端粒酶催化亚基活性中心hTERT的克隆及其真核表达

Cloning and Expression of cDNA for Human Telomerase Catalytic Subunit hTERT Active Site

本实验采用RT-PCR技术,应用高保真DNA聚合酶从人喉癌细胞中扩增端粒酶催化亚基活性中心基因片段,将克隆载体上hTERTcDNA用EcoRⅠ和HindⅢ双酶切,连入真核表达载体pCR3.1,利用pCR3.1载体上带有强的CMV启动子,高效表达活性中心蛋白,采用RT-PCR检测hTERT mRNA的表达情况,结果表明,在细胞内检测到了mRNA,证明该cDNA在细胞内确实得到了表达,并采用TRAP-ELISA方法检测端粒酶活性。

The RT-PCR was used to amplificate active site gene segment of telomerase catalytic subunit from the cells of human laryngeal carcinoma with DNA polymerase. The hTERTcDNA in the cloning vector was digested by EcoRⅠand HindⅢ, and connected with eukaryotic expression vector PCR3.1. The active site protein was efficiently expressed with strong CMV promoter in the vector PCR3.1 and the expression state of the hTERT mRNA was identified by RT-PCR. The result showed that mRNA was identified in the cell. It showed that hTERTcDNA was successfully expressed in the cell and telomerase activity was identified by the way of TRAP-ELISA.