人肠道病毒D68型微复制子的建立  

Construction of a Minireplicon System for EV-D68

作　　者：潘明磊 高帅 成金燕 徐亚楠 李显煌 段宇琴 王涛 PAN Minglei;GAO Shuai;CHENG Jinyan;XU Yanan;LI Xianhuang;DUAN Yuqin;WANG Tao;(School of Life Sciences,Tianjin University)

机构地区：[1]天津大学生命科学学院

出　　处：《病毒学报》2018年第2期153-158,共6页Chinese Journal of Virology

基　　金：国家重点研发计划(项目号:2017YFA0205102);题目:病毒特异性分子靶标的筛选与纳米标记;天津大学自主基金(项目号:2014XRX-0026);题目:病毒与免疫~~

摘　　要：建立人肠道病毒D68型（EV-D68）微复制子体系。利用增强绿色荧光蛋白（EGFP）或萤火虫荧光素酶（FLuc）报告基因替换病毒编码区,通过酶切连接,构建EV-D68 T7和PolⅠ系统微复制子体系,获得重组质粒pT7-miniEGFP、pT7-miniFLuc、pHH21-miniEGFP、pHH21-miniFLuc和pHH21-miniFLucΔpolyC。微复制子转染RD细胞,48h后荧光显微镜观察EGFP表达情况或用双报告系统检测荧光素酶表达水平。T7和PolⅠ系统微复制子均可表达报告基因,但PolⅠ系统荧光素酶表达水平是T7系统的5倍。并且病毒非编码区的PolyC区域对于病毒蛋白的表达起着重要的作用。本研究成功构建了EV-D68微复制子体系,为EV-D68非编码区功能研究提供工具。Human enterovirus D68（EV-D68）infection can cause diseases in the respiratory and central nervous systems.EV-D68 outbreaks worldwide have been documented in recent years.In the present study,an EV-D68 minireplicon system was established.EGFP or luciferase reporter gene was used to replace the virus coding region,and the T7 and Pol I minireplicon system was constructed.The minireplicon was transfected into RD cells.After 48 h,EGFP expression was observed by fluorescence microscopy,and luciferase expression was detected by dual-luciferase reporter assays.Both the T7 and Pol I minireplicon system could express the reporter genes,whereas the Fluc expression in the Pol I system was approximately fivefold that of the T7 system.Also,the PolyC sequence in the untranslated region played an important part in the expression of viral proteins.These data suggest that the EV-D68 minireplicon system was successfully constructed and could be used to study the function of the untranslated region of EV-D68.

关 键 词：人肠道病毒D68型(EV-D68) 反向遗传学 微复制子 RNA聚合酶Ⅰ 报告基因 

分 类 号：R373.2[医药卫生—病原生物学]