三种不同腺病毒基因组提取方法对全基因组克隆构建的影响分析

发布时间：2018-06-21 21:51

  本文选题：腺病毒 + 病毒核酸提取　； 参考：《中国医药导报》2016年36期

【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(HAd V-7)CQ1198株,收集病毒感染的细胞,分别用Hirt's改良法、试剂盒A和试剂B提取HAd V-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(20~50μg),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。
[Abstract]:Objective to establish a simple, rapid, effective and high purity genomic extraction method for human adenovirus. Methods A549 or AD293 cells were cultured in 60 mm cell culture dish. Human adenovirus type 7 (HAd V-7) CQ1198 strain was inoculated. The infected cells were collected. The whole genome of HAd V-7-CQ1198 strain was extracted by Hirtins modified method, kit A and reagent B, respectively. The extracted genomes were digested by restriction endonuclease, homologous recombination of bacteria and transfection of cells to save virus. Results High quality adenovirus genomes were successfully obtained by several methods, which could be used in PCR, sequencing, enzyme digestion, homologous recombination and transfection experiments. Genome extraction can be completed within 1 hour. The RNA content of the virus extracted from Kit A was the highest (20 ~ 50 渭 g), and RNase (RNase) was not needed. However, the improved method of HirtCs needed to add RNase to the lytic solution or the final product to remove the RNA components of the cells. Conclusion both Kit A and Kit B can be used for rapid extraction of high quality adenovirus genomes instead of the traditional HirttCys extraction methods. The extracted genomes can be used for enzyme digestion typing and transfection.
【作者单位】： 呼吸疾病国家重点实验室广州医科大学附属第一医院;广东省东莞市儿科研究所广东省东莞市第八人民医院;
【基金】：国家自然科学基金资助项目(31570163) 广东省广州市科技计划项目(201504010032)
【分类号】：R373
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本文编号：2050135