以Ad-Easy载体系统快速构建PTEN重组腺病毒表达载体  

Rapidly construction of PTEN recombinant adenovirus expression vector with Ad-easy system

作　　者：熊亮[1] 谢富华[2] 赵树勇[1] 阮杰[1] 马华谋[2] 林观平[1] 周克元[1] 

机构地区：[1]广东医学院生物化学与分子生物学教研室,广东湛江524023 [2]赣南医学院生物化学与分子生物学教研室,江西赣州341000

出　　处：《广东医学》2007年第4期516-518,共3页Guangdong Medical Journal

基　　金：广东省重点学科建设资助项目(编号:GX9307)

摘　　要：目的目的应用Ad-Easy腺病毒载体系统构建携带抑癌基因PTEN的重组腺病毒表达载体。方法用插入有抑癌基因PTEN和绿色荧光蛋白的穿梭载体pAdTrack-CMV转化已经含有腺病毒骨架载体pAdEasy1的E.coliBJ5183(Ad-1cells)进行同源重组,获得重组腺病毒质粒,经PacⅠ线性化后,转染293细胞。结果得到重组腺病毒,转染重组腺病毒DNA的293细胞出现细胞病变,经PCR对传代的Ad-PTEN分析证实得到目的基因,荧光显微镜能观察到293细胞内有绿色荧光。结论腺病毒Ad-Easy系统是一种较为简便、快速的构建重组腺病毒的好方法,且生成的病毒滴度高、转导效率好,为进一步研究PTEN基因应用于癌症基因治疗提供实验基础。Objective To construct a recombinant adenovirus expression vector containing an anti -oncogene, PTEN by using the Ad - Easy system. Methods The anti - oncogene PTEN was inserted into the shuttle vector pAdTraek -CMV which contains a separated expression box of green fluorescence protein. The shuttle vector was transformed into E. coli（ B J5183 ） which had already contained adenovirus backbone vector pAdEasy - 1 to achieve the homologous recombination and to obtain recombinant adenovirus genome plasmid. This recombinant construct was subsequently linearlized with Pae Ⅰ and transfected into 293 cells. Results Recombinant adenovirus named Ad - PTEN was obtained. The CPE of 293 cells was observed after being transfeeted. A gene integration of PTEN in continuously multiplied Ad ＇ PTEN was confirmed by PCR and the green fluorescence was observed in the 293 cells under fluorescent microscope. Conclusion The Ad - Easy system is a rapid and simple method to construct recombinant adenovirus with high titer and efficient transfection, which would provide experimental basis for studying gene therapy by PTEN.

关 键 词：重组腺病毒 PTEN 基因治疗 Ad—Easy 

分 类 号：R737.9[医药卫生—肿瘤]