三种不同腺病毒基因组提取方法对全基因组克隆构建影响分析.docVIP

三种不同腺病毒基因组提取方法对全基因组克隆构建影响分析 　　[摘要] 目的 建立一种简单、快速、有效、纯度高的人腺病毒全基因组提取的方法。 方法 用60 mm细胞培养皿培养A549或AD293细胞，接种培养人7型腺病毒（HAdV-7）CQ1198株，收集病毒感染的?胞，分别用Hirt’s改良法、试剂盒A和试剂B提取HAdV-7-CQ1198病毒株全基因组，提取的基因组进行限制性内切酶酶切实验、细菌内同源重组实验和转染细胞拯救病毒实验。 结果 几种方法都成功获得高质量的腺病毒全基因组，可以用于PCR、测序、酶切、同源重组和转染实验；两种试剂盒方法较传统的Hirt’s改良法更快，不需要特别步骤去除细胞基因组，可以在1 h内完成基因组提取。其中，试剂盒A提取的病毒基因组量最多（20～50 μg），RNA含量最少，不需要另外加入核糖核酸酶（RNase），而Hirt’s改良法需要在裂解液中或最终产物中加入RNase以去除细胞RNA成分。 结论 试剂盒A和试剂盒B均可替代传统Hirt’s提取方法用于快速提取高质量腺病毒基因组，提取的基因组能用于酶切分型、转染等实验操作 [关键词] 腺病毒；病毒核酸提取；酶切分析；病毒感染；Hirt’s方法 [中图分类号] R373 [文献标识码] A [文章编号] 1673-7210（2016）12（c）-0004-04 [Abstract] Objective To construct a simple， fast， efficient， high purity method for adenovirus genome extraction. Methods A549 or AD293 cells were cultured in 60 mm culture dish， and infected with human adenovirus type 7 （HAdv-7） CQ1198 strain. The infected cells were collected to extract adenovirus genome by using improved Hirt’s method， Extraction Kit A and Extraction Kit B. The extracted adenovirus genomes were detected by restriction endonuclease analysis， homologous recombination method， transfection and virus rescue experiment. Results High-quality adenovirus genomes were obtained by three different methods. The extracted DNA can be used for PCR， sequencing， restriction endonuclease analysis， homologous recombination and transfection experiment. The extraction process used two kinds of Kits were faster than improved Hirt’s method， and all the Kit methods had no additional process to remove host cell’s genome， the process can be finished in 1 hour. Compared all three kinds of methods， comprehensively， Extraction Kit A method obtained as most viral DNA as other methods （20-50 μg）， and it did not need RNase because the extracted product barely had RNA. Compare to the Kits， the improved Hirt’s method needed RNase to remove cellular RNA in the lysate or in the final production. Conclusion Compare to the traditional Hirt’s method， Extraction Kit A and Extraction Kit B can be used to extra