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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。
Objective To clone the human telomerase RNA (hTR) gene fragment of human gastric cancer cells and construct its sense and antisense eukaryotic expression vector, which lays a foundation for tumor gene therapy with telomerase as target. Methods Human hTR partial cDNA sequence was amplified from human gastric cancer cell line MKN-45 by RT-PCR. This fragment was inserted into the pEF6/V5-His-TOPO vector to construct the human telomerase RNA component (hTR) gene eukaryotic expression vector (pEF-hTR). The gene fragment of interest was then excised from pEF-hTR using EcoRV and SpeI and inserted back into the EcoRV/SpeI site on the pBluescriptII KS vector. Finally, the target gene fragment was excised from pBluescriptIIKS vector by KpnI and NotI and inserted forward into the KpnI/NotI restriction site of pEF-hTR to construct the antisense eukaryotic expression of human telomerase RNA component (hTR) gene. Vector (pEF-ahTR). The constructed vectors were identified by enzyme digestion and confirmed by sequencing. Results The cloned gene fragment had the same base sequence as reported in the literature, and the orientation of the inserted vector was completely correct. Conclusion This experiment has successfully cloned part of the human telomerase RNA (hTR) gene sequence and successfully constructed its sense and antisense eukaryotic expression vector, thereby studying antisense inhibition of telomerase activity in human gastric cancer cells on gastric cancer cells. The foundation of the influence of biological behavior.