灰飞虱内生病毒HiPV外壳蛋白VP1多克隆抗体的制备及在病毒检测中的应用
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摘要
【目的】前期发现水稻条纹病毒(rice stripe virus,RSV)可与介体灰飞虱Laodelphax striatellus体内的Hi PV病毒(Himetobi P virus,Hi PV)互作。本研究旨在制备Hi PV外壳蛋白VP1的多克隆抗体,并评估其在Hi PV病毒检测中的可用性,以为深入研究Hi PV-RSV和Hi PV-灰飞虱的互作机制提供技术支持。【方法】以RT-PCR方法从灰飞虱成虫体内扩增Hi PV主要外壳蛋白基因VP1,然后将VP1基因亚克隆至原核表达载体p ET-32a中,构建表达载体p ET-VP1。将重组质粒转化大肠杆菌Escherichia coli BL21 (DE3),经IPTG诱导、Ni2+-NTA亲和层析纯化,获得重组蛋白,免疫新西兰大白兔,制备抗体。【结果】从灰飞虱体内克隆到774 bp的Hi PV外壳蛋白基因VP1,经原核表达、纯化,获得分子量约47. 5 kD的融合蛋白,免疫新西兰大白兔后获得VP1多克隆抗体。该抗体间接ELISA效价达1∶819 200,与Hi PV外壳蛋白VP1有特异性反应,而与灰飞虱蛋白无交叉反应。利用该多克隆抗体建立了检测单头灰飞虱成虫体内Hi PV的Western blot和免疫捕获RT-PCR方法,检测结果显示Hi PV在携带和不携带RSV的灰飞虱高亲和性群体内均广泛存在。【结论】利用制备的Hi PV的VP1多克隆抗体可特异性检测灰飞虱体内Hi PV。本研究为Hi PV病毒的快速检测以及Hi PV-RSV互作、Hi PV-灰飞虱互作研究提供了技术支持。
【Aim】In our previous work,the interaction between rice stripe virus( RSV) and Himetobi P virus( Hi PV) of the small brown planthopper( SBPH),Laodelphax striatellus,was found. This study aims to prepare the polyclonal antibody( PAb) of the capsid protein VP1 of Hi PV and to assess its application in the detection of Hi PV,so as to provide a technical basis for further studying the interaction of Hi PV-RSV and Hi PV-SBPH. 【Methods】VP1 gene of Hi PV was amplified from SBPH adult via RTPCR,and subcloned into the prokaryotic expression vector pET-32 a to construct the vector p ET-VP1.The recombinant plasmid was transformed into Escherichia coli BL21( DE3),and the recombinant protein was obtained by IPTG induction and purified by Ni2 +-NTA affinity chromatography. The antibody was prepared by immunizing New Zealand white rabbit with the purified recombinant protein. 【Results】A 774 bp product of capsid protein gene VP1 of Hi PV was amplified from SBPH,and a 47. 5 kD VP1 fusion protein was obtained through prokaryotic expression and purification. The purified VP1 recombinant protein was used to immunize New Zealand white rabbit,and the PAb was prepared. The titer of PAb was 1∶ 819 200 in indirect ELISA,and the PAb could react specifically with Hi PV VP1 but not with SBPH proteins. Using PAb against Hi PV,methods of Western blot and immunocapture-RT-PCR( IC-RT-PCR) for the detection of Hi PV in single adult of SBPH were established. The detection results showed that Hi PV was prevalent in the RSV-infected and uninfected SBPH high-affinity populations.【Conclusion】Hi PV can be detected specifically in SBPH using the prepared VP1 polyclonal antibody.This study provides a technical support for the rapid detection of Hi PV and the study of Hi PV-RSV and Hi PV-SBPH interaction.
