应用两步氯化钙转化法高效制备双自杀基因重组腺病毒

目的 构建巨细胞病毒(CMV)启动子驱动下的双自杀基因重组腺病毒。方法 应用PCR扩增出CD和TK基因，经适当改造后插入pAdtrack CMV中构建成转移质粒pAdtrack CMV-CDTK。经酶切线性化后。转化用氯化钙制备的感受态AdEasier-1细菌，获得重组腺病毒质粒。最后将线性化的重组腺病毒质粒转染293细胞包装获得重组腺病毒。滴度测定后，体外感染血管内皮细胞ECV304。结果 PCR检测表明已成功构建了含CD、TK融合基因的重组腺病毒，为进一步开展肿瘤的双自杀基因治疗研究奠定了基础。结论 应用改进后的AdEasy系统构建腺病毒相比于传统AdEasy系统，方法更简便、成功率更高、实验周期更短。

Highly efficient construction of recombinant adenovirus containing double suicide gene driven by cytomegalovirus promoter using two-step CaCl2 transformation method]

OBJECTIVE: To construct recombinant adenovirus containing double suicide gene driven by cytomegalovirus (CMV) promoter. METHODS: CD and TK gene were amplified by PCR and cloned into the shuttle vector pAdtrack CMV, and the resultant plasmid pAdtrack CMV-CDTK was linearized with PmeI and transformed into competent AdEasier-1 cells prepared by CaCl2 method. PacI digestion of identified recombinant plasmid DNA was performed before it was transected into 293 cells to package adenovirus, which was used to infect endothelial cells in vitro after adenovirus titer determination. RESULTS: Chemical transformation of linearized transfer plasmid into AdEasier-1 cells resulted in very high ratio (20/20) of positive clones of recombinant adenovirus containing. CD and TK fusion gene as confirmed by PCR test. CONCLUSION: The modified AdEasy system is more convenient and efficient for constructing recombinant adenovirus, which may facilitate further study of anti-tumor therapy targeting at the double suicide gene.