越南水稻黄矮病毒多克隆血清抗体的制备（英文）
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摘要
【目的】制备用于检测水稻黄矮病毒(RYSV)的多克隆抗体,为水稻和黑尾叶蝉上的病毒诊断提供技术支持。【方法】利用两种不同来源的RYSV抗原免疫家兔,一种是从受感染的水稻病叶组织纯化获得RYSV病毒粒子;另一种是从越南RYSV分离株克隆出完整N蛋白编码基因,然后将其连接至pET-28a载体上,在大肠杆菌Rosetta(DE3)菌株中进行诱导表达获得N蛋白抗原。其中,N蛋白抗原又以两种形式(洗脱纯化N蛋白和N蛋白条带聚丙烯酰胺切片匀浆)对家兔进行免疫。最后,采用PTA-ELISA分析评估家兔抗体血清(多克隆抗体)对水稻叶片和黑尾叶蝉RYSV的特异性和敏感性。【结果】分离自越南RYSV分离株的N基因由1542个核苷酸组成,与来自我国和日本RYS分离株N基因序列进行比对,其核苷酸序列同源性分别为98.1%和97.9%,对应的推导氨基酸序列同源性为83.4%和99.4%。以RYSV病毒粒子、洗脱纯化N蛋白和N蛋白条带聚丙烯酰胺切片匀浆3种抗原免疫家兔获得的多克隆抗体均能有效检测出水稻植株中的RYSV,其中,感病植株的OD405分别为1.449、2.337和1.649,健康植株的OD405分别为0.375、0.294和0.283。PTA-ELISA检测结果表明,获得的多克隆抗体能从单个带病毒黑尾叶蝉中检测出RYSV,且该结论在RT-PCR检测中得到进一步验证。【结论】制备获得的多克隆抗体对RYSV具有较高特异性和敏感性,可用于水稻和黑尾叶蝉上的病毒诊断,同时表明以含有病毒植物蛋白的聚丙烯酰胺切片直接注射免疫模型动物制备多克隆抗体具有可行性。
【Objective】Polyclonal antibodies specific to rice yellow stunt virus(RYSV)was prepared to provide technique support for diagnosis of this virus in rice plants and green leafhopper vectors(Nephotettix spp.).【Method】Two sources of the RYSV antigens were prepared for the rabbit immunization. The first one was the RYSV virion purified from the infected rice leaf tissue. The second one was the complete N protein of one RYSV isolate from Vietnam,which was cloned in the pET28 a vector and expressed in Escherichia coli strain Rosetta(DE3). The N protein antigen was used in two forms,the eluted fraction of purified N protein and the homogenate of polyacrylamide slices containing N protein band. The specificity and sensitivity of the antisera and antibodies for detection of RYSV from rice leaves and rice green leafhopper vector individuals were evaluated by a plate trapped antigen enzyme linked immunosorbent assay(PTA-ELISA)method.【Result】In this study the N gene of the RYSV isolate from Vietnam was composed of 1542 nucleotides,shared sequence identities of 98.1 % and 97.9%(nucleotide level)and 83.4 % and 99.4 %(amino acid level)with the two RYSV isolates from China and Japan,respectively,available in the GenBank. All three antisera produced in rabbits immunized with the virion,purified N protein and homogenate of polyacrylamide slices containing N protein band were able to detect RYSV in infected rice plants with the PTA-ELISA absorbance values(OD405)were 1.449,2.337 and 1.649,respectively,while those values in healthy plants were 0.375,0.294 and 0.283,respectively. The antisera were able to detect RYSV in a single viruliferous green leafhopper vector in PTA-ELISA test as confirmed by reverse transcriptase PCR(RT-PCR)test.【Conclusion】In conclusion,the rabbit polyclonal antisera produced in this study had high specificity and sensitivity again RYSV,and therefore they can be used for diagnosis of this virus from rice plants and green leafhopper vectors. This study also indicates that the polyacrylamide slices containing a viral protein band can be used for direct injection of animal for production of diagnostic polyclonal antibody.
