重组真核表达质粒pHsp70-hsv-tk的构建及在细胞内的热诱导表达

Construction and identification of the recombinant eukaryotic expression plasmid pHsp70-hsv-tk

目的:构建用于靶向基因治疗的重组真核表达质粒pHsp70-hsv-tk(pHT),体外转染SMMC-7721肝癌细胞,观察热诱导基因表达情况。方法:设计1对引物,以pHSV-106质粒为模板进行PCR扩增,将扩增产物纯化所得目的基因片段与pD3SX载体连接,转化大肠杆菌E.coliDH5α,克隆PCR法筛选菌落,测序鉴定后大量抽提质粒。磁性转染法将pHT导入SMMC-7721,加热处理后,用RT-PCR法检测tk基因的整合。结果:获得重组的hsv-tk基因条带。结论:成功构建了pHT质粒,可用于进行靶向性基因治疗研究。

Objective： To construct the recombinant eukaryotic expression plasmid pHsp70-hsv-tk （pHT） for further target gene therapy research. Methods： The oligomers of primers were synthesized based on the reported sequences of hsv-tk and with the pHSV-106 plasmid as the template. Then the hsvtk gene fragment was amplified and inserted into the pD3SX vector. E. coli DH5α was transformed with the recombinant plasmids and the bacterial colonies containing the recombinant plasmids were identified by the PCR cloning method, and the recombinant plasmids were extracted and purified. Results： The recombinant eukaryotic expression plasmid pHT was obtained. The target gene obtained by PCR amplification had the same molecular size as predicted. The recombined pD3SX plasmid contained correct recombinant hsv-tk sequences. Conclusion： The plasmid pHT was successfully constructed, which can be used for further target gene therapy research.