马铃薯Y病毒单克隆抗体的制备及其检测应用  

Preparation and detection application of monoclonal antibodies against Potato virus Y

作　　者：宋革[1] 郭玉双[1,2] 饶黎霞[1] 周雪平[1] 吴建祥[1] 

机构地区：[1]浙江大学生物技术研究所,杭州310058 [2]贵州省烟草科学研究院,贵阳550081

出　　处：《浙江大学学报（农业与生命科学版）》2016年第5期517-526,共10页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家重点基础研究发展计划资助项目(2014CB138400)

摘　　要：马铃薯Y病毒(Potato virus Y,PVY)是危害农作物的重要病毒之一,在全球广泛传播并造成了严重的经济损失,而建立特异、灵敏的检测技术是防控PVY的关键。本研究以提纯PVYN病毒粒子为抗原免疫BALB/c小鼠,利用杂交瘤技术获得4株能分泌PVY单克隆抗体的杂交瘤细胞株(3B2、3E4、20B2和25C2),杂交瘤细胞分泌的腹水单抗的效价在10^(-6)~10^(-7)之间,Western blot分析发现3B2、3E4和20B2这3株单抗仅与感染PVY的叶片中1条分子质量约为30ku的蛋白有特异性反应。该蛋白的分子质量与PVY的外壳蛋白亚基大小相符,推测制备的这3个单抗能识别PVY的外壳蛋白亚基,而25C2单抗与感染PVY的病叶组织中约55ku的蛋白有特异性反应。进而以制备的单抗为核心建立了检测植株中PVY的ACP-ELISA、dot-ELISA、tissue blot-ELISA和IC-RTPCR这4种血清学方法。特异性分析结果表明,这4种血清学方法检测感染PVY的样品呈阳性反应,而检测健康的烟草和马铃薯及感染马铃薯S病毒、马铃薯X病毒、马铃薯卷叶病毒、马铃薯A病毒的样品呈阴性反应;灵敏度分析结果表明,ACP-ELISA和dot-ELISA检测马铃薯病叶的灵敏度分别达1∶81 920倍和1∶10 240倍稀释(g/mL),而具有最高检测灵敏度的IC-RT-PCR方法在感病植物组织稀释到1∶5 242 880倍(g/mL)时仍能检测到病毒。用建立的血清学方法对2015年采自云南的30个田间马铃薯样品进行检测,结果发现其中有20样品感染PVY,且血清学方法的检测结果与RT-PCR结果一致。抗PVY特异、灵敏单抗的制备及血清学检测方法的建立,为我国作物上PVY的检测和诊断、脱毒种薯生产、抗病育种和科学防控提供了物质和技术支撑。Potato virus Y（PVY）is one of the most important potato viruses causing serious economic loss in potato industry worldwide.Establishment of a sensitive and specific virus detection technique is the key to prevent and control PVY.To provide material and technical support for the diagnosis,scientific prevention and control of PVY in fields,four monoclonal antibodies（MAbs）against PVY were prepared with the hybridoma technique,and four serologicalassays were developed in this study.PVY particles with 730 nm×11 nm were obtained from PVYN-infected tobacco plants with a high yield purification method,and which were used as the immunogen.Four hybridoma cell lines（3B2,3E4,20B2and25C2）secreting sensitive and specific MAbs against PVY were prepared.Titers of the four MAbs in ascetic fluids secreted by prepared hybridoma cells were ranged 10^-6 to 10^-7 by an indirect ELISA.Western blot analyses indicated that three MAbs（3E4,3B2 and 20B2）could specifically react with 30 ku protein,which was supposed to be the coat protein of PVY based on the size of the protein.The MAb 25C2 could react with an approximately 55 ku protein,which was supposed to be HC-Pro based on the molecular mass of the protein.Based on the prepared MAbs,four serological assays, ACP-ELISA, dot-ELISA,tissue blot-ELISA and immunocapture-reverse transcription-polymerase chain reaction（IC-RT-PCR）were developed for detecting PVY in plants.Specificity analyses of the four developed serological assays demonstrated that all assays could accurately detect virus in PVYinfected plant tissues,and have negative detection results with the leaf tissue crude extracts from PVS,PVA,PVX,PLRV-infected plant tissues or healthy potato and tobacco plant tissues.The sensitivity analyses indicated that the developed ACP-ELISA,dot-ELISA could specifically detect virus in PVY-infected plant tissue crude extracts diluted at 1∶81 920 and 1∶10 240（g/mL）,respectively.The developed IC-RT-PCR was the most sensitive,and could successfully detect virus in PVY-infec

关 键 词：马铃薯Y病毒 单克隆抗体 ACP-ELISA DOT-ELISA TISSUE blot-ELISA IC-RT-PCR 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]