重组腺病毒方法的改建
A more effective and easier method for constructing recombinant adenovirus based on AdEasy system
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摘要:目的 对构建重组腺病毒的AdEasy载体系统的操作过程予以改进, 使其更加简便、高效。方法 将腺病毒骨架载体pAdEasy-1用电穿孔法转化大肠埃希菌BJ5183, 获得含pAdEasy-1的大肠埃希菌BJ5183/p菌株.氯化钙法制备大肠埃希菌BJ5183/p感受态细胞, 并转化含绿色荧光蛋白的穿梭载体, 通过同源重组获得重组腺病毒质粒.以限制性内切酶PacⅠ酶切, 得到重组腺病毒DNA分子, 转染293细胞。结果 293细胞出现细胞病变, 荧光显微镜观察有绿色荧光。结论 成功获得一种以AdEasy系统为基础的简便、高效构建重组腺病毒的新方法。Abstract:Objective To Obtain a more effective and easier method for constructing recombinant adenovirus by simplifying the protocol of the AdEasy system.Methods The replication-defective adenovirus type 5 backbone plasmid pAdEasy-1 was transformed into competent E. coli BJ5183 by electroporation and the E. coli BJ5183/p was obtained.Then E.coli BJ5183/p competent cells were prepared by CaCl 2 technique and the shuttle plasmid carrying green fluorescent protein(GFP)was transformed into E. coli BJ5183.The confirmed recombinant was linearlized with Pac I and transfected into 293 cell line.Results The CPE of 293 cells were observed and the green fluorescence was seen under fluorescent microscope.Conclusion A more effective and easier method for constructing recombinant adenovirus based on AdEasy was obtained successfully.
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