齿兰环斑病毒RdRp基因的原核表达及多克隆抗体制备
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摘要
采用RT-PCR法从感染齿兰环斑病毒(Odontoglossum ringspot virus,ORSV)贵州分离物的苋色藜叶片中扩增出病毒的依赖RNA的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)基因保守序列。测序结果显示,该保守片段长516bp,编码171个氨基酸残基。构建了该片段的原核表达载体pET32a-ORSV RdRp并转化BL21(DE3)菌株,在25℃以0.4mmol·L~(-1)IPTG诱导表达重组蛋白。SDS-PAGE分析表明,重组蛋白分子量约为36 kDa,与预测相符合。将该重组蛋白作为抗原免疫BABL/C小鼠,制备的多克隆抗体效价达1∶102 400。间接ELISA结果表明,该多抗具有较强的特异性,能检测到ORSV感染的病叶汁液中的RdRp,而与其它感染4种同属或不同属病毒的病叶汁液不发生血清学交叉反应,本实验为进一步研究RdRp的结构和功能以及从分子水平上探讨该病毒的致病机制奠定基础。
The conserved region of RdRp( RNA-dependent RNA polymerase) gene of Odontoglossum ringspot virus( ORSV) was amplified by RT-PCR from the Chenopodium amarantcolar leaves infected with ORSV Guizhou( China) isolate. DNA sequencing showed that the RdRp gene fragment was 516 bp in length and encoded 171 amino acid residues. The RdRp gene fragment was subcloned into a prokaryotic expression vector pET32a( +). By inducing with 0.4 mmol·L~(-1) IPTG at 25℃ the recombinant RdRp gene was expressed in E. coli BL21( DE3) and its product identified as a specific band of 36 kDa by SDS-PAGE. Polyclonal antibodies against the recombinant RdRp were obtained by hypodernal injection of mice. The titer of the prepared polyclonal antibodies was 1∶102 400,and the antibodies reacted specifically with ORSV infected leaf extract,but not with other 4 virus-infected samples.
The conserved region of RdRp( RNA-dependent RNA polymerase) gene of Odontoglossum ringspot virus( ORSV) was amplified by RT-PCR from the Chenopodium amarantcolar leaves infected with ORSV Guizhou( China) isolate. DNA sequencing showed that the RdRp gene fragment was 516 bp in length and encoded 171 amino acid residues. The RdRp gene fragment was subcloned into a prokaryotic expression vector pET32a( +). By inducing with 0.4 mmol·L~(-1) IPTG at 25℃ the recombinant RdRp gene was expressed in E. coli BL21( DE3) and its product identified as a specific band of 36 kDa by SDS-PAGE. Polyclonal antibodies against the recombinant RdRp were obtained by hypodernal injection of mice. The titer of the prepared polyclonal antibodies was 1∶102 400,and the antibodies reacted specifically with ORSV infected leaf extract,but not with other 4 virus-infected samples.
引文
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[7]Moraes L A,Krause-Sakate R,Pavan M A.Incidence and characterization of viruses infecting orchids in S2o Paulo state,Brazil[J],Tropical Plant Pathology,2017,42(2):126-131.
[8]Mahfut,Joko T,Daryono B S.Molecular characterization of Odontoglossum ringspot virus(ORSV)in Java and Bali,Indonesia[J],Asian Journal of Plant Pathology,2016,10(1):9-14.
[9]Wang C,Wu R,Liu G Q.Structure and functions of RNA-dependent RNA polymerase of positive-strand RNA viruses(in Chinese)[J].Chinese Journal of Animal Infectious Diseases(中国动物传染病学报),2011,19(5):76-82.
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[11]Zhan R J,Li Y K,Li J M,et al.Prokaryotic expression of movement protein(MP)gene of Odontoglossum ringspot virus and preparation of multiclonal antibodies against the recombinant MP(in Chinese)[J].Plant Quarantine(植物检疫),2013,27(6):60-63.
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[14]Tu X Y,Dong X Y,Guo C M,et al.Detection of three viruses CymMV,ORSV and CMV of Phalaenopsis by multiplex RT-PCR(in Chinese)[J].Journal of Jiangsu Agricultural Sciences(江苏农业科学),2017,45(5):91-93.