【Objective】Polyclonal antibodies specific to rice yellow stunt virus(RYSV)was prepared to provide technique support for diagnosis of this virus in rice plants and green leafhopper vectors(Nephotettix spp.).【Method】Two sources of the RYSV antigens were prepared for the rabbit immunization. The first one was the RYSV virion purified from the infected rice leaf tissue. The second one was the complete N protein of one RYSV isolate from Vietnam,which was cloned in the pET28 a vector and expressed in Escherichia coli strain Rosetta(DE3). The N protein antigen was used in two forms,the eluted fraction of purified N protein and the homogenate of polyacrylamide slices containing N protein band. The specificity and sensitivity of the antisera and antibodies for detection of RYSV from rice leaves and rice green leafhopper vector individuals were evaluated by a plate trapped antigen enzyme linked immunosorbent assay(PTA-ELISA)method.【Result】In this study the N gene of the RYSV isolate from Vietnam was composed of 1542 nucleotides,shared sequence identities of 98.1 % and 97.9%(nucleotide level)and 83.4 % and 99.4 %(amino acid level)with the two RYSV isolates from China and Japan,respectively,available in the GenBank. All three antisera produced in rabbits immunized with the virion,purified N protein and homogenate of polyacrylamide slices containing N protein band were able to detect RYSV in infected rice plants with the PTA-ELISA absorbance values(OD405)were 1.449,2.337 and 1.649,respectively,while those values in healthy plants were 0.375,0.294 and 0.283,respectively. The antisera were able to detect RYSV in a single viruliferous green leafhopper vector in PTA-ELISA test as confirmed by reverse transcriptase PCR(RT-PCR)test.【Conclusion】In conclusion,the rabbit polyclonal antisera produced in this study had high specificity and sensitivity again RYSV,and therefore they can be used for diagnosis of this virus from rice plants and green leafhopper vectors. This study also indicates that the polyacrylamide slices containing a viral protein band can be used for direct injection of animal for production of diagnostic polyclonal antibody.
引文
Amero S A,James T C,Elgin S C.2002.Production of antibodies using proteins in gel bands[M]//Waker J M.The protein protocols handbook.The 2ndEdition.Totowa:Humana Press.
Dietzgen R,Calisher C,Kurath G,Kuzmin I,Rodriguez L,Stone D.2011.The Family Rhadoviridae[R]//King A MQ,Lefkowitz E,Adams M J,Carstens E B.Virus taxonomy:Classification and nomenclature of viruses:Ninth Report of the International Committee on Taxonomy of Viruses.Amsterdam:Elsevier.
Fang R X,Wang Q,Xu B Y,Pang Z,Zhu H T,Mang K,Gao D M,Qin W S,Chua N H.1994.Structure of the nucleocapsid protein gene of rice yellow stunt rhabdovirus[J].Virology,204(1):367-375.
Gopal G J,Kumar A.2013.Strategies for the production of recombinant protein in Escherichia coli[J].The Protein Journal,32(6):419-425.
Ha V C,Le V H,Nguyen V H,Vu T M.2010.Identification of causative agent of rice yellow stunt disease in Bac Giang Province in summer season in 2010[J].Journal of Plant Protection(in Vietnamese),228:24-31.
Hibino H.1996.Biology and epidemiology of rice viruses[J].Annual Review of Phytopathology,34:249-274.
Hiraguri A,Hibino H,Hayashi T,Shimizu T,Uehara-Ichiki T,Omura T,Sasaya T.2010.Complete sequence analysis of rice transitory yellowing virus and its comparison to rice yellow stunt virus[J].Archives of Virology,155:243-245.
Huang Y W,Zhao H,Luo Z L,Chen X Y,Fang R X.2003.Novel structure of the genome of rice yellow stunt virus:I-dentification of the gene 6-encoded virion protein[J].Journal of General Virology,84:2259-2264.
Inoue H,Nojima H,Okayama H.1990.High efficiency transformation of Escherichia coli with plasmids[J].Gene,96(1900):23-28.
Jackson A O,Francki R I B,Zuidema D.1987.Biology,structure,and replication of plant rhabdoviruses[M]//Wagner R R.The rhabdoviruses.Berlin:Springer.
Jackson A O,Dietzgen R G,Goodin M M,Bragg J N,Deng M.2005.Biology of plant rhabdoviruses[J].Annual Review of Phytopathology,43:623-660.
Jackson A O,Dietzgen R G,Goodin M M,Li Z.2018.Development of model systems for plant rhabdovirus research[J].Advances in Virus Research,102:23-57.
Khow O,Suntrarachun S.2012.Strategies for production of active eukaryotic proteins in bacterial expression system[J].Asian Pacific Journal of Tropical Biomedicine,2(2):159-162.
Larkin M A,Blackshields G,Brown N P,Chenna R,Mc Gettigan P A,McWilliam H,Valetin F,Wallace I M,Wilm A,Lopez R,Thompson J D,Gibson T J,Higgins D G.2007.ClustalW and ClustalX version 2.0[J].Bioinformatics,23:2947-2948.