【Aim】In our previous work,the interaction between rice stripe virus( RSV) and Himetobi P virus( Hi PV) of the small brown planthopper( SBPH),Laodelphax striatellus,was found. This study aims to prepare the polyclonal antibody( PAb) of the capsid protein VP1 of Hi PV and to assess its application in the detection of Hi PV,so as to provide a technical basis for further studying the interaction of Hi PV-RSV and Hi PV-SBPH. 【Methods】VP1 gene of Hi PV was amplified from SBPH adult via RTPCR,and subcloned into the prokaryotic expression vector pET-32 a to construct the vector p ET-VP1.The recombinant plasmid was transformed into Escherichia coli BL21( DE3),and the recombinant protein was obtained by IPTG induction and purified by Ni2 +-NTA affinity chromatography. The antibody was prepared by immunizing New Zealand white rabbit with the purified recombinant protein. 【Results】A 774 bp product of capsid protein gene VP1 of Hi PV was amplified from SBPH,and a 47. 5 kD VP1 fusion protein was obtained through prokaryotic expression and purification. The purified VP1 recombinant protein was used to immunize New Zealand white rabbit,and the PAb was prepared. The titer of PAb was 1∶ 819 200 in indirect ELISA,and the PAb could react specifically with Hi PV VP1 but not with SBPH proteins. Using PAb against Hi PV,methods of Western blot and immunocapture-RT-PCR( IC-RT-PCR) for the detection of Hi PV in single adult of SBPH were established. The detection results showed that Hi PV was prevalent in the RSV-infected and uninfected SBPH high-affinity populations.【Conclusion】Hi PV can be detected specifically in SBPH using the prepared VP1 polyclonal antibody.This study provides a technical support for the rapid detection of Hi PV and the study of Hi PV-RSV and Hi PV-SBPH interaction.
引文
Bian G,Xu Y,Lu P,Xie Y,Xi Z,2010.The endosymbiotic bacterium Wolbachia induces resistance to dengue virus in Aedes aegypti.PLoSPathog.,6(4):e1000833.
Dutra HLC,Rocha MN,Dias FBS,Mansur SB,Caragata EP,Moreira LA,2016.Wolbachia blocks currently circulating Zika virus isolates in Brazilian Aedes aegypti mosquitoes.Cell Host Microbe,19(6):771-774.
Fang S,Yu J,Feng J,Han C,Li D,Liu Y,2001.Identification of rice black-streaked dwarf fijivirus in maize with rough dwarf disease in China.Arch.Virol.,146(1):167-170.
Gong ZX,Zheng QX,Peng H,Tsao TC,Eishiro S,1985.Study on relationship between WRSV in China and NCMV in Japan.Chin.J.Virol.,1(3):257-261.[龚祖埙,郑巧兮,彭海,曹天钦,四方英四郎,1985.中国小麦丛矮病毒与日本北方禾谷花叶病毒相关性的研究.病毒学报,1(3):257-261]
Guy PL,Toriyama S,Fuji S,1992.Occurrence of a picorna-like virus in planthopper species and its transmission in Laodelphax striatellus.J.Invertebr.Pathol.,59(2):161-164.
Jia D,Mao Q,Chen Y,Liu Y,Chen Q,Wu W,Zhang X,Chen H,Li Y,Wei T,2017.Insect symbiotic bacteria harbour viral pathogens for transovarial transmission.Nat.Microbiol.,2(5):17025.
Jiang JX,Chen ZX,Zhou XP,2003.Production of a monoclonal antibody to Sugarcane mosaic virus and its application for virus detection in China.J.Phytopathol.,151(6):361-364.
Le WJ,Shi WQ,Ji YH,Zhou YJ,2011.Relationship between Wolbachia and rice stripe virus infection in Laodelphax striatellus.J.Nanjing Agric.Univ.,34(1):46-50.[乐文静,史文琦,季英华,周益军,2011.灰飞虱体内Wolbachia的感染与其携带水稻条纹病毒的相关性分析.南京农业大学学报,34(1):46-50]
Li S,Ge S,Wang X,Sun L,Liu Z,Zhou Y,2015.Facilitation of rice stripe virus accumulation in the insect vector by Himetobi P virus VP1.Viruses,7(3):1492-1504.
