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中国小麦花叶病是引起小麦(Triticum aestivum)严重减产的中国小麦花叶病毒(Chinese wheat mosaic virus,CWMV)造成的。本研究旨在以制备的抗CWMV特异性单克隆抗体(monoclonal antibody,MAb)为核心建立检测CWMV的血清学方法,为该病毒病的诊断和科学防控提供技术支撑。用CWMV提纯病毒免疫小鼠(Mus musculus),经细胞融合、培养基筛选培养、阳性细胞的检测与单克隆化后,共获得4株能分泌抗CWMV单克隆抗体的杂交瘤细胞株5H5、7C2、9A7和12E5,并注射入BALB/c小鼠(Mus musculus)腹腔制备单抗腹水;间接酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)表明,4种单抗腹水效价均达到10-7,抗体类型及亚类均为Ig G1、kappa链。Western blot分析表明,4株单克隆抗体均能与CWMV的外壳蛋白亚基有特异性反应。利用制备的单抗建立检测田间小麦样品中CWMV的抗原包被酶联免疫吸附实验(antigen coating plate enzyme-linked immunosorbent assay,ACP-ELISA)和斑点酶联免疫吸附实验(dot-enzyme-linked immunosorbent assay,dot-ELISA),结果表明,该两种血清学方法检测感染CWMV小麦植物组织粗提液呈特异性阳性反应,而检测感染小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV)的小麦、感染大麦黄花叶病毒(Barley yellow mosaic virus,Ba YMV)的大麦(Hordeum vulgare)和健康小麦均呈阴性反应,说明建立的两种方法能特异性地检测小麦植物中的CWMV。灵敏度分析表明,以5H5和12E5单抗为核心建立的ACP-ELISA方法检测小麦病叶的灵敏度达到1∶81 920(W/V,g/m L),而以7C2和9A7单抗为核心建立的ACP-ELISA方法检测小麦病叶灵敏度达到1∶40 960(W/V,g/m L)倍稀释;以12E5和5H5单抗为核心建立的dot-ELISA方法检测小麦病叶的灵敏度达到1∶2 560倍稀释(W/V,g/m L),而以7C2和9A7单抗为核心建立的dot-ELISA方法的检测灵敏度达到1∶1 280倍稀释(W/V,g/m L)。田间样品检测结果表明,建立的ACP-ELISA、dot-ELISA方法能准确、可靠地用于小麦中CWMV病毒的检测,且进一步证明该病毒病在江苏和山东流行。CWMV单克隆抗体的制备及其血清学检测方法的建立为中国小麦CWMV的诊断、预测预报及科学防控提供了物质和技术支撑。
Chinese wheat mosaic disease is caused by the Chinese wheat mosaic virus (CWMV), which causes severe production of Triticum aestivum. The aim of this study was to establish a serological method for the detection of CWMV using the prepared anti-CWMV-specific monoclonal antibody (MAb) as the core to provide technical support for the diagnosis and scientific prevention and control of the virus disease. After the mice were immunized with CWMV (Mus musculus), four hybridoma cell lines 5H5 and 7C2 secreting anti-CWMV monoclonal antibodies were obtained after cell fusion, culture medium selection, detection of positive cells and monoclonal antibody. , 9A7 and 12E5 were injected into the peritoneal cavity of BALB / c mice (Mus musculus). The indirect ELISA was used to determine the ascites titer of the four McAbs, 7, antibody types and subclasses are Ig G1, kappa chain. Western blot analysis showed that all four monoclonal antibodies reacted specifically with the coat protein subunits of CWMV. The antigen coating plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot-enzyme-linked immunosorbent assay (Dot-enzyme-linked immunosorbent assay) were used to establish CWMV- , dot-ELISA). The results showed that the two serological methods were specific and positive for the detection of CWMV wheat plant tissue crude extracts. However, the detection of wheat infected with wheat yellow mosaic virus (WYMV) Both Hordeum vulgare and healthy wheat with Barley yellow mosaic virus (Ba YMV) showed negative reaction, indicating that the two established methods could specifically detect CWMV in wheat plants. Sensitivity analysis showed that the sensitivity of the ACP-ELISA method for detection of diseased leaves of wheat was 1:81 920 (W / V, g / m L) established by using 5H5 and 12E5 as the core, and the core was established by 7C2 and 9A7 monoclonal antibodies The sensitivity of the dot-ELISA method to detect the diseased leaves of wheat with the 12E5 and 5H5 monoclonal antibodies was 1, which was 1: 40 960 (W / V, g / m L) : 560-fold dilution (W / V, g / m L), while dot-ELISA established with 7C2 and 9A7 mAb as the core detection sensitivity of 1: 1 280 times dilution (W / V, ). The results of field samples showed that the established ACP-ELISA and dot-ELISA methods could accurately and reliably detect CWMV in wheat and further proved that the virus was endemic in Jiangsu and Shandong. The preparation of CWMV monoclonal antibody and the establishment of its serological detection methods have provided material and technical support for the diagnosis, prediction and prevention of wheat CWMV in China.