鸭坦布苏病毒感染对BHK-21细胞分泌胞外体的影响
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摘要
目的:探讨鸭坦布苏病毒感染BHK-21细胞对胞外体的影响,进一步研究鸭坦布苏病毒的致病机制。方法:利用鸭坦布苏病毒AH-F10株感染BHK-21细胞,并设置未感染病毒的对照组。用PEG法提取感染病毒的BHK-21细胞和对照组细胞上清中的胞外体,通过电镜观察、Western blot检测对胞外体进行鉴定,并对提取的两组胞外体进行质谱鉴定分析。结果:电镜观察到纯度高的内部色浅、边缘浓染的杯状囊泡,直径30~160 nm。感染组胞外体的平均粒径大小大于对照组胞外体的平均粒径大小。Western blot试验中,均检测出CD9、CD63分子。应用液相质谱法从胞外体中鉴定出106种蛋白,其中感染坦布苏病毒组共检测出84种蛋白,对照组49种蛋白,有27种共同蛋白分子。结论:鸭坦布苏病毒感染BHK-21细胞会影响细胞胞外体的分泌,影响胞外体粒径大小及其蛋白组成成分。本试验为进一步研究坦布苏病毒的感染和致病机制奠定了基础。
Objective: To explore the effect of duck Tembusu virus infection on secretion of exosomes in BHK-21 cells and the pathogenesis of the Tembusu virus. Methods: The exosomes were collected and purified from the culture supernatant of BHK-21 cells infected with duck Tembusu virus AH-F10 strain and the control BHK-21 cells by PEG precipitation method respectively. The purified exosomes were identified by electron microscopy,Western blot assay and mass spectrometry. Results: The classical exosome particle morphology was observed with hyperchromic cup-shaped vesicles and average particle size of 30-160 nm in diameter under transmission electron microscopy. The mean size of the exosome from the infected cells were bigger than the mean size of the exosome from the uninfected. Western blot assay demonstrated that CD9 and CD63 were detected in purified exosomes as exosome marker molecula. A total of106 proteins were identified by mass spectrometry assay,84 proteins of infected BHK-21 cells exosome,49 proteins of the uninfected,and the infected and the uninfected BHK-21 share 27 common proteins on exosomes. Conclusion: Duck Tembusu virus infection affect the exosome secretion of cells in connection to the particle size and protein molecular composition. This experiment can lay the foundation for further research of Tembusu virus infection and pathogenesis.
Objective: To explore the effect of duck Tembusu virus infection on secretion of exosomes in BHK-21 cells and the pathogenesis of the Tembusu virus. Methods: The exosomes were collected and purified from the culture supernatant of BHK-21 cells infected with duck Tembusu virus AH-F10 strain and the control BHK-21 cells by PEG precipitation method respectively. The purified exosomes were identified by electron microscopy,Western blot assay and mass spectrometry. Results: The classical exosome particle morphology was observed with hyperchromic cup-shaped vesicles and average particle size of 30-160 nm in diameter under transmission electron microscopy. The mean size of the exosome from the infected cells were bigger than the mean size of the exosome from the uninfected. Western blot assay demonstrated that CD9 and CD63 were detected in purified exosomes as exosome marker molecula. A total of106 proteins were identified by mass spectrometry assay,84 proteins of infected BHK-21 cells exosome,49 proteins of the uninfected,and the infected and the uninfected BHK-21 share 27 common proteins on exosomes. Conclusion: Duck Tembusu virus infection affect the exosome secretion of cells in connection to the particle size and protein molecular composition. This experiment can lay the foundation for further research of Tembusu virus infection and pathogenesis.
引文
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[26]葛菲菲,邱亚峰,杨耀武,等.乙脑病毒E-Hsp70与E-肽连接区对小鼠特异性免疫的比较[J].中国病毒学,2005,20(4):357-361.Ge FF,Qiu YF,Yang YW,et al.Compavison of JEV E-Hsp70and E-bindingdomain fusion protein on immune responses in mice[J].Virologica Sinca,2005,20(4):357-361.
[2]Gross JC,Chaudhary V,Bartscherer K,et al.Active Wnt proteins are secreted on exosomes[J].Nat Cell Biol,2012,14(10):1036-1045.
[3]Mathivanan S,Ji H,Simpson RJ.Exosomes:extracellular organelles important in intercellular communication[J].J Proteomics,2010,73(10):1907-1920.
[4]Schneider A,Simons M.Exosomes:vesicular carriers for intercellular communication in neurodegenerative disorders[J].Cell Tissue Res,2013,352(1):33-47.
[5]Sato-Kuwabara Y,Melo SA,Soares FA,et al.The fusion of two worlds:non-coding RNAs and extracellular vesicles--diagnostic and therapeutic implications(Review)[J].Int J Oncol,2015,46(1):17-27.
[6]袁生,李金平,张浩吉,等.我国发现的一种引起鸭产蛋下降综合征的新型黄病毒[J].中国畜牧兽医,2012,39(9):47-49.Yuan S,Li JP,Zhang HJ,et al.A new type of flavivirus found that causes duck egg drop syndrome in China[J].Chin J Veterinary Med,2012,39(9):47-49.
