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
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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.
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.
引文
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[2]Wensvoort G, Terpstra C, Pol J M, et al. Mystery swine disease in the Netherlands:the isolation of Lelystad virus[J]. Vet Q, 1991, 13(3):121-130.
[3]TerpstraC,WensvoortG,PolJM.Experimental reproduction of porcine epidemic abortion and respiratory syndrome(mystery swine disease)by infection with Lelystad virus:Koch's postulates fulfilled[J]. Vet Q, 1991,13(3):131-136.
[4]Collins J E, Benfield D A, Christianson W T, et al.Isolation of swine infertility and respiratory syndrome virus(isolate ATCC VR-2332)in North America and experimental reproduction of the disease in gnotobiotic pigs[J]. J Vet Diagn Invest, 1992, 4(2):117-126.
[5]郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国畜禽传染病, 1996(2):1-5.
[6]Tian K, Yu X, Zhao T, et al. Emegence of fatal PRRSV variants:unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J].PLoS One, 2007, 2(6):e526.
[7]ConzelmannKK,VisserN,VanWoenselP, etal.Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group[J]. Virology, 1993, 193(1):329-339.
[8]Snijder E J, Meulenberg J J. The molecular biology of arteriviruses[J]. J Gen Virol, 1998, 79(Pt5):961-979.
[9]Johnson C R, Griggs T F, Gnanandarajah J, et al. Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative ORF5 present in all arteriviruses[J]. J Gen Virol, 2011, 92(Pt5):1107-1116.
[10] Nauwynck H J, Duan X, Favoreel H W, et al. Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis[J]. J Gen Virol, 1999, 80(Pt 2):297-305.
[11]Tong G Z, Zhou Y J, Hao X F, et al. Highly pathogenic porcine reproductive and respiratory syndrome, China[J].Emerg Infect Dis, 2007, 13(9):1434-1436.
[12]Tian Z J, An T Q, Zhou Y J, et al. An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV)protects piglets against HP-PRRS[J]. Vet Microbiol, 2009, 138(1-2):34-40.
[13]曲泽慧,高飞,李丽薇,等.猪繁殖与呼吸综合征重组病毒非必需基因区的鉴定[J].中国预防兽医学报, 2015,37(4):246-249.
[14]刘欢,姜一峰,张文超,等.猪繁殖与呼吸综合征病毒N蛋白原核表达及多克隆抗体的制备[J].中国动物传染病学报, 2017, 25(2):82-86.
[15]韦天超,田志军,安同庆,等.猪繁殖与呼吸综合征病毒Taq Man-MGB荧光定量RT-PCR方法的建立和应用[J].中国预防兽医学报, 2008, 30(12):944-948, 991.
[16]Yang Q, Zhang Q, Tang J, et al. Lipid rafts both in cellular membrane and viral envelope are critical for PRRSV efficient infection[J]. Virology, 2015, 484:170-180.
[17]Huang L, Zhang Y P, Yu Y L, et al. Role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in MARC-145 cells[J]. Biochem Biophys Res Commun, 2011, 414(3):545-550.
[18]AlbornWE,CaoG,CareskeyHE, etal.Serum proprotein convertase subtilisin kexin type 9 is correlated directly with serum LDL cholesterol[J]. Clin Chem, 2007,53(10):1814-1819.
[19]WangTY,LiuYG,LiL, etal.Porcinealveolar macrophage CD163 abundance is a pivotal switch for porcine reproductive and respiratory syndrome virus infection[J]. Oncotarget, 2018, 9(15):12174-12185.
[20]Li Y, Wu Z, Liu K, et al. Proteomic analysis of the secretome of porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus[J].Proteomics, 2017, 17(21). doi:10.1002/pmic. 201700080.
[21]QuZ,GaoF,LiL, etal.Label-freequantitative proteomic analysis of differentially expressed membrane proteins of pulmonary alveolar macrophages infected with highly pathogenic porcine reproductive and respiratory syndrome virus and its attenuated strain[J]. Proteomics,2017, 17(23-24). doi:10.1002/pmic. 201700101.
