宿主细胞敲除G3BP2基因对塞内卡病毒复制的影响  

Effect of G3BP2 gene knockout on Seneca Valley virus replication in the cells

作　　者：郭承意 董钰蓓 温家成 延君芳 高雁怩 姜平 白娟[1,2] GUO Chengyi;DONG Yubei;WEN Jiacheng;YAN Junfang;GAO Yanni;JIANG Ping;BAI Juan(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Key Laboratory of Animal Bacteriology,Ministry of Agriculture and Rural Affairs,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]农业农村部动物细菌学重点实验室,江苏南京210095

出　　处：《畜牧与兽医》2025年第1期91-97,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划项目(2022YFD1800800);现代农业产业技术体系专项(CARS-35)。

摘　　要：利用CRISPR/Cas9基因编辑技术构建Ras GTP酶激活蛋白SH3结构域结合蛋白2(G3BP2)基因敲除的293T细胞系,并初步探讨了G3BP2基因缺失对于塞内卡病毒A(Senecavirus A,SVA)增殖的影响。采用激光共聚焦试验观察病毒感染是否引起应激颗粒的产生;设计、构建靶向G3BP2基因的px459-sgRNA表达载体并转染进293T细胞;通过嘌呤霉素压力筛选以及亚克隆得到细胞株,经过Western blot、基因测序的方式鉴定细胞株;CCK-8法测定细胞增殖速度;Western blot、RT-qPCR、噬斑试验分别检测SVA感染后病毒蛋白VP1的表达水平、VP1的拷贝数和细胞上清液的病毒滴度;设计并构建SVA核糖体进入位点(IRES)的双荧光素酶报告系统,双荧光素酶报告试验测定病毒启动子的启动活性。结果:病毒感染引起应激颗粒的产生;筛选得到1株G3BP2基因缺失10 bp的293T细胞系(HEK 293T G3BP2^(-/-)),其细胞活力与未敲除细胞相比无显著差异;Western blot、RT-qPCR、噬斑试验均显示G3BP2敲除后显著抑制了病毒的增殖;双荧光素酶报告试验表明G3BP2敲除后下调了病毒的翻译启动活性。综上,本研究获得了1株HEK 293T G3BP2^(-/-)细胞系,该细胞通过下调病毒的启动活性抑制病毒的增殖,为进一步探讨G3BP2对SVA复制的影响奠定基础。In This study,we used the CRISPR/CAS9 gene editing technology to construct a G3BP2 gene knockout 293T cell line and ini-tially explored the impact of G3BP2 gene deletion on the proliferation of Senecavirus A(SVA).Laser confocal microscopy was employed to see whether virus infection induced the formation of stress granules.The px459-sgRNA expression vector targeting the G3BP2 gene was de-signed and constructed,and transfected into 293T cells.Cell lines were obtained through puromycin pressure screening and subcloning,and were identified by western blot and gene sequencing.Cell proliferation rates were determined using the CCK-8 assay.Western blot,RT-qPCR,and plaque assay were used to detect the expression level of viral protein VP1,the copy number of VP1,and the virus titer in the cell supernatant after SVA infection.A dual luciferase reporter system targeting the SVA IRES was designed and constructed,and the promoter activity of the virus was measured using a dual luciferasereporter assay.The results showed that virus infection induced the formation of stress granules.A G3BP2 gene knockout 293T cell line(HEK 293T G3BP2^(-/-))with a 10 bp deletion was obtained,and there was no significant difference in cell viability,compared with non-knockout cells.Western blot,RT-qPCR,and plaque assay all demonstrated that G3BP2 knockout significantly inhibited virus proliferation.The dual luciferase reporter assay indicated that G3BP2 knockout downregulated virus pro-moter activity.In summary,this study obtained a HEK 293T G3BP2^(-/-)cell line that inhibits virus proliferation by downregulating virus pro-moter activity,which laid a foundation for further exploring the impact of G3BP2 on SVA replication.

关 键 词：G3BP2基因 基因敲除 塞内卡病毒A 复制 

分 类 号：S852[农业科学—基础兽医学]