小鼠端粒酶蛋白亚单位重组腺病毒载体的构建及鉴定

Construction and Identification of Mouse Telomerase Reverse Transcriptase Recombinant Adenovirus Vector

目的构建小鼠端粒酶蛋白亚单位(mousetelomerasereversetranscriptase,mTERT)基因重组腺病毒载体,为下一步研究其体外表达和动物实验研究提供基础。方法从肝癌细胞中提取总RNA,采用RT-PCR技术扩增mTERT的基因编码区序列,将序列定向克隆至真核表达载体pAC,将此表达质粒与腺病毒重组质粒pJM17共同转染293细胞,经同源重组产生重组腺病毒载体Ad-mTERT,纯化后的重组腺病毒载体Ad-mTERT在293细胞大量扩增并通过氯化铯密度梯度离心法纯化,测定病毒滴度。结果PCR及酶切证实:mTERTDNA正确克隆到穿梭质粒pAC中,带mTERTDNA的表达盒成功重组到腺病毒载体基因组E1A缺失区,并在293细胞中成功包装出具有感染活性的重组腺病毒Ad-mTERT。结论成功构建了小鼠TERT基因重组腺病毒载体,为研究其用于肿瘤的基因治疗奠定基础。

[Objective ] To construct the recombinant adenovirus vector expressing the mouse telomerase reverse transcriptase （mTERT） and provide the basis for further experiments in vivo and in vitro. [Methods] The cDNA of mTERT was amplified by RT-PCR. After purified, the gene fragment was cloned into a vector pAC. Recombinant adenovirus plasmid pAC-mTERT was co-transfected with pJM17 into 293 packaging cells and replication-deficient recombinant adenovirus Ad-mTERT by homologous recombination. The Ad-mTERT recombinant adenovirus was efficiently duplicated in 293 cells and was purified by CsCl density centrifugation and titer was measured. [Results] The recombinant plasmid was identified by PCR and digest with restriction enzyme. The recombinant adenovirus carrying mTERT gene was identified by PCR amplification. [Conclusion] The mouse TERT recombinant adenovirus vector was successfully constructed. The study lays foundation for tumor gene therapy by mTERT.