一种肠道病毒D68型感染性克隆的构建方法-北京疾病预防控制中心.PDFVIP

国际病毒学杂志 2018 年 4 月第 25 卷第 2 期　International Journal of Virology, April 2018, Vol. 25, No. 2 · 73 · ·论著· 一种肠道病毒 D68 型感染性克隆的构建方法 李凯琳　相子春 【摘要】 目的　构建肠道病毒 D68 型（EV-D68）cDNA 感染性克隆。 方法　利用聚合酶不 完全引物延伸（Polymerase Incomplete Primer Extension，PIPE）法分别扩增 EV-D68 基因组 cDNA 片 段与 pBR322 载体，得到的片段连接后测序，连接产物测序正确后作为载体与 EV-D68 的基因组的 第 2 段扩增连接。以此类推，边扩增边连接，并测序，直到全基因组全部连接到 pBR322 上，在 EV-D68 基因组前面插入 T7 启动子后，经体外转录后的 RNA 转染 RD 细胞，转染后显微镜下连续 观察是否出现细胞病变，并利用 EV-D68 的 VP1 特异性抗体进行免疫印迹确认。 结果　转染后 24 h 在显微镜下可以观察到典型的肠道病毒细胞病变，病变细胞裂解液的免疫印迹得到特异的 EV- D68 的 VP1 条带，这些结果显示包装出 EV-D68 病毒，这些包装的病毒经传代实验显示可以连续传 代。 结论　成功构建 EV-D68 cDNA 感染性克隆。 【关键词】 肠道病毒 D68 型；转染；RD 细胞；感染性克隆 基金项目： “艾滋病和病毒性肝炎等重大传染病防治”科技重大专项（2018Z； 国家自然科学基金 A construction method for full-length infectious cDNA clone of enterovirus D68 Li Kailin, Xiang Zichun. Institute of Pathogen Biology, Chinese Academy of Medical Sciences Peking Union Medical College, Beijing 100730, China Corresponding author: Xiang Zichun, Email: xiangzch@163.com Abstract Objective To develop the method for constructing a full-length infectious cDNA 【 】 clone of enterovirus D68 (EV-D68). Methods Using polymerase incomplete primer extension (PIPE) method, the first cDNA segment of the EV-D68 clinical strain and vector pBR322 were amplified and were linked together. After confirmation by sequencing, the correctly linked product was used as a new vector for the amplicon of the second cDNA segment. The process was repeated until all cDNA segments were cloned into vector pBR322. The T7 promoter was then inserted before the EV-D68 genome. After linearization and transcription in vitro, the synthesized RNA was transfected into RD cells. Cytopathic effect was continuously observed under microscope and was confirmed by western blot with specific antibody against VP1 of EV-D68. Results