摘要
【背景】猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)膜蛋白(M)在病毒粒子的组装、膜融合和病毒复制等方面具有重要的作用,但M蛋白与宿主细胞的互作机制尚不清楚。【目的】利用免疫沉淀技术和液质联用技术筛选细胞内与PEDVM蛋白相互作用的蛋白,为揭示M蛋白在病毒增殖过程中发挥的功能提供研究基础。【方法】将MOI=0.1的PEDV DR13疫苗株接种于长成单层的Vero细胞,感染36 h后,收集细胞并进行裂解。利用抗M的单克隆抗体沉淀与M相互作用蛋白复合物,通过液相色谱串联质谱(LC-MS/MS)进行鉴定并利用细胞功能富集分析(Gene ontology,GO)对感染组鉴定到的细胞蛋白进行分析,确定两个细胞内源性蛋白为候选蛋白,进行免疫共沉淀(Co-IP)验证和共定位分析。【结果】基于鉴定蛋白的肽段数的方法分析显示,感染组与对照组相比,鉴定了218个与M蛋白相互作用的细胞内源性蛋白,分别与蛋白质合成、代谢、细胞信号通路转导等密切相关,选择细胞分裂周期蛋白42 (Cell division cycle 42,CDC42)、真核翻译起始因子3亚基L蛋白(eIF3L)为候选蛋白进行Co-IP(Co-immunoprecipitation)验证和共定位分析,结果证实CDC42、eIF3L蛋白分别与M蛋白在细胞内存在相互作用。【结论】鉴定出PEDV M蛋白能够与宿主细胞CDC42和eIF3L蛋白相互作用,并鉴定出其他可能与M蛋白发生相互作用的宿主蛋白60个,为开展PEDV与宿主细胞蛋白相互作用研究提供了重要理论依据。
[Background] The membrane protein(M) of porcine epidemic diarrhea virus(PEDV) plays an important role in the viral assembly process, membrane fusion and viral replication, but the mechanism of interaction between M protein and host cells is still unclear.[Objective] Co-immunoprecipitation technique coupled with LC-MS/MS were used to screen cellular proteins interacting with PEDV M protein, which can provide a foundation for revealing the function of M in viral multiplication.[Methods]PEDV DR13 vaccine strain was inoculated into monolayer of Vero cells at a MOI of 0.1. After 36 hours of infection, the cells were collected and lysed. Host cellular proteins that interact with the M protein of PEDV were immunoprecipitated using the M monoclonal antibody, then identified by LC-MS/MS, and analyzed by gene ontology(GO) annotation. Among them, two interested cellular proteins were further confirmed with Co-IP and cellular colocalization.[Results] Based on the analysis of the number of peptide segments, 218 cellular proteins interacting with M protein were identified. These host cellular proteins are closely related to protein synthesis, metabolism and cell signaling pathway transduction. Cell division cycle 42(CDC42) and eukaryotic translation initiation factor 3 subunit L(eIF3 L) were chosen for reverse Co-IP reconfirmation and colocalization analysis. The results showed that both CDC42 and eIF3 L protein interact with M protein.[Conclusion] This study identified that PEDV M protein could interact with CDC42 and eIF3 L proteins in host cells, and identified 60 other host proteins that might interact with M protein. The study is to provide an important theoretical basis for the study of the interaction between PEDV and host cell proteins.
作者
王瑞阳
于瑞嵩
陈冰清
李凤平
谢春芳
司伏生
董世娟
李震
WANG Rui-Yang;YU Rui-Song;CHEN Bing-Qing;LI Feng-Ping;XIE Chun-Fang;SI Fu-Sheng;DONG Shi-Juan;LI Zhen(College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences,Shanghai 201106, China;Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;Shanghai Engineering Research Center of Breeding Pig, Shanghai 201106, China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2019年第6期1434-1442,共9页
Microbiology China
基金
国家自然科学基金(31602060)
上海市农业科学院学科领域建设专项(31602060-2016匹配)
国家重点研发计划(2016YFD0500101)
上海市科技兴农重点攻关项目(沪农科攻字(2015)第6-1-9号)~~
