番茄斑萎病毒核衣壳蛋白N及膜多糖蛋白Gn的多克隆抗体制备及应用
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摘要
由番茄斑萎病毒Tomato spotted wilt orthotospovirus (TSWV)侵染引起的番茄斑萎病毒病在我国蔬菜、花卉等作物上造成了严重的经济损失。本研究克隆了TSWV核衣蛋白N基因及膜多糖蛋白Gn基因,利用Gateway系统获得N和Gn基因的原核表达载体。将其转化到大肠杆菌表达菌株Rosetta (DE3)细胞内,IPTG诱导表达N蛋白和Gn蛋白,以此为抗原分别免疫新西兰大白兔,制备N蛋白和Gn蛋白的多克隆抗体。Western blot检测结果表明,N蛋白的多克隆抗体能够与N蛋白发生特异性结合反应,间接酶联免疫吸附(indirect enzyme-linked immunosorbent assay,in-ELISA)分析检测表明抗体滴度为1∶12 800,而Gn蛋白的多克隆抗体特异性差,效价未检出。本研究制备的N蛋白多克隆抗体可用于田间病株的病害诊断,同时有助于开展病毒在介体昆虫内定位、病毒和介体昆虫互作及功能分析的研究。
Tomato spotted wilt disease caused by tomato spotted wilt orthotospovirus(TSWV)has resulted seriously yield and quality losses in vegetables and flowers in China.In this study,nucleocapsid protein Ngene and membrane polysaccharide protein Gngene of TSWV were cloned into prokaryotic expression vectors,respectively.The plasmids were transformed into Rosetta(DE3)cells for expression of N or Gn proteins,which were used as immunogens to immunize New Zealand white rabbits,then the polyclonal antibodies against N and Gn proteins were successfully prepared.Western blot analysis showed that the polyclonal antibody against N protein could bind to the N protein specifically.Indirect ELISA showed that the titer of antiserum against N protein was about 1∶12 800.However,the specificity of antibody against Gn protein was poor,and the titer was not detected.The polyclonal antibody against N protein has been prepared successfully,and it can be a useful reagent for disease diagnosis in field,the research of virus localization in insect body and the interaction of virus and insect vector.
Tomato spotted wilt disease caused by tomato spotted wilt orthotospovirus(TSWV)has resulted seriously yield and quality losses in vegetables and flowers in China.In this study,nucleocapsid protein Ngene and membrane polysaccharide protein Gngene of TSWV were cloned into prokaryotic expression vectors,respectively.The plasmids were transformed into Rosetta(DE3)cells for expression of N or Gn proteins,which were used as immunogens to immunize New Zealand white rabbits,then the polyclonal antibodies against N and Gn proteins were successfully prepared.Western blot analysis showed that the polyclonal antibody against N protein could bind to the N protein specifically.Indirect ELISA showed that the titer of antiserum against N protein was about 1∶12 800.However,the specificity of antibody against Gn protein was poor,and the titer was not detected.The polyclonal antibody against N protein has been prepared successfully,and it can be a useful reagent for disease diagnosis in field,the research of virus localization in insect body and the interaction of virus and insect vector.
引文
[1]PARRELLA G,GOGNALONS P,GEBRE-SELASSIèK,et al.An update of the host range of tomato spotted wilt virus[J].Journal of Plant Pathology,2003,85(4):227-264.
[2]CHO J J,RFL M,GERMAN T L,et al.A multidisciplinary approach to management of tomato spotted wilt virus in Hawaii[J].Plant Disease,1989,73(5):375-383.
[3]SCHOLTHOF K B,ADKINS S,CZOSNEK H,et al.Top 10plant viruses in molecular plant pathology[J].Molecular Plant Pathology,2011,12(9):938-954.
[4]李洁,迟胜起,杨勤民,等.山东烟台地区发生番茄斑萎病毒病危害[J].植物保护,2017,43(1):228-232.
[5]张仲凯.番茄斑萎病毒属病毒粒体在寄主植物中的装配与运动机制研究[D].北京:中国农业科学院,2015.
[6]KING A M Q,CARSTENS E B,LEFKOWITZ E J.Ninth report of the international Committee on taxonomy of viruses[J].Elsevier,2011:774-796.
[7]DE H P,KORMELINK R,DE O R R,et al.Tomato spotted wilt virus L RNA eNCodes a putative RNA polymerase[J].Journal of General Virology,1991,72(9):2207-2216.