[15]Ni T Z,Leng Y,Qian W D,et al.Establishment of RT-PCR detection system for Odontoglossum ringspot virus in Dalian(in Chinese)[J].Molecular Plant Breeding(分子植物育种),2017,15(2):622-625.
[16]Koh K W,Lu H C,Chan M T.Virus resistance in orchids[J].Plant Science an International Journal of Experimental Plant Biology,2014,228:26-38.
[17]Peng B,Wei L,Yang K,et al.Genetic transformation and detection of the cymbidium hybridum modified by coat protein gene of ORSV(in Chinese)[J].Forest Research(林业科学研究),2016,29(2):234-237.
[18]Yang Y X,Yan Y H,Wei W Q,et al.Construction of hpRNAi expression vector of Cymbidium mosaic virus and Odontoglossum ringspot virus RdRp gene(in Chinese)[J].Jiangsu Journal of Agricultural Sciences(江苏农业学报),2013,29(1):76-80.
[19]Alexander M M,Cilia M.A molecular tug-of-war:Global plant proteome changes during viral infection[J].Current Plant Biology,2016,5:13-24.
[20]Lin P C,Hu W C,Lee S C,et al.Application of an integrated omics approach for identifying host proteins that interact with Odontoglossum ringspot virus capsid protein[J],Molecular Plant-Microbe Interactions,2015,28(6):711-726.
[21]Xu Y W.Immunoassay technology(2nd Ed.)(in Chinese)[M].Beijing:Science Press(北京:科学出版社),1991:158-183.
[22]Lewandowski D J,Dawson W O.Functions of the 126-and 183-kDa proteins of Tobacco mosaic virus[J].Virology,2000,271(1):90-98.
[23]Wu X,Shaw J G.Evidence that a viral replicase protein is involved in the disassembly of Tobacco mosaic virus in vivo[J].Virology,1997,239(2):426-434.
[24]Taylor D N,Carr J P.The GCD10 subunit of yeast eIF-3 binds the methyltransferase-like domain of 126-and183-kDa replicase proteins of Tobacco mosaic virus in the yeast two-hybrid system[J].Journal of General Virology,2000,81(6):1587-1591.
[25]Chen W H,Chen H H.Orchid Biotechnology III[M].Singapore:World Scientific Publishing Co.Pte.Ltd,2017:145-164.
[26]Hull R.Matthews’Plant Virology(4th Ed.)[M].Beijing:Science Press(北京:科学出版社),2007:342-343.
[27]Yuan L,Li G,Wang Y Q.Prokaryotic expression of amphioxus BRA protein and preparation of the polyclonal antibody(in Chinese)[J].Chinese Journal of Zoology(动物学杂志),2017,52(3):431-440.
[2]Yan W G,Tan Z,Cui J X.Detection of Odontoglossum ringspot virus and Cymbidium mosaic virus by real-time fluorescent RT-PCR(in Chinese)[J].Plant Protection(植物保护),2009,35(4):101-104.
[3]Luo J S,Wu Z J,Xie L H.Prokaryotic expression and antiserum preparation of the coat protein gene of Odontoglossum ringspot virus(in Chinese)[J].Chinese Agricultural Science Bulletin(中国农学通报),2011,27(19):115-120.
[4]Chng C G,Wong S M,Mahtani P H,et al.The complete sequence of a Singapore isolate of Odontoglossum ringspot virus and comparison with other tobamoviruses[J].Gene,1996,171(2):155-161.
[5]Wan Q J,Li Y K,Ma J,et al.Molecular identification of Odontoglossum ringspot virus in Guizhou(in Chineses)[J].Guizhou Agricultrual Sciences(贵州农业科学),2014,42(8):96-98.
[6]Canovas S E,Ballari M C,Nome C F.First report of Cymbidium mosaic virus and Odontoglossum ringspot virus in Argentina[J].Australasian Plant Disease Notes,2016,11(1):2.
[7]Moraes L A,Krause-Sakate R,Pavan M A.Incidence and characterization of viruses infecting orchids in S2o Paulo state,Brazil[J],Tropical Plant Pathology,2017,42(2):126-131.
[8]Mahfut,Joko T,Daryono B S.Molecular characterization of Odontoglossum ringspot virus(ORSV)in Java and Bali,Indonesia[J],Asian Journal of Plant Pathology,2016,10(1):9-14.