[2]Wensvoort G, Terpstra C, Pol J M, et al. Mystery swine disease in the Netherlands:the isolation of Lelystad virus[J]. Vet Q, 1991, 13(3):121-130.
[3]TerpstraC,WensvoortG,PolJM.Experimental reproduction of porcine epidemic abortion and respiratory syndrome(mystery swine disease)by infection with Lelystad virus:Koch's postulates fulfilled[J]. Vet Q, 1991,13(3):131-136.
[4]Collins J E, Benfield D A, Christianson W T, et al.Isolation of swine infertility and respiratory syndrome virus(isolate ATCC VR-2332)in North America and experimental reproduction of the disease in gnotobiotic pigs[J]. J Vet Diagn Invest, 1992, 4(2):117-126.
[5]郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国畜禽传染病, 1996(2):1-5.
[6]Tian K, Yu X, Zhao T, et al. Emegence of fatal PRRSV variants:unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J].PLoS One, 2007, 2(6):e526.
[7]ConzelmannKK,VisserN,VanWoenselP, etal.Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group[J]. Virology, 1993, 193(1):329-339.
[8]Snijder E J, Meulenberg J J. The molecular biology of arteriviruses[J]. J Gen Virol, 1998, 79(Pt5):961-979.
[9]Johnson C R, Griggs T F, Gnanandarajah J, et al. Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative ORF5 present in all arteriviruses[J]. J Gen Virol, 2011, 92(Pt5):1107-1116.
[10] Nauwynck H J, Duan X, Favoreel H W, et al. Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis[J]. J Gen Virol, 1999, 80(Pt 2):297-305.
[11]Tong G Z, Zhou Y J, Hao X F, et al. Highly pathogenic porcine reproductive and respiratory syndrome, China[J].Emerg Infect Dis, 2007, 13(9):1434-1436.
[12]Tian Z J, An T Q, Zhou Y J, et al. An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV)protects piglets against HP-PRRS[J]. Vet Microbiol, 2009, 138(1-2):34-40.
[13]曲泽慧,高飞,李丽薇,等.猪繁殖与呼吸综合征重组病毒非必需基因区的鉴定[J].中国预防兽医学报, 2015,37(4):246-249.
[14]刘欢,姜一峰,张文超,等.猪繁殖与呼吸综合征病毒N蛋白原核表达及多克隆抗体的制备[J].中国动物传染病学报, 2017, 25(2):82-86.
[15]韦天超,田志军,安同庆,等.猪繁殖与呼吸综合征病毒Taq Man-MGB荧光定量RT-PCR方法的建立和应用[J].中国预防兽医学报, 2008, 30(12):944-948, 991.
[16]Yang Q, Zhang Q, Tang J, et al. Lipid rafts both in cellular membrane and viral envelope are critical for PRRSV efficient infection[J]. Virology, 2015, 484:170-180.
[17]Huang L, Zhang Y P, Yu Y L, et al. Role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in MARC-145 cells[J]. Biochem Biophys Res Commun, 2011, 414(3):545-550.
[18]AlbornWE,CaoG,CareskeyHE, etal.Serum proprotein convertase subtilisin kexin type 9 is correlated directly with serum LDL cholesterol[J]. Clin Chem, 2007,53(10):1814-1819.
[19]WangTY,LiuYG,LiL, etal.Porcinealveolar macrophage CD163 abundance is a pivotal switch for porcine reproductive and respiratory syndrome virus infection[J]. Oncotarget, 2018, 9(15):12174-12185.
[20]Li Y, Wu Z, Liu K, et al. Proteomic analysis of the secretome of porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus[J].Proteomics, 2017, 17(21). doi:10.1002/pmic. 201700080.
[21]QuZ,GaoF,LiL, etal.Label-freequantitative proteomic analysis of differentially expressed membrane proteins of pulmonary alveolar macrophages infected with highly pathogenic porcine reproductive and respiratory syndrome virus and its attenuated strain[J]. Proteomics,2017, 17(23-24). doi:10.1002/pmic. 201700101.
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