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目的 :采用细菌内同源重组法高效制备含CD、TK融合自杀基因重组体腺病毒。方法 :CD、TK融合基因CDglyTK自载体 pWZLneoCDglyTK中切出 ,亚克隆至腺病毒穿梭质粒中 ,形成转移质粒 pAdtrackCMV CDglyTK ,将之PmeI酶切线性化后与腺病毒基因组质粒 pAdeasy 1共转化BJ5 183菌 ,抽提重组体腺病毒基因组质粒DNA ,PacI酶切后转染 2 93细胞包装成腺病毒颗粒 ,采用PCR方法对重组体腺病毒进行鉴定 ,利用报告GFP基因对病毒滴度和感染效率进行测定 ,WesternBlot检测感染腺病毒的肿瘤细胞中有无目的基因的表达。结果 :由pAdTrack CMV CDglyTK和 pAdeasy 1共转化BJ5 183菌 16～ 2 0h后 ,可获得 30 %左右的阳性重组体细菌克隆 ,由重组体克隆质粒DNA转染 2 93细胞包装产生的重组体腺病毒 ,经PCR检测表明已含有目的基因 ,且无复制能力腺病毒的存在。纯化所得腺病毒滴度约为 8× 10 12 pfu/L ,当MOI =10 0时 ,腺病毒感染HepG1细胞的效率 >75 % ,WesternBlot证实在感染重组体腺病毒的细胞中有相应基因产物的表达。结论 :细菌内同源重组法是一种高效、简便、快捷的重组体腺病毒载体制备方法。所制备的Ad CDglyTK在体外能有效表达相应的基因产物 ,为今后的深入研究奠定了基础。
Objective : To efficiently prepare recombinant adenoviruses containing CD and TK fusion suicide genes using homologous recombination in bacteria. METHODS: The CD and TK fusion gene CDglyTK was cut out from the vector pWZLneoCDglyTK and subcloned into the adenoviral shuttle plasmid to form the transfer plasmid pAdtrackCMVCDglyTK, which was linearized with PmeI and then co-transformed with the adenoviral plasmid pAdeasy 1 into the BJ5 183 strain. The plasmid DNA of the recombinant adenovirus genome was extracted, digested with PacI, transfected with 293 cells and packaged into adenoviral particles. The recombinant adenovirus was identified by PCR, and the virus titer and infection efficiency were determined using the reported GFP gene. Western Blot detects the expression of target genes in adenovirus-infected tumor cells. RESULTS: After co-transformation of BJ5 183 strains with pAdTrack CMV CDglyTK and pAdeasy 1 for 16 to 20 hours, approximately 30% positive recombinant bacterial clones were obtained, and the recombinant adenoviruses transfected with 293 cells were transfected with the recombinant clone plasmid DNA. PCR detection showed that the target gene was already present and there was no replication-competent adenovirus. The titer of the purified adenovirus was approximately 8×10 12 pfu/L. When MOI =10 0, the efficiency of adenovirus infection of HepG1 cells was >75%. Western Blot confirmed that there was a corresponding gene product in the cells infected with the recombinant adenovirus. expression. Conclusion : Homologous recombination in bacteria is a highly efficient, simple and rapid preparation method of recombinant adenovirus vector. The prepared Ad CDglyTK can effectively express the corresponding gene product in vitro, which lays a foundation for further research in the future.