科微学术
微生物学通报
2025年4月21日 周一
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)
WANG Rui-Yang
1 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;2 Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;3 Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;4 Shanghai Engineering Research Center of Breeding Pig, Shanghai 201106, ChinaYU Rui-Song
2 Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;3 Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;4 Shanghai Engineering Research Center of Breeding Pig, Shanghai 201106, ChinaCHEN Bing-Qing
2 Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;3 Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;4 Shanghai Engineering Research Center of Breeding Pig, Shanghai 201106, ChinaLI Feng-Ping
1 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;2 Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;3 Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;4 Shanghai Engineering Research Center of Breeding Pig, Shanghai 201106, ChinaXIE Chun-Fang
2 Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;3 Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;4 Shanghai Engineering Research Center of Breeding Pig, Shanghai 201106, ChinaSI Fu-Sheng
2 Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;3 Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;4 Shanghai Engineering Research Center of Breeding Pig, Shanghai 201106, ChinaDONG Shi-Juan
2 Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;3 Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;4 Shanghai Engineering Research Center of Breeding Pig, Shanghai 201106, ChinaLI Zhen
2 Institute of Animal Husbandry and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;3 Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;4 Shanghai Engineering Research Center of Breeding Pig, Shanghai 201106, China【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PEDV M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 (eIF3L) were chosen for reverse Co-IP reconfirmation and colocalization analysis. The results showed that both CDC42 and eIF3L protein interact with M protein. [Conclusion] This study identified that PEDV M protein could interact with CDC42 and eIF3L 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.
王瑞阳,于瑞嵩,陈冰清,李凤平,谢春芳,司伏生,董世娟,李震. 与猪流行性腹泻病毒M蛋白互作的宿主蛋白的鉴定[J]. 微生物学通报, 2019, 46(6): 1434-1442
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