[8]VAN K I,GOLDBACH R,KORMELINK R.Purified tomato spotted wilt virus particles support both genome replication and transcription in vitro[J].Virology,2002,303(2):278.
[9]KORMELINK R,STORMS M,VAN L J,et al.Expression and subcellular location of the NSM protein of tomato spotted wilt virus(TSWV),aputative viral movement protein[J].Virology,1994,200(1):56-65.
[10]STORMS M M,KORMELINK R,PETERS D,et al.The nonstructural NSm protein of tomato spotted wilt virus induces tubular structures in plant and insect cells[J].Virology,1995,214(2):485-493.
[11]LEWANDOWSKI D J,ADKINS S.The tubule-forming NSm protein from Tomato spotted wilt virus complements cell-tocell and long-distaNCe movement of Tobacco mosaic virus hybrids[J].Virology,2005,342(1):26.
[12]LI W,LEWANDOWSKI D J,HILF M E.Identification of domains of the Tomato spotted wilt virus NSm protein involved in tubule formation,movement and symptomatology[J].Virology,2009,390(1):110-121.
[13]KIKKERT M,VERSCHOOR A,KORMELINK R,et al.Tomato Spotted Wilt Virus Glycoproteins Exhibit Trafficking and Localization Signals That Are FuNCtional,in Mammalian Cells[J]. Journal of Virology, 2001, 75(2):1004-1012.
[14]SIN S H,MCNULTY B C,KENNEDY G G,et al.Viral genetic determinants for thrips transmission of Tomato spotted wilt virus[J].Proceedings of the National Academy of ScieNCes of the United States of America,2005,102(14):5168.
[15]TAKEDA A, SUGIYAMA K, NAGANO H, et al.Identification of a novel RNA sileNCing suppressor,NSs protein of Tomato spotted wilt virus[J].Febs Letters,2002,532(1-2):75.
[16]BUCHER E,SIJEN T,HAAN P D,et al.Negative-Strand Tospoviruses and Tenuiviruses Carry a Gene for a Suppressor of Gene SileNCing at Analogous Genomic Positions[J].Journal of Virology,2003,77(2):1329.
[17]DE H P,WAGEMAKERS L,PETERS D,et al.The S RNA segment of tomato spotted wilt virus has an ambisense character[J].Journal of General Virology,1990,71(5):1001-1007.
[18]KAINZ M,HILSON P,SWEENEY L,et al.Interaction between Tomato spotted wilt virus N protein monomers involves nonelectrostatic forces governed by multiple distiNCt regions in the primary structure[J].Phytopathology,2004,94(7):759-65.
[19]RICHMOND K E,CHENAULT K,SHERWOOD J L,et al.Characterization of the Nucleic Acid Binding Properties of Tomato Spotted Wilt Virus Nucleocapsid Protein[J].Virology,1998,248(1):6-11.
[20]RIBEIRO D,BORST J W,GOLDBACH R,et al.Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta[J].Virology,2009,383(1):121.
[21]UHRIG J F, BUCHER G L,SCHREIER P H. The Movement Protein NSm of Tomato Spotted Wilt Tospovirus(TSWV):RNA Binding,Interaction with the TSWV N Protein,and Identification of Interacting Plant Proteins[J].Proceedings of the National Academy of ScieNCes of the United States of America,2000,97(5):2373-2378.
[22]JOHN A L.TRBO:A High-Efficiency Tobacco Mosaic Virus RNA-Based over-expression Vector[J].Plant Physiology,2007(4):1232-1240.
[23]孙淼,荆陈沉,楚成茹,等.重庆辣椒上番茄斑萎病毒的血清学检测及分子鉴定[J].园艺学报,2017,44(3):487-494.
[24]于翠,邓凤林,杨翠云,等.番茄斑萎病毒外壳蛋白原核表达及Dot-blot ELISA检测方法的建立[J].浙江大学学报,2008,34(6):597-601.
[2]CHO J J,RFL M,GERMAN T L,et al.A multidisciplinary approach to management of tomato spotted wilt virus in Hawaii[J].Plant Disease,1989,73(5):375-383.
[3]SCHOLTHOF K B,ADKINS S,CZOSNEK H,et al.Top 10plant viruses in molecular plant pathology[J].Molecular Plant Pathology,2011,12(9):938-954.
[4]李洁,迟胜起,杨勤民,等.山东烟台地区发生番茄斑萎病毒病危害[J].植物保护,2017,43(1):228-232.
