中国小麦花叶病毒(CWMV)外壳蛋白(CP)特异性抗体的制备与应用
摘要：
中国小麦花叶病毒(CWMV)是浙江省农业科学院发现并鉴定的一种土传病毒,其RNA2编码的外壳蛋白(CP)在病毒侵染过程中发挥重要功能。为了检测该蛋白表达并分析其生物学功能,该研究采用RT-PCR方法从感染CWMV小麦病叶中扩增获得CP基因,并通过In-Fusion技术构建了CWMV CP重组表达载体,将其导入BL21(DE3)菌株诱导表达;原核表达的重组CP蛋白经镍柱亲和层析纯化后注射免疫家兔,收集抗血清经亲和层析纯化获得CP多克隆抗体。Dot-ELISA、ID-ELISA、Western blot显示,该抗体不仅对CWMV具有高度的特异性,而且其效价高达1∶2.048×107、灵敏度达6.25×10-2 ng。应用该抗体采用Dot-ELISA、Western blot方法检测田间小麦样品显示其可用于CWMV的准确诊断,这为CWMV病毒病检测、CP定量分析及其功能研究奠定了基础。
关键词: 中国小麦花叶病毒, 外壳蛋白, 重组蛋白, 多克隆抗体
Abstract:
Chinese wheat mosaic virus (CWMV) is a soil-borne virus discovered and identified by Zhejiang Academy of Agricultural Sciences, whose genomic segment RNA2 encodes a major coat protein (CP) that plays an important role in virus infection. To detect its expression and analyze its biological function, the gene was amplified by RT-PCR from CWMV-infected wheat leaves. The recombinant expression vector was constructed with In-Fusion method and introduced into BL21(DE3) strain for the induction of CP expression. The recombinant CP protein was purified by nickel column affinity chromatography and injected into the rabbits for immunization. The antisera were purified by affinity chromatography and the polyclonal antibodies against CP were obtained. Dot-ELISA, ID-ELISA and Western blot assays showed that the purified antibodies were highly specific to CWMV with a titer of 1∶2.048×107 and the sensitivity of 6.25×10-2 ng. Dot-ELISA and Western blot assays showed that the antibody could be used for accurate diagnosis of CWMV, which laid the foundation for CWMV detection, CP quantitation and its functional research.
Key words: Chinese wheat mosaic virus, coat protein, recombinant protein, polyclonal antibody
中图分类号:
S435.12
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