猪流行性腹泻病毒非结构蛋白Nsp15互作宿主蛋白的筛选及鉴定  

Screening and identification of host proteins interacting with the non-structural protein 15(Nsp15)of porcine epidemic diarrhea virus

作　　者：孙金磊 于瑞明 张莉萍 张中旺[2] 王永录[2] 潘丽[2] 张全伟[1] 刘新生[2] SUN Jinlei;YU Ruiming;ZHANG Liping;ZHANG Zhongwang;WANG Yonglu;PAN Li;ZHANG Quanwei;LIU Xinsheng(College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,Gansu,China;National Key Laboratory of Animal Disease Prevention and Control,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,Gansu,China)

机构地区：[1]甘肃农业大学生命科学技术学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室,甘肃兰州730000

出　　处：《生物工程学报》2024年第12期4533-4545,共13页Chinese Journal of Biotechnology

基　　金：国家生猪技术创新中心(NCTIP-XD/C 03);所级基本科研业务费项目(1610312021011)。

摘　　要：为了筛选和鉴定与猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)非结构蛋白15(non-structural protein 15,Nsp15)互作的关键宿主蛋白,通过IP/pull-down beads结合质谱技术筛选、鉴定与PEDV非结构蛋白Nsp15互作的宿主蛋白,并利用免疫共沉淀(co-immunoprecipitation)、激光共聚焦验证其互作关系,最后应用Western blotting和实时荧光定量多聚核苷酸链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)初步明确宿主蛋白SLC25a3与PEDV之间的作用关系。本研究成功构建了Nsp15重组真核表达载体pcDNA3.1(+)-Flag-Nsp15,并筛选出与PEDVNsp15互作的宿主蛋白SLC25a3。免疫共沉淀及激光共聚焦结果显示PEDVNsp15与宿主蛋白SLC25a3存在互作关系。宿主蛋白SLC25a3能够显著抑制PEDV复制且呈现剂量依赖性。本研究为进一步探究SLC25a3在抗PEDV免疫应答中的作用及机制提供了参考。To screen and identify the key host proteins interacting with the non-structural protein 15(Nsp15)of porcine epidemic diarrhea virus(PEDV).The IP/pull-down assay and mass spectrometry were employed to screen and identify the host proteins interacting with Nsp15.The interaction between the host protein and Nsp15 was studied by co-immunoprecipitation and laser scanning confocal microscopy.Finally,Western blotting and RT-qPCR were employed to examine the interaction between SLC25a3 and PEDV.The recombinant eukaryotic expression vector pcDNA3.1(+)-Flag-Nsp15 was successfully constructed,and the host protein SLC25a3 interacting with PEDV Nsp15 was screened out.An interaction existed between SLC25a3 and Nsp15,and SLC25a3 significantly inhibited PEDV replication in a dose-dependent manner.SLC25a3 inhibits PEDV replication.The results of this study provide a basis for deciphering the role and mechanism of SLC25a3 in the host immune response to PEDV infection.

关 键 词：猪流行性腹泻 非结构蛋白 蛋白互作 病毒复制 

分 类 号：S852.651[农业科学—基础兽医学]