猪流行性腹泻病毒Nsp8与宿主细胞互作蛋白的筛选及鉴定  

Screening and Identification of Host Proteins Interacting with Nsp8 of Porcine Epidemic Diarrhea Virus

作　　者：周书亭 丁思嘉[1] 凌悦 廖卓韵 王子泓 邹霭萱 罗渝宵 黄梓鑫 陈宇翔 袁聪俐[1] 朱建国[1] 崔立[1] 杨志彪[1] ZHOU Shuting;DING Sijia;LING Yue;LIAO Zhuoyun;WANG Zihong;ZOU Aixuan;LUO Yuxiao;HUANG Zixin;CHEN Yuxiang;YUAN Congli;ZHU Jianguo;CUI Li;YANG Zhibiao(Shanghai Key Laboratory of Veterinary Biotechnology,School of Agriculture and Biology,Shanghai Jiao Tbng University,Shanghai 200240,China)

机构地区：[1]上海交通大学农业与生物学院上海市兽医生物技术重点实验室,上海200240

出　　处：《中国动物传染病学报》2021年第1期1-8,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划(2016YFD0500100);国家自然科学基金(31472211);上海市科技兴农项目[沪农科推字(2017)第111号]。

摘　　要：猪流行性腹泻病毒Nsp8是RNA依赖的RNA聚合酶辅助因子,在病毒基因组复制中具有重要作用。为了从宿主细胞蛋白角度了解PEDV Nsp8蛋白的作用,本研究以PEDV感染的宿主细胞LLC-PK1为研究对象,提取LLC-PK1细胞总RNA,利用SMART技术经过反转录、扩增和纯化获得双链cDNAs,将其与pGADT7-Rec和carrier DNA共同转化至酵母Y187菌株,构建LLC-PK1 cDNA酵母文库,对该文库容量及质量进行鉴定。结果显示:本研究构建的LLC-PK1 cDNA文库库容量为2.1×10^7 CFU/mL,插入cDNA片段长度为500~2500 bp;将构建的诱饵质粒pGBKT7-Nsp8与LLC-PK1 cDNA酵母表达文库进行杂交,筛选阳性克隆菌株并测序分析,共获得了8个潜在互作蛋白;将8个蛋白分别与诱饵质粒pGBKT7-Nsp8进行酵母回复杂交验证,有4种宿主蛋白与Nsp8蛋白共转化后能在含X-α-Gal的DDO/X/A和QDO/X/A选择培养基中形成蓝色菌落;选择其中出现频率最高的LDHB蛋白与Nsp8,分别构建含有Flag标签和HA标签的真核表达载体,利用不同的标签抗体进行Co-IP鉴定,发现Nsp8与LDHB蛋白可以发生免疫共沉淀;通过激光共聚焦显微镜观察发现,Nsp8和LDHB蛋白在细胞质中存在明显共定位。本研究结果表明,Nsp8与宿主蛋白LDHB存在相互作用,推测LDHB蛋白可能通过与PEDV Nsp8蛋白在细胞质中的相互作用参与病毒复制的生物学过程。Nsp8 of Porcine epidemic diarrhea virus(PEDV)is a multifunctional protein,serving as an RNA primase for co-localization with RNA-dependent RNA polymerase(RdRp).To explore the interaction of host proteins with PEDV Nsp8 on viral replication,the host cell LLC-PK1 was used in the present study.Total RNA of LLC-PK1 was extracted and reverse-transcribed into the corresponding complementary DNA(cDNA)with SMART technology.LLC-PK1 cDNA library was then constructed by co-transfection of Y187 competent with PEDV Nsp8 on PEDV replication.Total RNA of LLC-PK1 was extracted and reverse-transcribed into the corresponding complementary DNA(cDNA)with SMART technology.LLC-PK1 cDNA library was constructed by co-transfected Y187 competent cells with pGADT7-Rec and carrier DNA.The results showed that the titer of the constructed LLC-PK1 cDNA library was 2.1×10^7 CFU/mLand the length of the inserted cDNA ranged from 500 to 2500 bp.The baited plasmid PGBKT7-NSP8 was hybridized with the cDNA yeast expression library of LLC-PK1 using yeast two-hybrid technology.The positive clones LDHB,SERTAD3,GMS2 and PSMB10 were verified by sequencing.The interaction of the obtained eight proteins and pGBKT7-Nsp8 were further investigated for clarify its role in viral replication.The results showed that four of these proteins co-transformed with Nsp8 and decomposed X-α-Gal in DDO/X/Aand QDO/X/A to form blue colonies.Meanwhile,the LDHB with the highest frequency was chosen to construct pCDNA4.0-NSP8-Flag and pCDNA3.1-LDHB-HA eukaryotic expression plasmids.Co-IP assay was conducted with antibodies against different tags.The results showed that Nsp8 co-immunoprecipitated with LDHB protein.Furthermore,LDHB co-localized with PEDV Nsp8 protein in the cytoplasms as observed using immunofluorescence assay.These results indicated that Nsp8 interacted with the host protein LDHB,therefore it was speculated LDHB protein might participate in the virus replication by interacting with PEDV Nsp8.

关 键 词：猪流行性腹泻病毒 Nsp8 CDNA文库 蛋白相互作用 

分 类 号：S852.659.6[农业科学—基础兽医学]