[9]Wang C,Wu R,Liu G Q.Structure and functions of RNA-dependent RNA polymerase of positive-strand RNA viruses(in Chinese)[J].Chinese Journal of Animal Infectious Diseases(中国动物传染病学报),2011,19(5):76-82.
[10]Liu Z X,Pan J S,Shao H S,et al.Cloning and sequence analysis of coat protein gene of Odontoglossum ringspot virus(in Chinese)[J].Chinese Journal of Virology(病毒学报),1998,14(1):87-90.
[11]Zhan R J,Li Y K,Li J M,et al.Prokaryotic expression of movement protein(MP)gene of Odontoglossum ringspot virus and preparation of multiclonal antibodies against the recombinant MP(in Chinese)[J].Plant Quarantine(植物检疫),2013,27(6):60-63.
[12]Shi N N,Xu Y,Yang K F,et al.Development of a sensitive diagnostic assay to detect Cymbidium mosaic virus and Odontoglossum ringspot virus in members of the Orchidaceae[J].Journal of Horticultural Science&Biotechnology,2011,86(1):69-73.
[13]Tsai C C,Huang C Y,Weng I S,et al.One-step RTLAMPanalysis for efficient detection of Odontoglossum ringspot virus from Oncidiinae hybrids(Orchidaceae)[J],Journal of General Plant Pathology,2016,82(2):89-95.
[14]Tu X Y,Dong X Y,Guo C M,et al.Detection of three viruses CymMV,ORSV and CMV of Phalaenopsis by multiplex RT-PCR(in Chinese)[J].Journal of Jiangsu Agricultural Sciences(江苏农业科学),2017,45(5):91-93.
[15]Ni T Z,Leng Y,Qian W D,et al.Establishment of RT-PCR detection system for Odontoglossum ringspot virus in Dalian(in Chinese)[J].Molecular Plant Breeding(分子植物育种),2017,15(2):622-625.
[16]Koh K W,Lu H C,Chan M T.Virus resistance in orchids[J].Plant Science an International Journal of Experimental Plant Biology,2014,228:26-38.
[17]Peng B,Wei L,Yang K,et al.Genetic transformation and detection of the cymbidium hybridum modified by coat protein gene of ORSV(in Chinese)[J].Forest Research(林业科学研究),2016,29(2):234-237.
[18]Yang Y X,Yan Y H,Wei W Q,et al.Construction of hpRNAi expression vector of Cymbidium mosaic virus and Odontoglossum ringspot virus RdRp gene(in Chinese)[J].Jiangsu Journal of Agricultural Sciences(江苏农业学报),2013,29(1):76-80.
[19]Alexander M M,Cilia M.A molecular tug-of-war:Global plant proteome changes during viral infection[J].Current Plant Biology,2016,5:13-24.
[20]Lin P C,Hu W C,Lee S C,et al.Application of an integrated omics approach for identifying host proteins that interact with Odontoglossum ringspot virus capsid protein[J],Molecular Plant-Microbe Interactions,2015,28(6):711-726.
[21]Xu Y W.Immunoassay technology(2nd Ed.)(in Chinese)[M].Beijing:Science Press(北京:科学出版社),1991:158-183.
[22]Lewandowski D J,Dawson W O.Functions of the 126-and 183-kDa proteins of Tobacco mosaic virus[J].Virology,2000,271(1):90-98.
[23]Wu X,Shaw J G.Evidence that a viral replicase protein is involved in the disassembly of Tobacco mosaic virus in vivo[J].Virology,1997,239(2):426-434.
[24]Taylor D N,Carr J P.The GCD10 subunit of yeast eIF-3 binds the methyltransferase-like domain of 126-and183-kDa replicase proteins of Tobacco mosaic virus in the yeast two-hybrid system[J].Journal of General Virology,2000,81(6):1587-1591.
[25]Chen W H,Chen H H.Orchid Biotechnology III[M].Singapore:World Scientific Publishing Co.Pte.Ltd,2017:145-164.
[26]Hull R.Matthews’Plant Virology(4th Ed.)[M].Beijing:Science Press(北京:科学出版社),2007:342-343.
[27]Yuan L,Li G,Wang Y Q.Prokaryotic expression of amphioxus BRA protein and preparation of the polyclonal antibody(in Chinese)[J].Chinese Journal of Zoology(动物学杂志),2017,52(3):431-440.