禽偏肺病毒感染Vero细胞的外泌体蛋白质组学分析  

Proteomic Analysis of Exosomes Secreted by Vero Cell Infected with Avian Metapneumovirus

作　　者：王丹[1] 侯磊 宋江伟 王菁[1] 全荣[1] 刘爵 WANG Dan;HOU Lei;SONG Jiangwei;WANG Jing;QUAN Rong;LIU Jue(Beijing Key Laboratory for Prevention and Control of Infectious Diseases in Livestock and Poultry,Institute of Animal Husbandry and Veterinary Medicine,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097;College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009)

机构地区：[1]北京市农林科学院畜牧兽医研究所,畜禽疫病防控技术北京市重点实验室,北京100097 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《中国家禽》2022年第10期24-30,共7页China Poultry

基　　金：北京市农林科学院科技创新能力建设专项(KJCX201914)。

摘　　要：为研究C型禽偏肺病毒(aMPV/C)感染Vero细胞后外泌体中蛋白质组分的变化,试验收集感染病毒后48 h的细胞培养液,用超速离心法分离外泌体。经透射电镜观察、纳米颗粒跟踪技术分析、蛋白质印迹法(Western blot)鉴定后,对外泌体进行示踪试验。利用非标记定量液相色谱-质谱联用(lable-free LC-MS/MS)技术和生物信息学方法分析外泌体的蛋白质组分。结果显示:分离的外泌体为杯状双层膜囊泡,具有特征性膜蛋白CD63、CD81、Tsg101,能够进入未感染的Vero细胞;aMPV/C感染组中170个蛋白表达上调,11个蛋白表达下调;差异表达蛋白大多来源于细胞器和细胞膜,主要参与细胞过程、生物调节、代谢过程等生物学过程,执行受体结合、酶催化、分子调节等功能,集中于内吞作用、病毒感染、细胞衰老等通路。研究表明,aMPV/C感染使Vero细胞外泌体蛋白质组分发生明显变化,差异表达蛋白主要集中于细胞膜信号传导通路、病毒感染相关信号通路、细胞增殖相关信号通路,被aMPV/C感染的Vero细胞能以外泌体形式向未感染细胞进行物质或信息传递。To investigate the proteome changes of exosomes secreted by Vero cells with or without a MPV/C infection,cell supernatant was collected 48 h post infection and exosomes were isolated by ultracentrifugation.The exosomes were identified by transmission electron microscopy,nanoparticle tracking analysis and Western blot.Label-free LC-MS/MS and bioinformatics methods were used to analyze the protein components in exosomes.The results showed that the isolated exosomes were cupshaped bilayer membranous vesicles with characteristic membrane proteins,CD63,CD81 and Tsg101,and could enter the uninfected Vero cells.There were 170 up-regulated proteins and 11 down-regulated proteins in exosomes.Most of the differentially expressed proteins were derived from organelles and cell membranes,mainly involved in cellular processes,biological regulation,metabolism and other biological processes,performing binding,catalytic activity and molecular regulation functions,mainly involved in endocytosis,virus infection,cellular senescence and other pathways.It′s suggested that aMPV/C infection significantly changed the protein composition in exosomes derived from Vero cells,the differentially expressed proteins were mainly involved in cell membrane signaling pathways,virus infection-associated signaling pathways and cell proliferation-associated signaling pathways,and exosomes might carry material or signal from aMPV/C-infected cell to uninfected cells.

关 键 词：禽偏肺病毒 VERO细胞 蛋白质组学 

分 类 号：S855.3[农业科学—临床兽医学]