猪源含缬酪肽蛋白(VCP)的真核表达及其活性鉴定  

Eukaryotic expression and activity identification of porcine valosin-containing protein(VCP)

作　　者：孙睿聪 胡家欢 李晓晗 陈婧[1] 周斌[1,2] SUN Ruicong;HU Jiahuan;LI Xiaohan;CHEN Jing;ZHOU Bin(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》2022年第8期66-72,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2021YFD1801300);企业横向课题(2020320122000022)。

摘　　要：含缬酪肽蛋白(VCP)作为细胞中的重要活性蛋白,参与多种细胞过程,也在许多病毒的感染过程中发挥重要作用。为了探究VCP对猪瘟病毒(CSFV)、日本脑炎病毒(JEV)和伪狂犬病病毒(PRV)复制的影响,试验扩增了猪源VCP的基因片段,并将其连接到pEGFP-C1载体中。经双酶切鉴定结合基因测序结果,成功构建真核表达重组载体pEGFP-VCP。通过间接免疫荧光(IFA)和蛋白免疫印迹(Western blot)证实,重组质粒EGFP-VCP能在猪肾细胞(PK-15)中成功表达。而在表达pEGFP-VCP的细胞中分别感染CSFV、JEV和PRV后,通过Western blot、实时荧光定量PCR(RT-qPCR)、半数细胞感染量(TCID_(50))以及IFA试验,检测到过表达VCP能够明显促进CSFV、JEV以及PRV病毒蛋白的表达,提高胞内病毒核酸的含量,并且使胞外病毒滴度增加,极大地促进了3种病毒在PK-15细胞中的复制,为深入研究VCP促进多种病毒感染宿主细胞的分子机制奠定了理论基础。Valosin-containing protein(VCP),as an important active protein in cells,is involved in a variety of cellular processes and also plays an important role in the infection process of many viruses.To investigate the effect of VCP on the replication of classical swine fever vi⁃rus(CSFV),Japanese encephalitis virus(JEV)and pseudorabies virus(PRV),we amplified a gene fragment of porcine-derived VCP and ligated it into a pEGFP-C1 vector.The eukaryotic expression recombinant vector pEGFP-VCP was successfully constructed by double diges⁃tion identification combined with the gene sequencing results.pEGFP-VCP was successfully expressed in PK-15 cells as confirmed by IFA and Western blot.And after the cells expressing pEGFP-VCP were infected with CSFV,JEV and PRV,respectively,the overexpression of VCP significantly promoted the expression of the viral proteins of CSFV,JEV and PRV and increased the viral nucleic acid content as detec⁃ted by Western blot,RT-qPCR,TCID_(50) and IFA,raised the intracellular viral nucleic acid content and the extracellular viral titer;which greatly promoted the replication of the three viruses in the PK-15 cells.The present results laid a theoretical foundation for in-depth research on the molecular mechanism of VCP in promoting the infection of host cells by various viruses.

关 键 词：含缬酪肽蛋白 猪瘟病毒 日本脑炎病毒 伪狂犬病毒 猪肾细胞 复制 

分 类 号：S852.65[农业科学—基础兽医学]