辣椒斑驳病毒CP蛋白多克隆抗体制备与应用

Preparation and Application of Polyclonal Antibodies Recognizing CP Protein in Pepper Mottle Virus

【目的】辣椒斑驳病毒(Pepper mottle virus,PepMoV)是近年来生产上主要侵染辣椒的新发病毒之一,目前有在我国快速扩展的趋势,因此,亟需开展该病毒的特异性快速检测技术,为明确该病毒在我国辣椒主产区的分布、发生致害规律及机制等研究提供科学手段。本研究以PepMoV编码的外壳蛋白CP为免疫源,制备特异性多克隆抗体,建立PepMoV的特异性快速检测方法,为PepMoV的分布和发生致害规律等研究奠定基础。【方法】采用特异性RT-PCR技术,从感染PepMoV辣椒的cDNA中扩增获得片段大小为822 bp的CP基因,并克隆到原核表达载体pET28α,转化E coli DH5α中进行诱导表达,采用Ni-NTA柱层析纯化。以纯化的重组CP蛋白作为抗原免疫新西兰白兔制备特异性多克隆抗体。制备的多克隆抗体采用ID-ELISA和Western blotting检测。【结果】获得原核表达的PepMoV重组CP蛋白,SDS-PAGE结果表明纯化蛋白为分子量约为37 kDa的单一条带。Western blotting和IDELISA检测结果表明,制备的多克隆抗体特异性高,仅识别PepMoV CP蛋白,不识别寄主蛋白和其他选择的马铃薯Y病毒;田间样本检测结果表明,PepMoV在湖南和贵州辣椒上的检出率为20.00%和43.33%。【结论】基于PepMoV CP蛋白特异性多克隆抗体建立的PepMoV快速检测方法,可为进一步深入研究该病毒在我国的分布和发生规律提供科学手段,也为该病毒CP蛋白的功能研究奠定基础。

【Objective】A specific,rapid method based on the polyclonal antibody prepared using purified recombinant CP protein for detecting a typical Potyvirus,pepper mottle virus(PepMoV),on Capsicum annuum L.was developed to assess the distribution,occurrence ratio,and pathogenicity of the disease in China.【Methods】The 822 bp of CP was amplified by specific RT-PCR using the total RNA of PepMoV-infected chili peppers.It was cloned into prokaryotic expressing plasmid pET28αand expressed in E.coli DH5α.The recombinant CP protein was purified by Ni-NTA chromatography and used as the antigen to prepare the polyclonal antibodies to be verified by ID-ELISA and western blotting.【Results】The purified recombinant CP protein was approximately 37 kDa,and the prepared polyclonal antibody verified to specifically recognize PepMoV CP protein.The ID-ELISA method detected 20.00%PepMoV infection on field chili pepper specimens in Hunan and43.33%in Guizhou.【Conclusion】The established specific and rapid detection method based on the polyclonal antibody against CP protein of PepMoV was applied to survey the disease spreading on chili peppers in China.It provided a tool for further studies on the CP protein.