摘要
本研究旨在探究TRIM7对口蹄疫病毒(FMDV)复制的影响。首先,针对猪源TRIM7基因外显子设计两条单向导RNA(single guide RNA,sgRNA),分别构建至载体PX459中,将成功构建的sgRNA重组质粒转染PK-15细胞,经嘌呤霉素筛选、有限稀释法挑取单克隆细胞,经测序及Western-blot分析鉴定TRIM7基因的敲除情况,获得敲除TRIM7基因的PK-15细胞系。将TRIM7基因敲除细胞和对照细胞中分别感染FMDV,用Western-blot、RT-qPCR和TCID50测定等方法综合评价敲除TRIM7基因后对FMDV复制的影响。同时,在PK-15细胞上过表达TRIM7检测其对病毒复制的影响。结果显示,成功构建TRIM7基因敲除PK-15细胞系,且FMDV在TRIM7基因敲除细胞系中的复制水平均显著低于在对照细胞中的复制水平,当过表达TRIM7时则会显著促进病毒的复制,表明TRIM7影响了FMDV的复制。本研究为深入探索TRIM7的生物学功能及其调控病毒复制的分子机制奠定了基础。
This study aimed to explore the effect of TRIM7 on the replication of foot-and-mouth disease virus(FMDV).Two single guide RNAs(sgRNAs)targeting the exons of porcine TRIM7 gene were designed and were respectively constructed into the vector PX459.The successfully constructed sgRNA recombinant plasmids were transfected into PK-15 cells.Monoclonal cells were selected by puromycin screening and the limited dilution method.The PK-15 cell lines with the knockout of the TRIM7 gene were identified by sequencing and Western-blot analysis.Subsequently,the TRIM7 gene-knockout cells and control cells were infected with FMDV,respectively.The effect of the knockout of the TRIM7 gene on the replication of FMDV was comprehensively evaluated by Western-blot,RT-qPCR,and TCID50 determination.Meanwhile,TRIM7 was overexpressed in PK-15 cells to detect its effect on virus replication.The results showed that the PK-15 cell line with the knockout of the TRIM7 gene was successfully constructed,and the replication level of FMDV in the TRIM7 gene-knockout celi line was significantly lower than that in the control cells.When TRIM7 was overexpressed,it significantly promoted the replication of the virus,indicating that TRIM7 affected the replication of FMDv.This study laid the foundation for further exploring the biological functions of TRIM7 and the molecular mechanisms of its regulation of viral replication.
作者
刘泓屹
张伟
蔡建涛
古涛鸣
王良锋
陈治彤
曹伟军
郑海学
杨帆
张桂红
LIU Hongyi;ZHANG Wei;CAI Jiantao;GU Taoming;WANG Liangfeng;HEN Zhitong;CAO Weijun;ZHENG Haixue;YANG Fan;ZHANG Guihong(Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control,College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;National Foot-and-Mouth Disease Reference Laboratory/State Key Laboratory for A nimal Disease Control and Prevention,College of Veterinary Medicine of Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of A gricultural Sciences,Lanzhou 730046,China;Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China)
出处
《中国兽医科学》
北大核心
2025年第7期870-876,共7页
Chinese Veterinary Science
基金
国家自然科学基金项目(32072831,32102639)
国家重点研发计划项目(2021YFD1800300)
甘肃省杰出青年基金项目(21JR7RA026)
中央高校基本科研业务费专项(lzujbky-2022-ey20)
甘肃省科技重大专项(22ZD6NA001)
