导　　师： 吴建祥

学科专业： 0904

授予学位： 硕士

作　　者： ;

机构地区： 浙江大学

摘　　要： 在我国小麦是仅次于水稻的第二大粮食作物,其安全生产关乎民生。近年来,小麦上的病毒病发生较重,尤其是大麦黄矮病毒/(Barley yellow dwarf virus, BYDV/)引起的小麦黄矮病和小麦黄花叶病毒/(Wheat yellow mosaic virus, WYMV/)引起的小麦黄花叶病,造成小麦严重减产和巨大经济损失。目前,侵染我国小麦的小麦黄矮病主要由BYDVGAV株系引起。为了了解这两种病毒病的发生、流行规律、建立早期监测和预警体系,急需建立快速、高效的病毒检测方法。以单克隆抗体为核心建立的血清学检测方法操作简单、特异性好、灵敏度高,且适合田间大规模样品检测,因此被广泛应用于植物病毒的诊断、流行规律的分析、预测预警、科学防控和抗病育种等方面。本论文分别制备了抗BYDV GAV株系和WYMV的单克隆抗体/(Monoclonal antibodies, MAbs/),并以单抗为核心建立了相应的血清学检测方法。 /(1/)抗BYDV GAV单克隆抗体的制备及其检测应用：用提纯的BYDV GAV病毒粒子免疫BALB//c小鼠,经细胞融合、筛选、克隆获得8株能稳定传代并分泌抗BYDV GAV单克隆抗体的杂交瘤细胞株/(18A1、18A9、12A11、7H11、1G7、1F1、27E11和3F11/)。8株杂交瘤细胞分泌的腹水单抗的间接ELISA效价均达到10-6以上,抗体类型及亚类均为IgG1、kappa链。8株单抗具有特异性好、灵敏度高的特点。以制备的单抗为核心建立了特异性检测BYDV GAV的Antigen-coated plate enzyme-linked immunosorbent assay /(ACP-ELISA/)和dot enzyme-linked immunosorbent assay /(dot-ELISA/)两种血清学方法。建立的血清学方法检测感染BYDV GAV的小麦组织呈特异性阳性反应,而检测感染其他病毒的小麦和健康小麦组织呈阴性反应。用dot-ELISA方法检测携带BYDV GAV的麦蚜呈特异性阳性反应,而检测携带其他病毒的麦蚜和健康麦蚜呈阴性反应。灵敏度分析表明:ACP-ELISA和dot-ELISA检测小麦病叶的灵敏度分 Wheat is the second food crop after rice in China, whose production safety related to people's livelihood closely.In recent years, virus diseases on wheat are serious in China. Especially, wheat yellow dwarf disease caused by Barley yellow dwarf virus /(BYDV/) and wheat yellow mosaic disease caused by Wheat yellow mosaic virus /(WYMV/) cause severe yield and huge economic losses. At present, BYDV GAV has become a mainstream strain in China. In order to understand the occurrences and epidemic regularities of two wheat virus diseases and establish early detection and warning systems, rapid and efficient detection methods should be urgently developed. Monoclonal antibody-based serological methods for plant virus detection are simple, rapid, sensitive, specific and suitable to detect large-scale field samples. So it has been widely used in plant virus disease diagnosis, epidemic rule analyses, prediction and early warning, scientific prevention and control and resistive breeding. In this study, Monoclonal antibodies /(MAbs/) against BYDV GAV and WYMV were respectively produced, and two serological methods were developed for rapid and reliable virus detection. /(1/) MAbs against BYDV GAV and its application:The purified BYDV GAV virions were used as an immunogen to produce MAbs. Via cell fusion, cell culture, antibody detection and cell cloning, eight sensitive and specific murine MAbs against BYDV GAV /(18A1,18A9,12A11,7H11.1G7,1F1,27E1and3F11/) were produced. The titers of ascitic fluids of MAbs were up to10'6by indirect-ELISA. All MAbs were isotyped as IgG1, kappa light chain. Two serological methods of antigen-coated plate enzyme-linked immunosorbent assay /(ACP-ELISA/) and dot enzyme-linked immunosorbent assay /(dot-ELISA/) were established for detecting BYDV GAV in field wheat and aphid samples. Specificities of the ACP-ELISA and dot-ELISA were confirmed by a positive reaction of detection with BYDV GAV-infected wheat plant tissues and negative reactions of detection with wheat plant infected by other detecte

关 键 词： 小麦 大麦黄矮病毒 小麦黄花叶病毒 单克隆抗体 麦蚜

领　　域： [农业科学] [农业科学]