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目的 对腺病毒AdEasy载体系统进行方法改良 ,使之成为能被一般实验室采用的快速构建重组腺病毒的系统。方法 用插入有G1VP7基因和绿色荧光蛋白的穿梭载体pAdTrackCMV与腺病毒骨架载体pAdEasy 1共转化E .coliBJ5 183并进行同源重组 ,获得重组腺病毒质粒 ,经PacⅠ线性化后 ,转染 2 93细胞。为便于操作 ,对AdEasy载体系统的某些操作过程进行了适当改进。结果 得到重组腺病毒rvAdG1VP7(G)。转染重组腺病毒DNA的 2 93细胞出现细胞病变 ,经PCR对传代的rvAdG1VP7(G)分析证实 ,有特异性的G1VP7基因整合 ,荧光显微镜能观察到 2 93细胞内有绿色荧光。结论 腺病毒AdEasy载体系统是一种较为简便、快速的构建重组腺病毒的好方法 ,经过适当改进后更适合于国内实验室的研究和应用
OBJECTIVE To improve the adenovirus AdEasy vector system and make it a rapid construction of recombinant adenovirus which can be used by general laboratories. Methods The shuttle vector pAdTrackCMV with G1VP7 gene and green fluorescent protein inserted into adenovirus backbone vector pAdEasy 1 was co-transformed into E. coli BJ5 183 and homologously recombined to obtain a recombinant adenovirus plasmid. After PacⅠ linearization, 293 cells . For ease of operation, some improvements to the AdEasy carrier system have been made. As a result, the recombinant adenovirus rvAdG1VP7 (G) was obtained. 2 93 cells transfected with recombinant adenovirus DNA showed cytopathic effect. Analysis of rvAdG1 VP7 (G) by PCR showed that there is specific integration of G1VP7 gene, and green fluorescence in 293 cells can be observed by fluorescence microscopy. Conclusion The adenovirus AdEasy vector system is a simple and rapid method for constructing recombinant adenovirus. After appropriate improvement, it is more suitable for the research and application of domestic laboratories