[5]张仲凯.番茄斑萎病毒属病毒粒体在寄主植物中的装配与运动机制研究[D].北京:中国农业科学院,2015.
[6]KING A M Q,CARSTENS E B,LEFKOWITZ E J.Ninth report of the international Committee on taxonomy of viruses[J].Elsevier,2011:774-796.
[7]DE H P,KORMELINK R,DE O R R,et al.Tomato spotted wilt virus L RNA eNCodes a putative RNA polymerase[J].Journal of General Virology,1991,72(9):2207-2216.
[8]VAN K I,GOLDBACH R,KORMELINK R.Purified tomato spotted wilt virus particles support both genome replication and transcription in vitro[J].Virology,2002,303(2):278.
[9]KORMELINK R,STORMS M,VAN L J,et al.Expression and subcellular location of the NSM protein of tomato spotted wilt virus(TSWV),aputative viral movement protein[J].Virology,1994,200(1):56-65.
[10]STORMS M M,KORMELINK R,PETERS D,et al.The nonstructural NSm protein of tomato spotted wilt virus induces tubular structures in plant and insect cells[J].Virology,1995,214(2):485-493.
[11]LEWANDOWSKI D J,ADKINS S.The tubule-forming NSm protein from Tomato spotted wilt virus complements cell-tocell and long-distaNCe movement of Tobacco mosaic virus hybrids[J].Virology,2005,342(1):26.
[12]LI W,LEWANDOWSKI D J,HILF M E.Identification of domains of the Tomato spotted wilt virus NSm protein involved in tubule formation,movement and symptomatology[J].Virology,2009,390(1):110-121.
[13]KIKKERT M,VERSCHOOR A,KORMELINK R,et al.Tomato Spotted Wilt Virus Glycoproteins Exhibit Trafficking and Localization Signals That Are FuNCtional,in Mammalian Cells[J]. Journal of Virology, 2001, 75(2):1004-1012.
[14]SIN S H,MCNULTY B C,KENNEDY G G,et al.Viral genetic determinants for thrips transmission of Tomato spotted wilt virus[J].Proceedings of the National Academy of ScieNCes of the United States of America,2005,102(14):5168.
[15]TAKEDA A, SUGIYAMA K, NAGANO H, et al.Identification of a novel RNA sileNCing suppressor,NSs protein of Tomato spotted wilt virus[J].Febs Letters,2002,532(1-2):75.
[16]BUCHER E,SIJEN T,HAAN P D,et al.Negative-Strand Tospoviruses and Tenuiviruses Carry a Gene for a Suppressor of Gene SileNCing at Analogous Genomic Positions[J].Journal of Virology,2003,77(2):1329.
[17]DE H P,WAGEMAKERS L,PETERS D,et al.The S RNA segment of tomato spotted wilt virus has an ambisense character[J].Journal of General Virology,1990,71(5):1001-1007.
[18]KAINZ M,HILSON P,SWEENEY L,et al.Interaction between Tomato spotted wilt virus N protein monomers involves nonelectrostatic forces governed by multiple distiNCt regions in the primary structure[J].Phytopathology,2004,94(7):759-65.
[19]RICHMOND K E,CHENAULT K,SHERWOOD J L,et al.Characterization of the Nucleic Acid Binding Properties of Tomato Spotted Wilt Virus Nucleocapsid Protein[J].Virology,1998,248(1):6-11.
[20]RIBEIRO D,BORST J W,GOLDBACH R,et al.Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta[J].Virology,2009,383(1):121.
[21]UHRIG J F, BUCHER G L,SCHREIER P H. The Movement Protein NSm of Tomato Spotted Wilt Tospovirus(TSWV):RNA Binding,Interaction with the TSWV N Protein,and Identification of Interacting Plant Proteins[J].Proceedings of the National Academy of ScieNCes of the United States of America,2000,97(5):2373-2378.
[22]JOHN A L.TRBO:A High-Efficiency Tobacco Mosaic Virus RNA-Based over-expression Vector[J].Plant Physiology,2007(4):1232-1240.
[23]孙淼,荆陈沉,楚成茹,等.重庆辣椒上番茄斑萎病毒的血清学检测及分子鉴定[J].园艺学报,2017,44(3):487-494.
[24]于翠,邓凤林,杨翠云,等.番茄斑萎病毒外壳蛋白原核表达及Dot-blot ELISA检测方法的建立[J].浙江大学学报,2008,34(6):597-601.