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

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

目的克隆人胃癌细胞端粒酶RNA组分(hTR)基因片段并构建其正义和反义真核表达载体,为以端粒酶为靶目标的肿瘤基因治疗奠定基础。方法采用RT-PCR方法从人胃癌细胞株MKN-45中扩增出人hTR部分cDNA序列。将该片段插入pEF6/V5-His-TOPO载体后构建人端粒酶RNA组分(hTR)基因正义真核表达载体(pEF-hTR)。随后采用EcoRV和SpeⅠ从pEF-hTR上切下该目的基因片段并反向插入pBluescriptⅡKS载体上的EcoRV/SpeⅠ酶切位点上。最后采用KpnⅠ和NotⅠ从pBluescriptⅡKS载体上切下该目的基因片段并正向插入pEF-hTR的KpnⅠ/NotⅠ酶切位点上从而构建出人端粒酶RNA组分(hTR)基因反义真核表达载体(pEF-ahTR)。对所构建的载体均进行酶切鉴定和测序确认。结果所克隆的基因片段其碱基序列与文献报道完全一致,且插入载体的方向完全正确。结论本实验已成功克隆了人端粒酶RNA组分(hTR)基因的部分序列并成功构建其正义、反义真核表达载体,从而为研究反义抑制人胃癌细胞端粒酶活性对胃癌细胞生物学行为的影响奠定了基础。

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.