猪繁殖与呼吸综合征病毒感染肺泡巨噬细胞后差异表达膜蛋白的鉴定与分析  

CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED MEMBRANE PROTEINS OF PULMONARY ALVEOLAR MACROPHAGES INFECTED WITH PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

作　　者：张玉娇 高飞[1,2] 曲泽慧 姜一峰[1,2] 周艳君[1] 虞凌雪 李丽薇[1] 赵款[1] 童光志[1,2] ZHANG Yu-jiao;GAO Fei;QU Ze-hui;JIANG Yi-feng;ZHOU Yan-jun;YU Ling-xue;LI Li-wei;ZHAO Kuan;TONG Guang-zhi(Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonose, Yangzhou University, Yangzhou 225009, China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国动物传染病学报》2019年第3期1-8,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划政府间国际科技创新合作重点专项(2016YFE0112500);973计划项目(2014CB542701);国家科技支撑计划项目(2015BAD12B01);国家自然科学基金(31670158)

摘　　要：猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是危害养猪业发展的重要传染病之一。2006年我国首次爆发高致病性PRRS(highly-pathogenicPRRS,HP-PRRS),与经典的PRRS相比,HP-PRRS发病率和死亡率均显著升高。目前,对高致病性猪繁殖与呼吸综合征病毒(highly-pathogenic porcine reproductive and respiratory syndrome virus,HP-PRRSV)引起的高发病率和高死亡率的机制仍不清楚。本研究利用1MOI的EGFP标记的HP-PRRSV毒株HuN4(rHuN4-EGFP)和其体外传代致弱毒株HuN4-F112(rHuN4-F112-EGFP)分别感染肺泡巨噬细胞(pulmonaryalveolarmacrophage,PAM),24h后通过流式细胞术,从FITC通道收集被PRRSV强弱毒感染的PAM,提取膜蛋白进行Shotgun质谱分析。试验最终获得82个在强弱毒感染PAM过程中存在差异表达的膜蛋白。感染rHuN4-EGFP的样品与未感染样品相比,有72个蛋白特异表达;感染rHuN4-F112-EGFP的样品与未感染样品相比,有12个蛋白特异表达。通过GO分析发现,这些蛋白主要参与代谢、生物调节与细胞加工过程,大多数蛋白具有结合、催化活性。进一步在MARC-145细胞上过表达部分差异膜蛋白,发现其中前蛋白转化酶枯草杆菌蛋白酶9(proprotein coverteases subtilism/kexin 9,PCSK9)能明显抑制PRRSV强毒株和弱毒株的复制,Clusterin、Apoliprotein C-II能促进PRRSV强毒株复制。本研究表明利用Shotgun质谱技术鉴定出的差异表达膜蛋白对PRRSV复制有影响,深入分析PRRSV感染后差异表达的膜蛋白变化将有利于进一步明确其致病机制。Porcine reproductive and respiratory syndrome(PRRS) is one of the most important infectious diseases that threaten swine industry. In 2006, a highly pathogenic PRRS(HP-PRRS) emerged in China, which caused high morbidity and mortality in comparison with classical PRRS. However, the mechanism is still unclear. In this study, pulmonary alveolar macrophages(PAMs) were infected with1 MOI EGFP labeled HP-PRRS virus HuN4(rHuN4-EGFP) and its attenuated strain HuN4-F112(rHuN4-F112-EGFP), respectively.The infected PAMs were collected through FITC channel using flow cytometry at 24 hours post infection. Then the membrane proteins of the infected PAMs were extracted for shotgun proteomics(liquid chromatography-tandem mass spectrometry, LS-MS/MS) analysis.Total 82 differentially expressed membrane proteins were identified, of which 72 proteins were from rHuN4-EGFP infected PAMs and12 proteins were from rHuN4-F112-EGFP infected PAMs. These proteins mostly participated in metabolic process, cellular process and biological regulation that possessed characteristics of combination and catalytic activities. Using over expression method, we confirmed that PCSK9 inhibited replication of PRRSV while Clusterin and Apoliprotein C-II promoted PRRSV growth. This study demonstrated that the differentially expressed membrane proteins identified by Shotgun mass spectra had an effect on PRRSV replication. Therefore, further analysis of these differentially expressed membrane proteins expressed on infected PAMs would help clarify the pathogenesis of PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒 肺泡巨噬细胞 膜蛋白 

分 类 号：S852.659.6[农业科学—基础兽医学]