西瓜花叶病毒抗血清制备及其应用
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摘要
西瓜花叶病毒(Watermelon mosaic virus,WMV)是危害葫芦科作物的重要病毒。制备特异性强、效价高的抗血清对快速准确检测和鉴定WMV具有重要意义。本研究将WMV外壳蛋白(Coat protein,CP)基因克隆到原核表达载体pEHISTEV获得pEHISTEV-WMV-CP。将pEHISTEV-WMV-CP转化大肠杆菌Rosetta,经IPTG诱导,成功表达出分子量约为36 kDa的蛋白,与预期WMV CP大小一致。切胶回收WMV CP,与等体积弗氏不完全佐剂充分乳化后免疫健康新西兰大白兔。Western blotting结果表明,制备的抗血清与WMV CP有反应,与同属的番木瓜环斑病毒(Papaya ringspot virus,PRSV)、马铃薯Y病毒(Potato virus Y,PVY)、小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)和烟草花叶病毒属的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)均无反应。酶联免疫吸附测定(PTA-ELISA)结果表明,本研究制备的WMV抗血清效价为1∶8192,并且能够检测稀释512倍的病毒汁液。利用该抗血清对田间采集的10个RT-PCR检测为阳性的样品进行检测,全部呈现阳性。本研究利用大肠杆菌表达的CP制备的WMV抗血清具有较高的特异性和灵敏度。研究结果为WMV的快速检测和鉴定奠定了基础。
Watermelon mosaic virus( WMV; genus Potyvirus) is an economically important virus infecting cucurbit crops. Preparation of antiserum with high specificity and sensitivity is essential for rapid and accurate detection and identification of WMV. In this study,WMV coat protein( CP) gene was cloned into E. coli expression vector pEHISTEV,named pEHISTEV-WMV-CP. pEHISTEV-WMV-CP was transformed and expressed into E. coli Rosetta. A protein with expected molecular weight of 36 kDa was obtained after IPTG induction. The band was cut and used to immune New Zealand white rabbit. Western blotting results showed that the antiserum could specifically recognize WMV CP expressed in E. coli and WMV from infected leaves. There was no cross reaction to closely related Papaya ringspot virus( PRSV),Potato virus Y( PVY),Zucchini yellow mosaic virus( ZYMV) from the genus Potyvirus,neither the Cucumber green mottle mosaic virus( CGMMV) of the genus Tobamovirus. The antiserum had a titer of 1 ∶ 8192 in PTA-ELISA and could detect WMV infected samplesdiluted up to 512 times. Among the 10 samples showed positive reaction in RT-PCR,all of them were positive when detected with this antiserum via PTA-ELISA. These results indicated that the antiserum obtained in our study had high specificity and sensitivity and laid solid foundation for rapid and accurate detection and identification of WMV.
Watermelon mosaic virus( WMV; genus Potyvirus) is an economically important virus infecting cucurbit crops. Preparation of antiserum with high specificity and sensitivity is essential for rapid and accurate detection and identification of WMV. In this study,WMV coat protein( CP) gene was cloned into E. coli expression vector pEHISTEV,named pEHISTEV-WMV-CP. pEHISTEV-WMV-CP was transformed and expressed into E. coli Rosetta. A protein with expected molecular weight of 36 kDa was obtained after IPTG induction. The band was cut and used to immune New Zealand white rabbit. Western blotting results showed that the antiserum could specifically recognize WMV CP expressed in E. coli and WMV from infected leaves. There was no cross reaction to closely related Papaya ringspot virus( PRSV),Potato virus Y( PVY),Zucchini yellow mosaic virus( ZYMV) from the genus Potyvirus,neither the Cucumber green mottle mosaic virus( CGMMV) of the genus Tobamovirus. The antiserum had a titer of 1 ∶ 8192 in PTA-ELISA and could detect WMV infected samplesdiluted up to 512 times. Among the 10 samples showed positive reaction in RT-PCR,all of them were positive when detected with this antiserum via PTA-ELISA. These results indicated that the antiserum obtained in our study had high specificity and sensitivity and laid solid foundation for rapid and accurate detection and identification of WMV.
引文
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[4]Xie H,Sun Y,Wei X W,et al. Studies on melon seeds infected by WMV-2(in Chinese)[J]. Xinjiang Farmland Reclamation Science&Technology(新疆农垦科技),1989,4:31-32.