[7]袁生,李金平,张浩吉,等.新型黄病毒在种鹅中垂直传播的研究[J].南方农业学报,2012,43(1):99-102.Yuan S,Li JP,Zhang HJ,et al.Vertical transmission of new type flavivirus in egg-laying goose[J].J Southern Agriculture,2012,43(1):99-102.
[8]赵圆圆,李传峰,王桂军,等.鸭产蛋下降-死亡综合征的临床诊断与病原的初步鉴定[J].扬州大学学报(农业与生命科学版),2011,32(3):11-14.Zhao YY,Li CF,Wang GJ,et al.Clinical diagnosis and pathogenic identification of egg drop-death syndrome in duck[J].J Yangzhou University(Agriculturalan Life Science Edition),2011,32(3):11-14.
[9]胡旭东,路浩,刘培培,等.鸭传染性产蛋减少症(暂定名)的病原分离初报[J].中国兽医杂志,2011,7(3):43-47.Hu XD,Lu H,Liu PP,et al.The primary study on virus isolation of duck Infectious egg failings[J].Chin J Animal Infectious Diseases,2011,7(3):43-47.
[10]廖敏,牟小东,耿阳,等.鹅源黄病毒的分离和初步鉴定[J].中国动物传染病学报,2011,19(1):22-26.Liao M,Mu XD,Geng Y,et al.Isolation and Identification of Goose flavivirus[J].China Animal Husbandry&Veterinary Medicine,2011,19(1):22-26.
[11]Cao Z,Zhang C,Liu Y,et al.Tembusu virus in ducks,China[J].Emerg Infect Dis,2011,17(10):1873-1875.
[12]Thery C,Amigorena S,Raposo G,et al.Isolation and characterization of exosomes from cell culture supernatants and biological fluids[J].Curr Protoc Cell Biol,2006,3(22):1-29.
[13]Chahar H,Bao X,Casola A.Exosomes and their role in the life cycle and pathogenesis of RNA viruses[J].Viruses,2015,7(6):3204-3225.
[14]Taylor DD,Shah S.Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes[J].Methods,2015,87:3-10.
[15]Rider MA,Hurwitz SN,Meckes DG.Extra PEG:a polyethylene glycol-based method for enrichment of extracellular vesicles[J].Scientific Reports,2016,6(1):1-14.
[16]Narayanan A,Iordanskiy S,Das R,et al.Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA[J].J Biol Chem,2013,288(27):20014-20033.
[17]Purushothaman A,Bandari SK,Liu J,et al.Fibronectin on the surface of myeloma cell-derived exosomes mediates exosome-cell interactions[J].J Biol Chem,2016,291(4):1652-1663.
[18]Mansilla C,Gorraiz M,Martinez M,et al.Immunization against hepatitis C virus with a fusion protein containing the extra domain A from fibronectin and the hepatitis C virus NS3 protein[J].JHepatol,2009,51(3):520-527.
[19]Zhang F,Sodroski C,Cha H,et al.Infection of hepatocytes with HCV increases cell surface levels of heparan sulfate proteoglycans,uptake of cholesterol and lipoprotein,and virus entry by upregulating SMAD6 and SMAD7[J].Gastroente-rology,2016,152(1):257-270.
[20]Kaushik DK,Gupta M,Kumawat KL,et al.NLRP3 inflammasome:key mediator of neuroinflammation in murine Japanese encephalitis[J].PLo S One,2012,7(2):1-12.
[21]Negash AA,Ramos HJ,Crochet N,et al.IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease[J].PLo S Pathog,2013,9(4):1-14.
[22]Gaudieri S,Rauch A,Pfafferott K,et al.Hepatitis C virus drug resistance and immune-driven adaptations:relevance to new antiviral therapy[J].Hepatology,2009,49(4):1069-1082.
[23]Liu S,Peng N,Xie J,et al.Human hepatitis B virus surface and e antigens inhibit major vault protein signaling in interferon induction pathways[J].J Hepatol,2015,62(5):1015-1023.
[24]张占东,杨巍,马飞,等.休克蛋白27,60和90在胃癌中表达及其临床价值[J].中国免疫学杂志,2016,32(7):1042-1049.Zhang ZD,Yang W,Ma F,et al.Expression of heat shock protein27,60 and 90 in gastric cancer and its clinical value[J].Chin JImmunol,2016,32(7):1042-1049.
[25]王云龙,李娜,李玉林,等.热休克蛋白90α荧光免疫层析检测方法的建立[J].中国免疫学杂志,2017,33(5):712-720.Wang YL,Li N,Li YL,et al.Establishment of quantitative fluroscence immunochromatographic assay detection method for heat shock protein 90α(HSP90α)[J].Chin J Immunol,2017,33(5):712-720.
[26]葛菲菲,邱亚峰,杨耀武,等.乙脑病毒E-Hsp70与E-肽连接区对小鼠特异性免疫的比较[J].中国病毒学,2005,20(4):357-361.Ge FF,Qiu YF,Yang YW,et al.Compavison of JEV E-Hsp70and E-bindingdomain fusion protein on immune responses in mice[J].Virologica Sinca,2005,20(4):357-361.