表达狂犬病病毒G蛋白嵌合型重组水疱性口炎病毒的构建及鉴定

Construction and verification of recombinant chimeric vesicular stomatitis virus expressing rabies virus G protein

目的以水疱性口炎病毒(vesicular stomatitis virus,VSV)为载体,构建表达狂犬病病毒(rabies virus,RV)G蛋白的嵌合型重组VSV。方法将VSV骨架质粒G基因替换为狂犬病疫苗CTN-1株G基因,与分别表达T7聚合酶及VSV N、P、L蛋白的辅助质粒共转染293T细胞,获得重组病毒。通过RT-PCR、免疫荧光法和Western blot检测RV G基因和G蛋白表达。将重组病毒在Vero细胞上传代培养,检测不同代次病毒滴度并绘制病毒生长曲线。结果成功获得了重组病毒VSV-RVG,RT-PCR结果表明重组病毒基因组中成功插入了RV G基因,免疫荧光法和Western blot可检测到RV G蛋白的表达。重组病毒连续传代5次,病毒滴度稳定在7.5~8.5 lgTCID_(50)/mL之间。结论成功构建了表达RV G蛋白的嵌合型重组VSV,重组病毒具有良好的遗传稳定性,为以VSV为载体的反向遗传学技术平台的构建奠定了基础。

Objective To construct recombinant chimeric vesicular stomatitis virus(VSV)expressing G protein of rabies virus(RV)using VSV as vector.Methods To rescue the recombinant virus,G gene of VSV antigenome was replaced with G gene of RV vaccine CTN-1 strain,and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and proteins N,P and L of VSV.The expression of RV G gene and G protein was detected by RT-PCR,immunofluorescence assay and Western blot.The recombinant virus was subcultured in Vero cells,the virus titer of different generations was detected and the virus growth curve was drawn.Results The recombinant virus VSV-RVG was successfully rescued.RTPCR results demonstrated that the RV G gene was successfully inserted into the genome of the recombinant virus,and the expression of RVG protein was detected by immunofluorescence assay and Western blot.The recombinant virus was continuously passaged for 5 generations,and the virus titer was stable within 7.5~8.5 lgTCID_(50)/mL.Conclusion The recombinant chimeric VSV expressing RV G protein was successfully constructed with good genetic stability,which lays a foundation of the construction of reverse genetics technology platform based on VSV vector.