新型冠状病毒水貂变异株重组水泡性口炎病毒载体假病毒的拯救及鉴定  

Rescue and identification of a recombinant vesicular stomatitis virus-vectored pseudovirus of SARS-CoV-2 mink variant

作　　者：王申 王玮琦 闫飞虎 冯娜[1] 赵永坤[1] 王铁成 高玉伟 杨松涛 夏咸柱[1,2] WANG Shen;WANG Weiqi;YAN Feihu;FENG Na;ZHAO Yongkun;WANG Tiecheng;GAO Yuwei;YANG Songtao;XIA Xianzhu(Jilin Key Laboratory of Zoonosis Prevention and Control,Institute of Changchun Veterinary Medicine,Chinese Academy of Agricultural Sciences,Changchun 130122,China;Key Laboratory of Zoonosis Research,Ministry of Education,College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]中国农业科学院长春兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [2]吉林大学动物医学学院人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《中国兽医学报》2022年第4期668-674,共7页Chinese Journal of Veterinary Science

基　　金：解放军总后勤部卫生部科研基金资助项目(YT230)。

摘　　要：为拯救新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)水貂变异株重组水泡性口炎病毒(vesicular stomatitis virus,VSV)载体假病毒,并对该假病毒进行鉴定。基于本实验室构建的VSV反向遗传系统,使用SARS-CoV-2的刺突蛋白(S)基因替换全长cDNA克隆P3.1-VSV-eGFP中的VSV糖蛋白(G)基因,得到重组cDNA克隆P3.1-VSV△G-S-eGFP。将P3.1-VSV△G-S-eGFP与分别能表达VSV核蛋白N、磷蛋白P、聚合酶L及G的4个辅助质粒共转染细胞以拯救携带绿色荧光标签的重组假病毒VSV△G-S-eGFP,并通过荧光显微镜观察;生长动力学曲线检测;RT-PCR鉴定;Western blot鉴定及透射电镜观察对重组假病毒进行鉴定。经鉴定重组假病毒VSV△G-S-eGFP拯救成功并可在细胞中有效增殖;VSV△G-S-eGFP的最高生长滴度可达10^(6.8)TCID_(50)/mL。本研究拯救的重组假病毒VSV△G-S-eGFP为SARS-CoV-2水貂变异株抗体筛选、疫苗评估提供了有力工具。To rescue and identify SARS-CoV-2 mink variant recombinant vesicular stomatitis virus-vectored pseudovirus.Based on the established VSV reverse genetic system,the VSV glycoprotein(G)gene of full-length cDNA clone(P3.1-VSV-eGFP)was replaced by SARS-CoV-2 spike(S)gene,the recombinant cDNA clone was named P3.1-VSV△G-S-eGFP.P3.1-VSV△G-S-eGFP was cotransfected with four support plasmids expressing VSV nucleoprotein N,phosphoprotein P,polymerase L and G respectively to rescue the recombinant pseudovirus carrying green fluorescent tag(VSV△G-S-eGFP).Then the recombinant pseudovirus was identified by fluorescence microscope,growth kinetic curve,RT-PCR,Western blot and transmission electron microscope.According to the above identification,the recombinant pseudovirus was rescued successfully and effectively replicated in cells;the growth titer of VSV△G-S-eGFP was up to 10^(6.8)TCID_(50)/mL.VSV△G-S-eGFP provides a powerful tool for antibody screening and vaccine evaluation of SARS-CoV-2 mink variant.

关 键 词：新型冠状病毒 水貂变异株 水泡性口炎病毒 假病毒 

分 类 号：S852.65[农业科学—基础兽医学]