表达马尔堡病毒囊膜糖蛋白重组水泡性口炎病毒的构建  

Construction of recombinant vesicular stomatitis virus expressing Marburg virus envelope glycoprotein

作　　者：梁博 王申 王琪 王玮琦[1,3] 贺文文 李政蓉 冯娜 赵永坤[1] 王铁成[1] 闫飞虎 杨松涛 夏咸柱 LIANG Bo;WANG Shen;WANG Qi;WANG Wei-qi;HE Wen-wen;LI Zheng-rong;FENG Na;ZHAO Yong-kun;WANG Tie-cheng;YAN Fei-hu;YANG Song-tao;XIA Xian-zhu(Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China;College of Animal Science and Technology,Shihezi University,Shihezi 832099,China;College of Veterinary Medicine,Jilin University,Changchun 130062,China;The Second Affiliated Clinical Hospital,Hengyang Medical College,University of South China,Hengyang 421001,China)

机构地区：[1]中国农业科学院长春兽医研究所,吉林长春130122 [2]石河子大学动物科技学院,新疆石河子832099 [3]吉林大学动物医学学院,吉林长春130062 [4]南华大学衡阳医学院附属第二临床医院,湖南衡阳421001

出　　处：《中国兽医科学》2022年第11期1397-1404,共8页Chinese Veterinary Science

基　　金：国家重点研发计划青年科学家项目(2021YFF0703600)。

摘　　要：为了构建携带e GFP标签并表达马尔堡病毒(Marburg virus,MARV)囊膜糖蛋白(GP)的复制型水泡性口炎病毒(Vesicular stomatitis v,iVrSuVs)假型病毒,通过将VSV感染性克隆全长质粒中的G基因替换成马尔堡病毒的GP基因,与4个辅助质粒p3.1-VSV-N、p3.1-VSV-P、p3.1-VSV-L、p3.1-VSV-G共转染BSR细胞,拯救成功的重组VSV病毒传5代后,进行Western-blot、间接免疫荧光试验、RT-PCR鉴定和生长动力学曲线检测。显微镜检测结果显示,3个重组全长质粒分别与4个辅助质粒共转染BSR细胞培养48 h后均能使细胞发生合胞体病变并伴有绿色荧光蛋白表达;Western-blot、间接免疫荧光试验鉴定结果显示,3株马尔堡病毒的囊膜糖蛋白在重组VSV病毒中正确表达;生长动力学曲线显示,3株重组VSV病毒的毒力相较于母本VSV病毒均降低,但病毒滴度依旧能达到1×10^(6)~1×10^(7)TCID_(50)mL^(-1)。结论:本研究成功构建3株携带e GFP标签表达MARV GP的复制型VSV假型病毒,为后续MARV疫苗与抗体的效果评价等奠定了基础。To construct replication-competent vesicular stomatit(iVsSVvi)r pusseudotyped viruses expressing Marburg virus(MARV)glycoprotein(GP),the G gene in the full-length plasmid of the VSV cloning vector was replaced with the GP gene of Marburg vi,raunsd the recombinant full-length plasmid and four helper plasmids p3.1-VSV-N,p3.1-VSV-P,p3.1-VSV-L,p3.1-VSV-G were co-transfected into BSR cells to rescue the recombinant VSV virus,and then the recombinant virus were identified by Wester,ni-nbdliortect immunofluoresce,nce RT-PCR,and growth kinetic curve detection after 5 passages.The microscope results indicated that BSR cells co-transfected with the recombinant full-length plasmid and four helper plasmids exhibited syncytial lesions w green fluorescence in 48 h.Western-blot and indirect immunofluorescence indicated that the MARV GP was correctly expressed in the recombinant VSV virus.The growth kinetic curve showed that the titer of the recombinant virus was lower than that of the parent,vwihriucsh could reach from1×10^(6)to 1×10^(7)TCID_(50)mL^(-1)3 days post infection.In conclus,itohnree replicating VSV pseudotyped viruses carrying e GFP and expressing MARV GP were successfully construc,twehdich laid a foundation for the subsequent evaluation of the effects of MARV vaccines and antibodies.

关 键 词：马尔堡病毒 水泡性口炎病毒 假型病毒 

分 类 号：S852.659.5[农业科学—基础兽医学]