含融合自杀基因CDglyTK复制型重组腺病毒的构建与鉴定

目的:构建含融合自杀基因CDglyTK复制型重组腺病毒,并在肺腺癌细胞A549中验证其复制能力和溶瘤特性。方法:建立重叠延伸PCR方法,用该方法制备的E1A-IRES片段插入AdEasy载体构建含融合自杀基因CDglyTK复制型重组腺病毒rAdE1A-CDglyTK。并用该病毒与复制缺陷型重组腺病毒rAd-CDglyTK分别感染肿瘤细胞A549,从荧光蛋白表达、细胞病变及病毒增殖等方面进行比较,以确定其复制能力及溶瘤特性。结果:成功构建了含融合自杀基因CDglyTK的复制型重组腺病毒rAdE1A-CDglyTK,该病毒感染肿瘤细胞A549后报告基因绿色荧光蛋白(GFP)的表达,能大量增殖并出现明显的细胞病变效应(CPE)。结论:构建的复制型重组腺病毒在肿瘤细胞内的复制能力、溶瘤特性和外源基因的表达明显优于目前广泛应用的复制缺陷型重组腺病毒。

Construction and Identification of Replicative Recombinant Adenovirus containing Fusion Suicide Gene CDglyTK

Objective: To construct replicative recombinant adenovirus containing fusion suicide gene CDglyTK and verify its replicative ability and oncolytic ability in the tumor cell A549. Methods: Establish overlap-extension PCR,and construct replicative recombinant adenovirus containing fusion suicide gene CDglyTK by E1A-IRES fragment from overlap-extension PCR and the modified AdEasy system of competent cell prepared using calcium chloride and homologous recombination in bacteria. Using the replicative recombinant adenovirus rAdE1A-CDglyTK and the replication defective adenovirus rAd-CDglyTK infect the tumor cell A549 respectively. Compare two kinds of adenovirus from the expression of gene green fluorescent protein(GFP),the cytopathic effect(CPE) and proliferation of adenovirus. Results: Successfully constructed replicative recombinant adenovirus containing fusion suicide gene CDglyTK. After infecting the tumor cell A549,the replicative recombinant adenovirus proliferated enormously,the CPE was very obvious and the expression of gene GFP was plentiful. Conclusion: The replicative recombinant adenovirus is more effective than the replication defective adenovirus used generally at present in replicative ability,oncolytic ability and the expression of exogenous gene in the tumor cell.