脂滴相关蛋白Rab18对猪血凝性脑脊髓炎病毒复殖和转运的影响  

Effects of lipid droplet associated protein Rab18on replication and transport of porcine haemagglutinating encephalomyelitis virus

作　　者：韦寒露 母少倩 田轶涵 邱瑞召 陆慧君 兰云刚 李姿 高丰 贺文琦 WEI Hanlu;MU Shaoqian;TIAN Yihan;QIU Ruizhao;LU Huijun;LAN Yungang;LI Zi;GAO Feng;HE Wenqi(College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2024年第4期774-780,共7页Chinese Journal of Veterinary Science

基　　金：教育部重点实验室开放课题资助项目(JLAU2021003);吉林省科技厅农业重点科技攻关资助项目(20210202041NC);吉林省创新平台资助项目(20230508002RC)。

摘　　要：通过分子克隆技术、RNAi技术及共聚焦显微镜技术检测Rab18蛋白对猪血凝性脑脊髓炎病毒(porcine haemagglutinating encephalomyelitis virus,PHEV)复殖和转运的影响。PCR技术扩增目的基因Rab18,将其插入pCMVC-EGFP质粒中,构建野生型Rab18-EGFP过表达质粒;利用点突变盒子定点突变Rab18-EGFP,构建活化型Rab18和失活型Rab18过表达质粒。将成功构建的Rab18过表达质粒或者Rab18siRNA转染小鼠神经瘤母细胞(neuro-2a cells)后接种PHEV,通过Western blot和qPCR检测PHEV蛋白和基因表达。结果显示,活化型Rab18促进PHEV的增殖和释放,失活型Rab18抑制PHEV的增殖;敲低Rab18后PHEV增殖及病毒的胞外释放受到抑制;利用共聚焦显微技术观察到PHEV、Rab18和LDs在细胞质中有明显共定位,且PHEV感染后Rab18与脂肪-甘油三酯-脂肪酶(ATGL)的共定位增强。结果表明,Rab18通过脂质代谢中LDs形成相关途径调控PHEV复殖与转运。Molecular cloning,RNAi and confocal microscopy were used in this study to clarify the role of Rab18inthe proliferation and transport of PHEV.The target gene Rab18was amplified by PCR and inserted into pCMV-C-EGFP plasmid to construct wild-type Rab18-EGFP overexpression plasmid;activated Rab18(Rab18Q67L)and inactivated Rab18(Rab18S22N)overexpression plasmid were constructed by site-mutating RAB18-EGFP with mutation kit.The Rab18overexpression plasmids or Rab18siRNA was transfected into N2a cells and then inoculated with PHEV.The protein and gene expression of PHEV were detected by Western blot and qPCR,it was found that Rab18Q67L promoted the and secretion of extracellular of PHEV,while overexpression of Rab18S22Nsignificantly suppressed the replication of PHEV.In addition,knock-down of Rab18 significantly suppressed the replication and secretion of extracellular of PHEV.Furthermore,confocal fluorescence microscopy observed the colocalization of Rab18with LDs and PHEV,which was enhanced after PHEV infection.Take together these results demonstrate that Rab18regulates the replication and transport of PHEV through the pathway related to LDs formation in lipid metabolism,providing a theoretical basis to elucidate the intracellular transport mechanism in PHEV.

关 键 词：猪血凝性脑脊髓病毒 Rab18 脂滴 胞内运输 

分 类 号：S852.3[农业科学—基础兽医学]