[5]Xie H,Sun Y,Wei X W,et al. Confirmation of sweet melon seeds infected by WMV-2(in Chinese)[J]. Acta Phytopathologica Sinica(植物病理学报),1988,18(4):28.
[6]Purcifull D,Hiebert E,Edwardson J. Watermelon mosaic virus 2 CMI/AAB[Z]. Description of plant viruses,1984:293.
[7]Grafton-Cardwell E E,Perring T M,Smith R F,et al.Occurrence of mosaic viruses in melons in central valley of California[J]. Plant Disease,1996,80(10):1092-1097.
[8]Li X D,Zhu H C,Guo X Q. Research processes on viruses infecting watermelon(in Chinese)[J]. Plant Protection(植物保护),1997,23(4):35-37.
[9]Wei N S,Wu Y F. Studies on virus diseases infecting watermelon and melon in Shaanxi province(in Chinese)[J]. Shaanxi Agricultural Science(陕西农业科学),1993,2:11-12.
[10]Towbin H,Staehelin T,Gordon J,Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications[J].Proceedings of the National Academy of Sciences of the United States of America,1979,76(9):4350-4354.
[11]Chen Z,Song G,Zhou X P,et al. Preparation and application of monoclonal antibodies against Watermelon mosaic virus(in Chinese)[J]. Chinese Agricultural Science(中国农业科学),2016,49(14):2711-2724.
[12]Zhang X J,Liu Q L,Wu Y F. Cloning and prokaryotic expression coat protein gene from Watermelon mosaic virus(in Chinese)[J]. Journal of Henan Normal University(Natural Science)(河南师范大学学报)(自然科学版),2008,36(4):117-119.
[13]Liang X M,Li G F,Ma J,et al. Preparation of the monoclonal antibody against Watermelon mosaic virus and TAS-ELISA kit(in Chinese)[J]. Plant Quarantine(植物检疫),2013,27(2):53-56.
[2]Li X D,Zhu H C,Yan D Y,et al. Identification of viruses infecting watermelon(Citrullus vulgaris)in Shandong province(in Chinese)[J]. Journal of Shandong Agricultural University(山东农业大学学报),1995,3:299-306.
[3]Xie H. The study of new vector of WMV-2 and CMVAphis umbrella(Borner)(in Chinese)[J]. Journal of Plant Protection(植物保护学报),1989,16(1):72.
[4]Xie H,Sun Y,Wei X W,et al. Studies on melon seeds infected by WMV-2(in Chinese)[J]. Xinjiang Farmland Reclamation Science&Technology(新疆农垦科技),1989,4:31-32.
[5]Xie H,Sun Y,Wei X W,et al. Confirmation of sweet melon seeds infected by WMV-2(in Chinese)[J]. Acta Phytopathologica Sinica(植物病理学报),1988,18(4):28.
[6]Purcifull D,Hiebert E,Edwardson J. Watermelon mosaic virus 2 CMI/AAB[Z]. Description of plant viruses,1984:293.
[7]Grafton-Cardwell E E,Perring T M,Smith R F,et al.Occurrence of mosaic viruses in melons in central valley of California[J]. Plant Disease,1996,80(10):1092-1097.
[8]Li X D,Zhu H C,Guo X Q. Research processes on viruses infecting watermelon(in Chinese)[J]. Plant Protection(植物保护),1997,23(4):35-37.
[9]Wei N S,Wu Y F. Studies on virus diseases infecting watermelon and melon in Shaanxi province(in Chinese)[J]. Shaanxi Agricultural Science(陕西农业科学),1993,2:11-12.
[10]Towbin H,Staehelin T,Gordon J,Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications[J].Proceedings of the National Academy of Sciences of the United States of America,1979,76(9):4350-4354.
[11]Chen Z,Song G,Zhou X P,et al. Preparation and application of monoclonal antibodies against Watermelon mosaic virus(in Chinese)[J]. Chinese Agricultural Science(中国农业科学),2016,49(14):2711-2724.
[12]Zhang X J,Liu Q L,Wu Y F. Cloning and prokaryotic expression coat protein gene from Watermelon mosaic virus(in Chinese)[J]. Journal of Henan Normal University(Natural Science)(河南师范大学学报)(自然科学版),2008,36(4):117-119.
[13]Liang X M,Li G F,Ma J,et al. Preparation of the monoclonal antibody against Watermelon mosaic virus and TAS-ELISA kit(in Chinese)[J]. Plant Quarantine(植物检疫),2013,27(2):53-56.