肠道病毒71型EGFP标记重组病毒的构建与拯救  

Construction and rescue of EGFP-enterovirus type 71 recombinant virus

作　　者：张海陆 宋绍霞[2] 王锴 王欣 李书晗 赵丽 王志玉 温红玲 Zhang Hailu;Song Shaoxia;Wang Kai;Wang Xin;Li Shuhan;Zhao Li;Wang Zhiyu;Wen Hongling(Department of Microbiological Laboratory Technology,School of Public Health,Cheeloo College of Medicine,Key Laboratory of Infectious Disease Control and Prevention in Universities of Shandong,Shandong University,Jinan 250012,China;Shandong Center for Disease Control and Prevention,Shandong Provincial Key Laboratory of Infectious Disease Prevention and Control,Jinan 250012,China)

机构地区：[1]山东大学齐鲁医学院公共卫生学院微生物检验学系,山东省"十三五"高等学校感染性疾病防控重点实验室,济南250012 [2]山东省疾病预防控制中心,山东省传染病预防控制重点实验室,济南250012

出　　处：《中华实验和临床病毒学杂志》2020年第5期511-515,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：山东省自然科学基金面上项目(ZR2018MH035);山东省重点研发计划(2019GSF107067)。

摘　　要：目的构建并拯救增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)标记的EGFP-EV-A71重组病毒。方法利用重叠PCR技术,以pMD19T-SDLY107全长质粒为模板,将2A蛋白酶识别序列加入到结构蛋白VP4的5′端;以pEGFP-N1质粒作为模板,扩增获得EGFP目的片段,将其插入到上述重组质粒中,得到重组质粒pMD19T-107-EGFP。经酶切和体外转录后,转染横纹肌细胞肉瘤(RD),拯救获得重组病毒EGFP-EV-A71。采用Karber法计算细胞培养半数感染量(CCID50)以测定病毒滴度。利用实时荧光定量PCR(qRT-PCR)检测不同时间点的病毒复制水平,并绘制病毒复制曲线,比较重组病毒与母本病毒的复制力有无差异。乳酸脱氢酶(LDH)和细胞增殖(CCK-8)实验分别测定被感染细胞的细胞损伤率和存活率。结果包含EGFP的重组EV-A71感染性cDNA克隆被成功构建;转染RD细胞后,出现典型的细胞病变并且表达出较强的绿色荧光;重组病毒与母本病毒具有相似的复制动力学特征和致细胞病变效应。结论成功拯救了EGFP标记的重组病毒EGFP-EV-A71。Objective To construct and rescue enhanced green fluorescent protein(EGFP)-labeled EGFP-EV-A71 recombinant virus.Methods pMD19T-SDLY107 full-length plasmid was used as a template,and 2A protease recognition sequence was added to the 5′end of the structural protein VP4.EGFP gene was amplified using the pEGFP-N1 plasmid as a template and inserted into the above-mentioned recombinant plasmid.RD cells were transfected to rescue the recombinant virus EGFP-EV-A71 after enzymatic digestion and in vitro transcription.The cell culture infectious dose 50%endpoint(CCID50)was determined.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the virus replication level at different time points,and the virus replication curve was drawn to compare the replication ability of the recombinant virus and the parent virus.Lactate dehydrogenase(LDH)and cell proliferation(CCK-8)experiments were used to determine the cell injury rate and survival rate of infected cells,respectively.Results Recombinant EV-A71 infectious cDNA clones containing specific restriction sites and EGFP were successfully constructed.Typical cytopathic effect and green fluorescence was observed.The qRT-PCR replication curve showed that the recombinant virus had similar replication kinetics to the parent virus.Conclusions EGFP-labeled enterovirus type 71 recombinant virus EGFP-EV-A71 was successfully rescued.

关 键 词：肠道病毒71型 增强型绿色荧光蛋白 病毒拯救 

分 类 号：R373.2[医药卫生—病原生物学]