侵染西番莲的三种马铃薯Y病毒属病毒的广谱单克隆抗体制备及检测应用  

Preparation and Detection Application of Broad-spectrum Monoclonal Antibody Against Three Potyviruses Infecting Chinese Passionflower(Passiflora edulis)

作　　者：李雪[1] 李晋海 王兰花 戚朵 郭萌萌 周雪平[1] 吴建祥[1] LI Xue;LI Jin-Hai;WANG Lan-Hua;QI Duo;GUO Meng-Meng;ZHOU Xue-Ping;WU Jian-Xiang(State Key Laboratory of Rice Biology/Institute of Biotechnology,Zhejiang University,Hangzhou 310058,China;Agricultural Green Development Service Center of Qiubei County,Qiubei 663200,China)

机构地区：[1]浙江大学生物技术研究所/水稻生物学国家重点实验室,杭州310058 [2]丘北县农业绿色发展服务中心,丘北663200

出　　处：《农业生物技术学报》2022年第9期1845-1854,共10页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划项目(2019YFD1001800);国家自然科学基金(31772125)。

摘　　要：马铃薯Y病毒属(Potyvirus)的夜来香花叶病毒(Telosma mosaic virus,TelMV)、东亚西番莲病毒(East Asian passiflora virus,EAPV)和西番莲重型斑驳相关病毒(Passion fruit severe mottle-associated virus,PFSMaV)严重危害我国西番莲(Passiflora edulis)产业。迄今植物病毒没有有效的防治药剂,而病毒检测技术是植物病毒防控手段建立的关键。本研究利用田间采集的TelMV、EAPV和PFSMaV三种病毒复合侵染的西番莲,从病叶提纯病毒粒子,以混合病毒粒子作为BALB/c小鼠(Mus musculus)的免疫原,通过杂交瘤技术获得能特异、广谱检测这3种病毒的单克隆抗体。Western blot检测发现,研制的4株单克隆抗体能同时识别TelMV、EAPV和PFSMaV的衣壳蛋白。进一步以制备的病毒单抗为检测抗体,建立用于快速检测这3种病毒的血清学检测方法,即斑点酶联免疫吸附试验(Dot-ELISA)和组织印迹酶联免疫吸附试验(Tissue print-ELISA)。特异性和广谱性测定结果表明,建立的2种血清学方法均能简单快速、特异灵敏、广谱高效地检测西番莲中的TelMV、EAPV和PFSMaV,而检测感染同属的李痘病毒(Plumpox virus,PPV)、马铃薯Y病毒(Potato Virus Y,PVY)、马铃薯A病毒(Potato Virus A,PVA)和芜菁花叶病毒(Turnip mosaic virus,TuMV)病叶、感染黄瓜花叶病毒(Cucumber mosaic virus,CMV)和西番莲潜隐病毒(Passiflora latent virus,PLV)的病叶及健康西番莲叶片均呈阴性反应。灵敏度测定结果表明,Dot-ELISA方法检测病叶粗提液的灵敏度高达1∶20480(W/V,g/mL)倍稀释,即绝对检出量为0.0976 ng感病植物组织。田间样品的检测结果表明,建立的2种血清学方法能准确、广谱、灵敏地用于检测侵染西番莲的3种马铃薯Y病毒属病毒,其结果与RT-PCR的符合率达到100%。本研究为TelMV、EAPV和PFSMaV三种病毒的检测诊断、无毒种子种苗生产及其科学防控提供技术支持。Telosma mosaic virus(TelMV),East Asian passiflora virus(EAPV),and Passion fruit severe mottleassociated virus(PFSMaV)belonging to the genus Potyvirus have seriously damaged the passionflower(Passiflora edulis)industry in China.So far,there are no effective agents for preventing and curing plant viruses.Thus,Developments of viral detection techniques are crucial for establishing scientific prevention and control system of plant viral diseases.In this study,field-collected passionflower leaves,co-infected with TelMV,EAPV and PFSMaV were used for virus particle purification.Purified and mixed virions of these 3potyviruses were used as the immunogen to immunize 4 BALB/c mice(Mus musculus).Four hybridoma strains(11A12,6D8,6D12 and 17E4)secreting specific and broad-spectrum monoclonal antibodies against above 3infecting passionflower plant potyviruses were obtained by the hybridoma technique including cell fusion,hybridoma cell screening,antibody detection,cell cloning and ascites preparation.Western blot assay showed that the 4 monoclonal antibodies could simultaneously recognize capsid proteins of TelMV,EAPV and PFSMaV.Further,the produced monoclonal antibodies was used as the detection antibodies to develop two serological assays,i.e.dot enzyme-linked immunosorbent assay(Dot-ELISA)and Tissue print-ELISA for the broad-spectrum,specific and sensitive detection of these 3 potyviruses.The two established serological methods could simply,quickly,specifically,sensitively,broadly and efficiently detect TelMV,EAPV and PFSMaV in passionflower samples.Meanwhile,there were negative reactions when they detected the diseased leaves infected with Plumpox virus(PPV),Potato virus Y(PVY),or Potato virus A(PVA),Turnip mosaic virus(TuMV),Passiflora latent virus(PLV),Cucumber mosaic virus(CMV)or uninfected passionflower leaves.Sensitivity analysis assays showed that the virus could be detected in infected plant crude extracts diluted up to1∶20480(W/V,g/mL),i.e.in 0.0976 ng virus-infected plant tissues of the absolute detection amount

关 键 词：马铃薯Y病毒属 夜来香花叶病毒(TelMV) 东亚西番莲病毒(EAPV) 西番莲重型斑驳相关病毒(PFSMaV) 单克隆抗体 斑点ELISA(Dot-ELISA) 组织印迹ELISA(Tissue print-ELISA) 

分 类 号：S643.6[农业科学—蔬菜学]