Li S,Li X,Zhou Y,2018.Ribosomal protein L18 is an essential factor that promote rice stripe virus accumulation in small brown planthopper.Virus Res.,247:15-20.
Li S,Zhou C,Chen G,Zhou Y,2017.Bacterial microbiota in small brown planthopper populations with different rice viruses.J.Basic Microbiol.,57(7):590-596.
Liu HJ,Cheng ZB,Wang Y,Wei BQ,Ren CM,Zhou YJ,Fan YJ,2007.Preliminary study on transmission of rice stripe virus by small brown planthopper.Jiangsu J.Agric.Sci.,23(5):492-494.[刘海建,程兆榜,王跃,魏邦庆,任春梅,周益军,范永坚,2007.灰飞虱传递水稻条纹病毒研究初报.江苏农业学报,23(5):492-494]
Morin S,Ghanim M,Zeidan M,Czosnek H,Verbeek M,van den Heuvel JFJM,1999.A Gro EL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus.Virology,256(1):75-84.
Nakashima N,Sasaki J,Toriyama S,1999.Determining the nucleotide sequence and capsid-coding region of Himetobi P virus:a member of a novel group of RNA viruses that infect insects.Arch.Virol.,144(10):2051-2058.
Shang HL,Zhou XP,Wu JX,2010.Polyclonal antibody-based dotELISA and immunocapture-RT-PCR for Cucumber green mottle mosaic virus detection.J.Zhejiang Univ.(Agric.Life Sci.),36(5):485-490.[尚海丽,周雪平,吴建祥,2010.免疫斑点法和免疫捕获RT-PCR检测黄瓜绿斑驳花叶病毒.浙江大学学报(农业与生命科学版),36(5):485-490]
Suzuki Y,Toriyama S,Matsuda I,Kojima M,1993.Detection of a picorna-like virus,Himetobi P virus,in organs and tissues of Laodelphax striatellus by immunogold labeling and enzyme-linked immunosorbent assay.J.Invertebr.Pathol.,62(1):99-104.
van den Heuvel JFJM,Bruyère A,Hogenhout SA,Ziegler-Graff V,Brault V,Verbeek M,van den Wilk F,Richards K,1997.The N-terminal region of the luteovirus readthrough domain determines virus binding to Buchnera Gro EL and is essential for virus persistence in the aphid.J.Virol.,71(10):7258-7265.
Wei TY,Li Y,2016.Rice reoviruses in insect vectors.Annu.Rev.Phytopathol.,54:99-120.
Zhou YJ,Li S,Cheng ZB,Zhou T,Fan YJ,2012.Research advances in rice stripe disease in China.Jiangsu J.Agric.Sci.,28(5):1007-1015.[周益军,李硕,程兆榜,周彤,范永坚,2012.中国水稻条纹叶枯病研究进展.江苏农业学报,28(5):1007-1015]
Dutra HLC,Rocha MN,Dias FBS,Mansur SB,Caragata EP,Moreira LA,2016.Wolbachia blocks currently circulating Zika virus isolates in Brazilian Aedes aegypti mosquitoes.Cell Host Microbe,19(6):771-774.
Fang S,Yu J,Feng J,Han C,Li D,Liu Y,2001.Identification of rice black-streaked dwarf fijivirus in maize with rough dwarf disease in China.Arch.Virol.,146(1):167-170.
Gong ZX,Zheng QX,Peng H,Tsao TC,Eishiro S,1985.Study on relationship between WRSV in China and NCMV in Japan.Chin.J.Virol.,1(3):257-261.[龚祖埙,郑巧兮,彭海,曹天钦,四方英四郎,1985.中国小麦丛矮病毒与日本北方禾谷花叶病毒相关性的研究.病毒学报,1(3):257-261]
Guy PL,Toriyama S,Fuji S,1992.Occurrence of a picorna-like virus in planthopper species and its transmission in Laodelphax striatellus.J.Invertebr.Pathol.,59(2):161-164.
