一种肠道病毒D68型感染性克隆的构建方法

A construction method for full-length infectious cDNA clone of enterovirus D68

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 PrimerExtension，PIPE）法分别扩增EV—D68基因组eDNA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24h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—D68eDNA感染性克隆。

Objective To develop the method for Constructing a full-length infectious eDNA clone of enterovirus D68 （EV-D68）. Methods Using polymerase incomplete primer extension （PIPE） method, the first eDNA 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 eDNA segment. The process was repeated until all eDNA 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 ILD cells. Cytopathie effect was continuously observed under microscope and was confirmed by western blot with specific antibody against VP1 of EV-D68. Results Typical CPE ofenterovirus was observed at 24h after transfeetion. Western blot analysis showed specific VP1 band of EV-D68. The packaged viruses have been shown to pass through in succession in serial culture experiments. Conclusions The full-length infectious eDNA clone was constructed successfully.