稳定表达脑心肌炎病毒先导蛋白N_2a细胞系的建立与鉴定
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摘要
为建立稳定表达脑心肌炎病毒先导蛋白的N_2a细胞系,本研究应用脂质体介导DNA转染法,将表达脑心肌炎病毒(EMCV)先导(L)蛋白的重组质粒pNTAP-B-L转染至N_2a细胞中,通过筛选G418抗性单克隆细胞,建立能够稳定表达融合蛋白TAP tag-L的N_2a细胞系;利用免疫印迹试验(Western blotting)和间接免疫荧光试验(IFA)对融合蛋白TAP tag-L的表达及细胞内定位情况进行鉴定检测。结果表明EMCV的L基因已经整合进N_2a细胞基因组DNA中且L蛋白在获得的阳性N_2a细胞中稳定表达,表达产物TAP tag-L主要分布在细胞浆内。建立的稳定表达L蛋白的N_2a细胞系,为进一步研究先导蛋白功能及EMCV感染过程中与宿主细胞(N_2a)相互作用蛋白的筛选奠定了基础。
We wished to construct a cell line stably expressing the leader protein(L)of the encephalomyocarditis virus(ECMV).Recombinant plasmids pNTAP-L expressing the L protein of the EMCV were transfected into N_2a cells mediated by liposomes.The cell line with stable expression of the fusion protein TAP tag-L gene was selected by the antibiotic G418.Expression and localization of exogenous TAP tag-L were detected by Western blotting and an indirect immunofluorescence assay.Results showed that the TAP tag-L gene was integrated into the genome of N_2a cells,and was expressed stably.Consequently,the N_2a cell line with stable expression of the L protein was established.The present study provides a cell model and potential foundation for future research on the function of the L protein and its interaction with the host cell(N_2a)in EMCV infection.
We wished to construct a cell line stably expressing the leader protein(L)of the encephalomyocarditis virus(ECMV).Recombinant plasmids pNTAP-L expressing the L protein of the EMCV were transfected into N_2a cells mediated by liposomes.The cell line with stable expression of the fusion protein TAP tag-L gene was selected by the antibiotic G418.Expression and localization of exogenous TAP tag-L were detected by Western blotting and an indirect immunofluorescence assay.Results showed that the TAP tag-L gene was integrated into the genome of N_2a cells,and was expressed stably.Consequently,the N_2a cell line with stable expression of the L protein was established.The present study provides a cell model and potential foundation for future research on the function of the L protein and its interaction with the host cell(N_2a)in EMCV infection.
引文
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[10]Basta H A,Palmenberg A C.AMP-activated protein kinase phosphorylates EMCV,TMEV and SafV leader proteins at different sites[J].Virology,2014,462-463c:236-240.
[11]Ichinose-Asakura K,Himeda N T T,Nojiri M,Okuwa T,Ohara Y.Leader(L)of Theiler's murine encephalomyelitis virus(TMEV)is required for virus growth in a murine macrophage-like cell line[J].Virus Res,2010,147(2):224-230.
[12]Prabakaran M,Tan S Z K.Neurotrophic potential of Saffold virus and the effects of its leader protein in the central nervous system of a mouse model[J].J Clin Virol,2016,82:S19-S19.
[13]Grubman M J,Moraes M P,Diaz-San Segundo F,Pena L,de los Santos T.Evading the host immune response:how foot-and-mouth disease virus has become an effective pathogen[J].FEMS Immunol Med Microbiol,2008,53(1):8-17.
[14]De Los Santos T,de Avila Botton S,Weiblen R,Grubman M J.The leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mRNA and blocks the host innate immune response[J].J Virol,2006,80(4):1906-1914.
[15]De Los Santos T,Diaz-San Segundo F,Grubman M J.Degradation of nuclear factor kappa B during foot and mouth disease virus infection[J].J Clin Virol,2007,81(23):12803-12815.
[16]De Los Santos T,Diaz-San Segundo F,Zhu J,Koster M,Camila C.A.Dias,Marvin J.Grubman.A conserved domain in the leader proteinase of foot and mouth disease virus is required for proper subcellular localization and function[J].J Clin Virol,2009,83(4):1800-1810.
[17]Rigaut G,Shevchenko A,Rutz B,Wilm M,Mann M,Séraphin B.A generic protein purification method for protein complex characterization and proteome exploration[J].Nat Biotechnol,1999,17(10):1030-1032.
[18]Goodfellow I,Bailey D.Detection of protein-protein interactions using tandem affinity purification[J].Methods Mol Biol,2014,1177(1177):121-133.
[19]Kosobokova E N,Skrypnik K A,Kosorukov V S.Overview of fusion tags for recombinant proteins[J].Biochem,2016,81(3):187-200.