Jia D,Mao Q,Chen Y,Liu Y,Chen Q,Wu W,Zhang X,Chen H,Li Y,Wei T,2017.Insect symbiotic bacteria harbour viral pathogens for transovarial transmission.Nat.Microbiol.,2(5):17025.
Jiang JX,Chen ZX,Zhou XP,2003.Production of a monoclonal antibody to Sugarcane mosaic virus and its application for virus detection in China.J.Phytopathol.,151(6):361-364.
Le WJ,Shi WQ,Ji YH,Zhou YJ,2011.Relationship between Wolbachia and rice stripe virus infection in Laodelphax striatellus.J.Nanjing Agric.Univ.,34(1):46-50.[乐文静,史文琦,季英华,周益军,2011.灰飞虱体内Wolbachia的感染与其携带水稻条纹病毒的相关性分析.南京农业大学学报,34(1):46-50]
Li S,Ge S,Wang X,Sun L,Liu Z,Zhou Y,2015.Facilitation of rice stripe virus accumulation in the insect vector by Himetobi P virus VP1.Viruses,7(3):1492-1504.
Li S,Li X,Zhou Y,2018.Ribosomal protein L18 is an essential factor that promote rice stripe virus accumulation in small brown planthopper.Virus Res.,247:15-20.
Li S,Zhou C,Chen G,Zhou Y,2017.Bacterial microbiota in small brown planthopper populations with different rice viruses.J.Basic Microbiol.,57(7):590-596.
Liu HJ,Cheng ZB,Wang Y,Wei BQ,Ren CM,Zhou YJ,Fan YJ,2007.Preliminary study on transmission of rice stripe virus by small brown planthopper.Jiangsu J.Agric.Sci.,23(5):492-494.[刘海建,程兆榜,王跃,魏邦庆,任春梅,周益军,范永坚,2007.灰飞虱传递水稻条纹病毒研究初报.江苏农业学报,23(5):492-494]
Morin S,Ghanim M,Zeidan M,Czosnek H,Verbeek M,van den Heuvel JFJM,1999.A Gro EL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus.Virology,256(1):75-84.
Nakashima N,Sasaki J,Toriyama S,1999.Determining the nucleotide sequence and capsid-coding region of Himetobi P virus:a member of a novel group of RNA viruses that infect insects.Arch.Virol.,144(10):2051-2058.
Shang HL,Zhou XP,Wu JX,2010.Polyclonal antibody-based dotELISA and immunocapture-RT-PCR for Cucumber green mottle mosaic virus detection.J.Zhejiang Univ.(Agric.Life Sci.),36(5):485-490.[尚海丽,周雪平,吴建祥,2010.免疫斑点法和免疫捕获RT-PCR检测黄瓜绿斑驳花叶病毒.浙江大学学报(农业与生命科学版),36(5):485-490]
Suzuki Y,Toriyama S,Matsuda I,Kojima M,1993.Detection of a picorna-like virus,Himetobi P virus,in organs and tissues of Laodelphax striatellus by immunogold labeling and enzyme-linked immunosorbent assay.J.Invertebr.Pathol.,62(1):99-104.
van den Heuvel JFJM,Bruyère A,Hogenhout SA,Ziegler-Graff V,Brault V,Verbeek M,van den Wilk F,Richards K,1997.The N-terminal region of the luteovirus readthrough domain determines virus binding to Buchnera Gro EL and is essential for virus persistence in the aphid.J.Virol.,71(10):7258-7265.
Wei TY,Li Y,2016.Rice reoviruses in insect vectors.Annu.Rev.Phytopathol.,54:99-120.
Zhou YJ,Li S,Cheng ZB,Zhou T,Fan YJ,2012.Research advances in rice stripe disease in China.Jiangsu J.Agric.Sci.,28(5):1007-1015.[周益军,李硕,程兆榜,周彤,范永坚,2012.中国水稻条纹叶枯病研究进展.江苏农业学报,28(5):1007-1015]
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