人胃癌细胞端粒酶RNA组分hTR基因片段的克隆及其正反义真核表达载体的构建

目的:克隆人胃癌细胞端粒酶RNA组

hTR gene cloning from human gastric cancer cells and the construction of its sense and antisense eukaryotic expression vector

AIM To clone human telomerase RNA component (hTR) gene from human gastric cancer cells and construct its eukaryotic sense and antisense expression vector. METHODS The hTR cDNA was cloned from human gastric cancer cell line ( MKN-45) through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO vector) to construct hTR gene sense eukaryotic expression vector (pEF-hTR). The inserted hTR cDNA was then cut from EcoRV/Spel sites of pEF-hTR with restriction endonuclease and subcloned into the pBluescript II KS vector in trans-direction. Finally, the inserted hTR cDNA was cut from Kpnl/Notl sites of pBluescript II KS vector with restriction endonuclease and subcloned into the pEF- hTR in cis-direction to construct hTR gene antisense eukaryotic expression vector (pEF-ahTR). The constructed hTR sense and antisense expression vector were both confirmed through restriction endonuclease analysis and sequencing. RESULTS The sequence of cloned hTR cDNA was confirmed by comparison with the database of the Genbank. The constructed hTR sense and antisense eukaryotic expression vector (pEF-hTR and pEF-ahTR)was proVed to be the same as original design by restriction endonuclease analysis and sequencing. CONCLUSIONS We succeeded in cloning hTR cDNA and constructing its ense and antisense eukaryotic expression vector,which laid the solid foundation for further studying the influence of the inhibition of telomerase activity on the biological behaviors of human gastric cancer cells.