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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

潘慧 赵忠豪 章松柏 张德咏 张洁 李俊凯 郑立敏

潘慧, 赵忠豪, 章松柏, 张德咏, 张洁, 李俊凯, 郑立敏. 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[J]. 福建农业学报, 2018, 33(9): 963-968. doi: 10.19303/j.issn.1008-0384.2018.09.013
引用本文: 潘慧, 赵忠豪, 章松柏, 张德咏, 张洁, 李俊凯, 郑立敏. 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[J]. 福建农业学报, 2018, 33(9): 963-968. doi: 10.19303/j.issn.1008-0384.2018.09.013
PAN Hui, ZHAO Zhong-hao, ZHANG Song-bai, ZHANG De-yong, ZHANG Jie, LI Jun-kai, ZHENG Li-min. Preparation and Application of Polyclonal Antibodies Against Nucleocapsid Protein N and Envelope Membrane Glycoprotein Gn of Tomato Spotted Wilt Orthotospovirus[J]. Fujian Journal of Agricultural Sciences, 2018, 33(9): 963-968. doi: 10.19303/j.issn.1008-0384.2018.09.013
Citation: PAN Hui, ZHAO Zhong-hao, ZHANG Song-bai, ZHANG De-yong, ZHANG Jie, LI Jun-kai, ZHENG Li-min. Preparation and Application of Polyclonal Antibodies Against Nucleocapsid Protein N and Envelope Membrane Glycoprotein Gn of Tomato Spotted Wilt Orthotospovirus[J]. Fujian Journal of Agricultural Sciences, 2018, 33(9): 963-968. doi: 10.19303/j.issn.1008-0384.2018.09.013

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

doi: 10.19303/j.issn.1008-0384.2018.09.013
基金项目: 

国家自然科学基金 31571981

国家自然科学基金 31501609

国家自然科学基金 31560499

国家自然科学基金 31871935

公益性行业(农业)科研专项 201303028

云南省应用基础研究计划重点项目 2018FA020

湖南省农业科学院科技创新项目 2016QN23

详细信息
    作者简介:

    潘慧(1994-), 女, 硕士研究生, 研究方向:植物病毒与分子生物学(E-mail: 1252085867@qq.com)

    通讯作者:

    李俊凯(1969-), 男, 教授, 博士生导师, 研究方向:农药学(E-mail:junkaili@sina.com)

    郑立敏(1985-), 女, 助理研究员, 研究方向:植物病毒与分子生物学(E-mail:lmzheng66@126.com)

  • 中图分类号: S436.412

Preparation and Application of Polyclonal Antibodies Against Nucleocapsid Protein N and Envelope Membrane Glycoprotein Gn of Tomato Spotted Wilt Orthotospovirus

  • 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NGn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。
  • 图  1  TSWV N(A)和Gn(B)的RT-PCR扩增结果

    注:M为DNA分子标记Trans2k Plus;A为TSWV N基因RT-PCR扩增产物;B为TSWVGn基因RT-PCR扩增产物。

    Figure  1.  RT-PCR amplifications of TSWV N(A) and Gn (B)

    图  2  SDS-PAGE分析TSWV N和Gn蛋白的原核表达

    注: M为10~180 kDa蛋白分子标记; A为TSWV N蛋白的原核表达, 1和2为经IPTG诱导的N蛋白, 3和4为未经IPTG诱导的N蛋白; B为TSWB Gn蛋白的原核表达, 1为经IPTG诱导的Gn蛋白, 2为未经IPTG诱导的Gn蛋白。

    Figure  2.  SDS-PAGE analysis of TSWV N and Gn protein expression

    图  3  Western blot检测N蛋白多克隆抗体的特异性

    注: M为10~180 kDa蛋白分子标记; 1为感染TSWV的辣椒; 2为健康辣椒。

    Figure  3.  Specificity of N polyclonal antibodies detected by western blot

    图  4  N抗体效价的检测

    Figure  4.  Detection of N antibody titer

    图  5  利用N(A)和Gn(B)抗体Dot-ELISA检测辣椒中的TSWV

    注: A和B分别是N和Gn抗体建立的Dot-ELISA检测感病辣椒; 1~3为TSWV感病辣椒; 4~6为健康辣椒。

    Figure  5.  Dot-ELISA analysis of TSWV in pepper by using N(A) or Gn(B) antibody

    表  1  PCR引物

    Table  1.   PCR primers

    引物名称 引物序列5'-3' 引物用途
    N-F GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GTC TAA GGT TAA GCT CAC N的扩增
    N-R GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA AGC AAG TTC TGC AAG TTT TG N gene cloning
    Gn-F GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGA TCA TCC TGA GGT TTA TG Gn的扩增
    Gn-R GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC ACC AGG TTT TTT TAT CAA ATA AC Gn gene cloning
    下载: 导出CSV
  • [1] PARRELLA G, GOGNALONS P, GEBRE-SELASSIè K, et al. An update of the host range of tomato spotted wilt virus[J]. Journal of Plant Pathology, 2003, 85(4):227-264. http://www.jstor.org/stable/41998156
    [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. doi: 10.1094-PD-73-0375/
    [3] SCHOLTHOF K B, ADKINS S, CZOSNEK H, et al. Top 10 plant viruses in molecular plant pathology[J]. Molecular Plant Pathology, 2011, 12(9):938-954. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=6fcee62f0c982d24b26117a9fd857d06
    [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. doi: 10.3969/j.issn.0529-1542.2017.01.043
    [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. doi: 10.1099/0022-1317-72-9-2207
    [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. http://europepmc.org/abstract/MED/12490389
    [9] KORMELINK R, STORMS M, VAN L J, et al. Expression and subcellular location of the NSM protein of tomato spotted wilt virus (TSWV), a putative viral movement protein[J]. Virology, 1994, 200(1):56-65. doi: 10.1006/viro.1994.1162
    [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. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=4a7fe9353a0ae3c440853c1b95c1fccb
    [11] LEWANDOWSKI D J, ADKINS S. The tubule-forming NSm protein from Tomato spotted wilt virus complements cell-to-cell and long-distaNCe movement of Tobacco mosaic virus hybrids[J]. Virology, 2005, 342(1):26. doi: 10.1016/j.virol.2005.06.050
    [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. doi: 10.1016/j.virol.2009.04.027
    [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. http://d.old.wanfangdata.com.cn/OAPaper/oai_pubmedcentral.nih.gov_113997
    [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. doi: 10.1073/pnas.0407354102
    [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. doi: 10.1016-S0014-5793(02)03632-3/
    [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. doi: 10.1128-JVI.77.2.1329-1336.2003/
    [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. doi: 10.1099/0022-1317-71-5-1001
    [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. doi: 10.1094/PHYTO.2004.94.7.759
    [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. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=9359c254d3c0bb2aac59e067cc3d19f6
    [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. doi: 10.1016/j.virol.2008.09.028
    [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. doi: 10.1073/pnas.030548397
    [22] JOHN A L. TRBO:A High-Efficiency Tobacco Mosaic Virus RNA-Based over-expression Vector[J]. Plant Physiology, 2007(4):1232-1240. http://d.old.wanfangdata.com.cn/NSTLQK/NSTL_QKJJ02703031/
    [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. http://d.old.wanfangdata.com.cn/Periodical/yyxb201703008
    [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. http://d.old.wanfangdata.com.cn/Periodical/zjdxxb-nyysm200806003
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  • 收稿日期:  2018-05-18
  • 修回日期:  2018-08-20
  • 刊出日期:  2018-09-01

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