[20]Gerace E,Moazed D.Affinity purification of protein complexes using TAP tags[J].Methods Enzymol,2015,559:37-52.
[2]Murnane T G,Craighead J E,Mondragon H,Shelokov.Fatal disease of swine due to encephalomyocarditis virus[J].Sci,1960,131(3399):498-499.
[3]张海霞,冯若飞,王丹,凡静静,李向茸,杨妍梅.脑心肌炎病毒的体外细胞增殖培养及其冻干保存试验[J].中国兽医科学,2012,42(11):1128-1132.Zhang H X,Feng R F,Wang D,Fan J J.Proliferation in vitro and freeze-drying of encephalomyocarditis virus[J].Vet Sci China,2012,42(11):1128-1132.(in Chinese)
[4]Liu H,He X,Song X,Xu L,Zhang Y,Zhou Y,Zhu W J,Chen C,Yin Z,Shi Y H,Wang C Q,Chang H T.Isolation and molecular and phylogenetic analyses of encephalomyocarditis virus from wild boar in central China[J].Infect Genet Evol,2016,40:67-72.
[5]盖新娜,杨汉春,郭鑫,王芳,陈艳红,查振林.猪脑心肌炎病毒的分离与鉴定[J].畜牧兽医学报,2007,38(1):59-65.
[6]Zhang G Q,Ge X N,Guo X,Yang H C.Genomic analysis of two porcine encephalomyocarditis virus strains isolated in China[J].Arch Virol,2007,152(6):1209-1213.
[7]Hato S V,Sorgeloos F,Ricour C,Zoll J,Melchers W J,Michiels T,van Kuppeveld F J.Differential IFN-α/βproduction suppressing capacities of the leader proteins of mengovirus and foot and mouth disease virus[J].Cell Microbiol,2010,12(3):310-317.
[8]Tan S Z,Chua K B,Xu Y,Prabakaran M.The pathogenesis of saffold virus in AG129mice and the effects of its truncated L protein in the central nervous system[J].Viruses,2016,8(2):63-70.
[9]Xu Y,Victorio C B,Ng Q,Prabakaran M,Tan Y J,Chua K.Intracellular localization of Saffold virus Leader(L)protein differs in Vero and HEp-2cells[J/OL].EMI,2016,5(10):e109.
[10]Basta H A,Palmenberg A C.AMP-activated protein kinase phosphorylates EMCV,TMEV and SafV leader proteins at different sites[J].Virology,2014,462-463c:236-240.
[11]Ichinose-Asakura K,Himeda N T T,Nojiri M,Okuwa T,Ohara Y.Leader(L)of Theiler's murine encephalomyelitis virus(TMEV)is required for virus growth in a murine macrophage-like cell line[J].Virus Res,2010,147(2):224-230.
[12]Prabakaran M,Tan S Z K.Neurotrophic potential of Saffold virus and the effects of its leader protein in the central nervous system of a mouse model[J].J Clin Virol,2016,82:S19-S19.
[13]Grubman M J,Moraes M P,Diaz-San Segundo F,Pena L,de los Santos T.Evading the host immune response:how foot-and-mouth disease virus has become an effective pathogen[J].FEMS Immunol Med Microbiol,2008,53(1):8-17.
[14]De Los Santos T,de Avila Botton S,Weiblen R,Grubman M J.The leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mRNA and blocks the host innate immune response[J].J Virol,2006,80(4):1906-1914.
[15]De Los Santos T,Diaz-San Segundo F,Grubman M J.Degradation of nuclear factor kappa B during foot and mouth disease virus infection[J].J Clin Virol,2007,81(23):12803-12815.
[16]De Los Santos T,Diaz-San Segundo F,Zhu J,Koster M,Camila C.A.Dias,Marvin J.Grubman.A conserved domain in the leader proteinase of foot and mouth disease virus is required for proper subcellular localization and function[J].J Clin Virol,2009,83(4):1800-1810.
[17]Rigaut G,Shevchenko A,Rutz B,Wilm M,Mann M,Séraphin B.A generic protein purification method for protein complex characterization and proteome exploration[J].Nat Biotechnol,1999,17(10):1030-1032.
[18]Goodfellow I,Bailey D.Detection of protein-protein interactions using tandem affinity purification[J].Methods Mol Biol,2014,1177(1177):121-133.
[19]Kosobokova E N,Skrypnik K A,Kosorukov V S.Overview of fusion tags for recombinant proteins[J].Biochem,2016,81(3):187-200.
[20]Gerace E,Moazed D.Affinity purification of protein complexes using TAP tags[J].Methods Enzymol,2015,559:37-52.