Le D T,Netsu O,Uehara-Ichiki T,Shimizu T,Choi I R,Omura T,Sasaya T.2010.Molecular detection of nine rice viruses by a reverse-transcription loop-mediated isothermal amplification assay[J].Journal of Virological Methods,170(1-2):90-93.
Lima J A A,Nascimento A K Q,Radaelli P,Purcifull D E.2012.Serology applied to plant virology[M]//Al-Moslih M.Serological diagnosis of certain human,animal and plant diseases.London:IntechOpen.
Lockhart B E L,Khaless N,Maataoui M E L,Lastra R.1985.Cynodon chlorotic streak virus,a previously undescribed plant rhabdovirus infecting Bermuda grass and maize in the Mediterranean area[J].Phytopathology,75:1094-1098.
Luo Z,Chen X,Gao D.Fang R.1998.The gene 4 of rice yellow stunt rhabdovirus encodes the matrix protein[J].Virus Genes,16(3):277-280.
Luo Z L,Fang R X.1998.Structure analysis of the rice yellow stunt rhabdovirus glycoprotein gene and its mRNA[J].Archives of Virology,143(12):2453-2459.
Mowat W P,Dawson S.1987.Detection and identification of plant viruses by ELISA using crude sap extracts and unfractionated antisera[J].Journal of Virological Methods,15:233-247.
Ou S H.1985.Rice diseases[M].The 2ndEdition.Surrey:Commonwealth Mycological Institute Publication.
Rosano G L,Ceccarelli E A.2014.Recombinant protein expression in Escherichia coli:Advances and challenges[J].Frontiers in Microbiology,5:172.
Rybicki E,von Wechmar M,Burger J.1990.Monospecific antibody preparation for use in the detection of viruses[M]//Burnett P A.World Perspectives on Barley Yellow Dwarf.Mexico:International Maize and Wheat Improvement Center.
Souiri A,Zemzami M,Amzazi S,Ennaji M M.2014.Polyclonal and monoclonal antibody-based methods for detection of plant viruses[J].European Journal of Scientific Research,123:281-295.
Takahashi Y,Omura T,Hayashi T,Shohara K,Tsuchizaki T.1988.Detection of rice transitory yellowing virus(RTYV)in infected rice plants and insect vectors by simplified ELISA[J].Annals of the Phytopathological Society of Japan,54:217-219.
Tamura K,Stecher G,Peterson D,Filipski A,Kumar S.2013.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,30:2725-2729.
Uehara-Ichiki T,Shiba T,Matsukura K,Ueno T,Hirae M,Sasaya T.2013.Detection and diagnosis of rice-infecting viruses[J].Frontiers in Microbiology,4:289.
van Regenmortel M H.2014.Specificity,polyspecificity,and heterospecificity of antibody-antigen recognition[J].Journal of Molecular Recognition,27(11):627-639.
Wang Q,Chen X,Luo Z,Fang R.1999.Sequence analysis of leader and trailer regions of rice yellow stunt rhabdovirus and characterization of their in vivo transcripts[J].Science in China.Series C:Life sciences,42:50-56.
Dietzgen R,Calisher C,Kurath G,Kuzmin I,Rodriguez L,Stone D.2011.The Family Rhadoviridae[R]//King A MQ,Lefkowitz E,Adams M J,Carstens E B.Virus taxonomy:Classification and nomenclature of viruses:Ninth Report of the International Committee on Taxonomy of Viruses.Amsterdam:Elsevier.
Fang R X,Wang Q,Xu B Y,Pang Z,Zhu H T,Mang K,Gao D M,Qin W S,Chua N H.1994.Structure of the nucleocapsid protein gene of rice yellow stunt rhabdovirus[J].Virology,204(1):367-375.
Gopal G J,Kumar A.2013.Strategies for the production of recombinant protein in Escherichia coli[J].The Protein Journal,32(6):419-425.
Ha V C,Le V H,Nguyen V H,Vu T M.2010.Identification of causative agent of rice yellow stunt disease in Bac Giang Province in summer season in 2010[J].Journal of Plant Protection(in Vietnamese),228:24-31.
Hibino H.1996.Biology and epidemiology of rice viruses[J].Annual Review of Phytopathology,34:249-274.
Hiraguri A,Hibino H,Hayashi T,Shimizu T,Uehara-Ichiki T,Omura T,Sasaya T.2010.Complete sequence analysis of rice transitory yellowing virus and its comparison to rice yellow stunt virus[J].Archives of Virology,155:243-245.
