酶切插入PG载体 ,得到pcDNA3.1 (+) -GFP -hTERT(PGT)载体。将PGT载体转染人食管癌EC970 6细胞系 ,经G4 1 8筛选 ,荧光显微镜下观察。结果 :克隆得到的启动子序列与文献相符 ,重组载体经酶切鉴定方向正确 ,含有人端粒酶逆转录酶基因启动子序列。经转染人食管癌EC970 6细胞系 ,在荧光显微镜下观察到绿色的阳性克隆。结论 :克隆的人端粒酶逆转录酶基因启动子序列在真核细胞中具有启动子活性。

Aim: To construct an eukaryotic expression vector containing human telomerase reverse transcriptase (hTERT) gene promoter and reporter green fluorescent protein(GFP). Methods: PCR amplification was performed using primers based on hTERT gene sequence from GenBank and human genomic DNA as a template. PCR product was directedly ligated into pMD18-T vector for sequencing. The promoter fragment was cloned into a pcDNA3.1(+)-GFP plasmid after it had been identified correctly. A human esophageal cancer cell line EC9706 was transfected with the plasmid containing hTERT promoter and reporter gene GFP, and the transfected cells were observed under fluorescent microscope 36 hours later. Results: A 733bp DNA fragment was amplified. Sequensing and restriction endonuclease digestion demonstrated that the fragment was cloned into the pcDNA3.1(+)-GFP-hTERT vector. Positive transfected cells with green fluorescence were detected under fluorescent microscope. Conclusion: The cloned hTERT promoter fragment in pcDNA3.1(+)-GFP-hTERT plasmid vector is active in human esophageal cancer cell line EC9706.