摘要
构建促红细胞生成素肝细胞受体A2(Erythropoietin-producing hepatocellular receptor A2,EphA2)基因敲除和过表达的BHK-21细胞系,探究其对口蹄疫病毒(FMDV)复制的影响。通过CRISPR/Cas9基因编辑技术构建Eph A2基因敲除的BHK-21细胞系,利用慢病毒感染构建Eph A2基因过表达的BHK-21细胞系。经嘌呤霉素筛选及单克隆挑取,成功获得稳定细胞系。随后,以野生型BHK-21细胞为对照,将FMDV分别感染上述细胞系,采用实时荧光定量PCR、蛋白质免疫印迹等方法分别检测病毒核酸和蛋白水平。结果表明,Eph A2基因敲除后,FMDV在BHK-21细胞中的复制显著受到抑制,病毒核酸和蛋白表达量明显降低;而Eph A2基因过表达时,FMDV复制增强。本研究揭示了Eph A2基因在FMDV复制过程中具有重要作用,为深入理解FMDV的感染机制及防控策略提供了理论依据。
The aim of this study was to construct erythropoietin-producing hepatocellular receptor A2(EphA2)gene knockout and overexpression BHK-21 cell lines to investigate their effects on foot-and-mouth disease virus(FMDV)replication.The EphA2 gene knockoout BHK-21 cell line was constructed by CRISPR/Cas9 gene editing technology,and the EphA2 gene overexpression BHK-21 cell line was constructed using lentivirus infection.After puromycin screening and monoclonal picking,stable cell lines were successfully obtained.Subsequently,wild-type BHK-21 cells were used as the control,and FMDV was used to infect the above cell lines respectively,and the viral nucleic acid and protein levels were detected by real-time fluorescence quantitative PCR and immunoblotting respectively.The results showed that the replication of FMDV in BHK-21 cells was significantly inhibited by EphA2 knockout,and the expression of viral nucleic acids and proteins was significantly reduced;while FMDV replication was enhanced when EphA2 gene was overexpressed.This study reveals that EphA2 gene has an important role in the replication process of FMDV,which provides a theoretical basis for an in-depth understanding of the infection mechanism and prevention and control strategies of FMDV.
作者
李熙忠
范许许
朱兆宇
裴丹诗
王一卓
何路
任青峰
郑海学
李伟伟
郜原
LI Xizhong;FAN Xuxu;ZHU Zhaoyu;PEI Danshi;WANG Yizhuo;HE Lu;REN Qingfeng;ZHENG Haixue;LI Weiwei;GAO Yuan(College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory for Animal Disease Control and Prevention/College of Veterinary Medicine/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)
出处
《中国兽医科学》
北大核心
2025年第10期1291-1298,共8页
Chinese Veterinary Science
基金
国家自然科学基金青年项目(32202781,32302857)
国家自然科学基金重点项目(32330107)
国家重点研发计划项目(2021YFD1800300)
中国博士后科学基金项目(2023M743830)
国家生猪产业技术体系项目(CARS-35,CARS-39-13)
国家生猪技术创新中心先导科技项目(NCTIP-XD/C03)
中国农业科学院创新计划项目(CAASCSLPDCP-2023002,CAAS-ASTIP-2023-LVRI)
“十四五”广东省农业科技创新十大主攻方向“揭榜挂帅”项目(2024KJ14)
兰州兽医研究所基本科研业务费项目(161031202-1009)。