Huang Y W,Zhao H,Luo Z L,Chen X Y,Fang R X.2003.Novel structure of the genome of rice yellow stunt virus:I-dentification of the gene 6-encoded virion protein[J].Journal of General Virology,84:2259-2264.
Inoue H,Nojima H,Okayama H.1990.High efficiency transformation of Escherichia coli with plasmids[J].Gene,96(1900):23-28.
Jackson A O,Francki R I B,Zuidema D.1987.Biology,structure,and replication of plant rhabdoviruses[M]//Wagner R R.The rhabdoviruses.Berlin:Springer.
Jackson A O,Dietzgen R G,Goodin M M,Bragg J N,Deng M.2005.Biology of plant rhabdoviruses[J].Annual Review of Phytopathology,43:623-660.
Jackson A O,Dietzgen R G,Goodin M M,Li Z.2018.Development of model systems for plant rhabdovirus research[J].Advances in Virus Research,102:23-57.
Khow O,Suntrarachun S.2012.Strategies for production of active eukaryotic proteins in bacterial expression system[J].Asian Pacific Journal of Tropical Biomedicine,2(2):159-162.
Larkin M A,Blackshields G,Brown N P,Chenna R,Mc Gettigan P A,McWilliam H,Valetin F,Wallace I M,Wilm A,Lopez R,Thompson J D,Gibson T J,Higgins D G.2007.ClustalW and ClustalX version 2.0[J].Bioinformatics,23:2947-2948.
Le D T,Netsu O,Uehara-Ichiki T,Shimizu T,Choi I R,Omura T,Sasaya T.2010.Molecular detection of nine rice viruses by a reverse-transcription loop-mediated isothermal amplification assay[J].Journal of Virological Methods,170(1-2):90-93.
Lima J A A,Nascimento A K Q,Radaelli P,Purcifull D E.2012.Serology applied to plant virology[M]//Al-Moslih M.Serological diagnosis of certain human,animal and plant diseases.London:IntechOpen.
Lockhart B E L,Khaless N,Maataoui M E L,Lastra R.1985.Cynodon chlorotic streak virus,a previously undescribed plant rhabdovirus infecting Bermuda grass and maize in the Mediterranean area[J].Phytopathology,75:1094-1098.
Luo Z,Chen X,Gao D.Fang R.1998.The gene 4 of rice yellow stunt rhabdovirus encodes the matrix protein[J].Virus Genes,16(3):277-280.
Luo Z L,Fang R X.1998.Structure analysis of the rice yellow stunt rhabdovirus glycoprotein gene and its mRNA[J].Archives of Virology,143(12):2453-2459.
Mowat W P,Dawson S.1987.Detection and identification of plant viruses by ELISA using crude sap extracts and unfractionated antisera[J].Journal of Virological Methods,15:233-247.
Ou S H.1985.Rice diseases[M].The 2ndEdition.Surrey:Commonwealth Mycological Institute Publication.
Rosano G L,Ceccarelli E A.2014.Recombinant protein expression in Escherichia coli:Advances and challenges[J].Frontiers in Microbiology,5:172.
Rybicki E,von Wechmar M,Burger J.1990.Monospecific antibody preparation for use in the detection of viruses[M]//Burnett P A.World Perspectives on Barley Yellow Dwarf.Mexico:International Maize and Wheat Improvement Center.
Souiri A,Zemzami M,Amzazi S,Ennaji M M.2014.Polyclonal and monoclonal antibody-based methods for detection of plant viruses[J].European Journal of Scientific Research,123:281-295.
Takahashi Y,Omura T,Hayashi T,Shohara K,Tsuchizaki T.1988.Detection of rice transitory yellowing virus(RTYV)in infected rice plants and insect vectors by simplified ELISA[J].Annals of the Phytopathological Society of Japan,54:217-219.
Tamura K,Stecher G,Peterson D,Filipski A,Kumar S.2013.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,30:2725-2729.
Uehara-Ichiki T,Shiba T,Matsukura K,Ueno T,Hirae M,Sasaya T.2013.Detection and diagnosis of rice-infecting viruses[J].Frontiers in Microbiology,4:289.
van Regenmortel M H.2014.Specificity,polyspecificity,and heterospecificity of antibody-antigen recognition[J].Journal of Molecular Recognition,27(11):627-639.
Wang Q,Chen X,Luo Z,Fang R.1999.Sequence analysis of leader and trailer regions of rice yellow stunt rhabdovirus and characterization of their in vivo transcripts[J].Science in China.Series C:Life sciences,42:50-56.
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