内质网分子伴侣GRP94对伪狂犬病毒增殖的调节作用  

Regulation of endoplasmic reticulum molecular chaperone GRP94 on Pseudorabies virus replication

作　　者：倪敏舒 陈丽[2,3,4] 鲍熹 徐悦[2,3,4] 庄腾寒 冯磊[1,2,3,4] NI Minshu;CHEN Li;BAO Xi;XU Yue;ZHUANG Tenghan;FENG Lei(School of Pharmacy,Jiangsu University,Zhenjiang 212013,Jiangsu,China;Institute of Veterinary Immunology&Engineering,National Research Center of Engineering and Technology for Veterinary Biologicals,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,Jiangsu,China;Jiangsu Key Laboratory of Food Quality and Safety,State Key Laboratory Cultivation Base Jointly Built by Provinces and Ministries,Nanjing 210014,China)

机构地区：[1]江苏大学药学院,江苏镇江212013 [2]江苏省农业科学院动物免疫工程研究所,国家兽用生物制品工程技术研究中心,江苏南京210014 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [4]江苏省食品质量安全重点实验室省部共建国家重点实验室培育基地,江苏南京210014

出　　处：《浙江农业学报》2022年第11期2386-2394,共9页Acta Agriculturae Zhejiangensis

基　　金：江苏省重点研发计划(SBE2022780057)。

摘　　要：为探究葡萄糖调节蛋白94(GRP94)对伪狂犬病毒(Pseudorabies virus,PRV)增殖的影响,以及GRP94与PRV结构蛋白之间的相互作用。利用慢病毒转染构建GRP 94基因过表达的BHK-21细胞,以及CRISPR/Cas9技术构建GRP 94基因敲除的BHK-21细胞,检测PRV感染不同细胞的增殖水平,比较内质网应激相关蛋白表达水平,探索GRP94与PRV结构蛋白之间的互作关系。结果表明,GRP 94基因过表达细胞株显著有利于PRV增殖,而GRP 94基因缺失细胞株对PRV增殖无显著影响。蛋白免疫印迹结果显示,在GRP 94基因缺失细胞株中,GRP78表达水平显著上调。免疫共沉淀结果表明,GRP94与gB、gD、gL具有相互作用。GRP 94基因过表达有利于PRV增殖,检测未折叠蛋白反应(unfolded protein response,UPR)相关蛋白,研究GRP94在病毒增殖过程中的具体作用,可为PRV与内质网应激提供分子方面的研究基础,于对抗PRV病毒的感染具有重要意义。To explore the effect of glucose-regulated protein 94(GRP94)on Pseudorabies virus(PRV)replication and interaction between GRP94 and PRV structural proteins.BHK-21 cells with GRP 94 overexpression were constructed by recombinant lentiviruses transfection and BHK-21 cells with GRP94 coding sequences knockout were constructed by CRISPR/Cas9 system.Progeny PRV titers were measured to compare the changes of virus proliferation efficiency in different cell lines.Western blot was performed to detect the expression level of unfolded protein response(UPR)related proteins in different cell lines.The interaction between virus structural proteins and GRP94 was determined by immunoprecipitation analysis.PRV replication was significantly increased in BHK-21-GRP94 cells,while no significant effect on PRV replication was occurred in BHK-21-GRP94-KO cells.The expression level of GRP78 was significantly up-regulated in BHK-21-GRP94-KO cells and interactions between GRP94 and gB,gD,gI were detected by Co-IP.The overexpression of GRP 94 was conducive to the proliferation of PRV.By analyzing UPR related proteins,it was of great significance to study the specific role of GRP94 in the process of virus replication,so as to provide a molecular research basis for PRV and ER stress.

关 键 词：葡萄糖调节蛋白94 葡萄糖调节蛋白78 伪狂犬病毒 蛋白互作 

分 类 号：S859.1[农业科学—临床兽医学]