中国小麦花叶病毒富含半胱氨酸蛋白多克隆抗体的制备与应用  

Preparation and application of polyclonal antibody against a cysteine rich protein encoded by Chinese wheat mosaic virus

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作  者:戴远兴 郭留明 何婧 沈峥嵘 耿艳飞 吕明芳[2] 袁正杰[2] 李静[2] 张恒木[2] DAI Yuanxing;GUO Liuming;HE Jing;SHEN Zhengrong;GENG Yanfei;LÜMingfang;YUAN Zhengjie;LI Jing;ZHANG Hengmu(College of Life Science,Zhejiang Normal University,Jinhua 321004,Zhejiang,China;Institute of Virology and Biotechnology,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,Zhejiang,China)

机构地区:[1]浙江师范大学生命科学学院,浙江金华321004 [2]浙江省农业科学院病毒学与生物技术研究所,浙江杭州310021

出  处:《浙江大学学报(农业与生命科学版)》2023年第5期677-686,共10页Journal of Zhejiang University:Agriculture and Life Sciences

基  金:浙江省“万人计划”科技创新领军人才培养项目(2019R52033);中国博士后科学基金项目(2019M662114)。

摘  要:中国小麦花叶病毒(Chinese wheat mosaic virus,CWMV)是小麦花叶病的重要病原体之一,长期威胁小麦的产量和品质;CWMV富含半胱氨酸蛋白(cysteine-rich protein,CRP)在病毒侵染过程中具有重要而复杂的功能。为了深入研究CRP的功能和CWMV侵染机制,本研究采用反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)从CWMV侵染的小麦叶片中获得CRP基因编码区,将其克隆至原核表达载体pET-32a上,并将重组质粒pET-CRP转化至大肠埃希菌BL21(DE3)感受态细胞中进行诱导表达;通过镍柱亲和层析法纯化CRP重组蛋白,并用作抗原免疫新西兰白兔,制备多克隆抗体。蛋白质印迹法、间接酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和斑点ELISA分析结果显示:纯化的CRP抗体不仅具有高度的特异性,而且效价高达1∶4096000,是未纯化抗体效价的4倍;该抗体能识别0.5 ng抗原,显示出较高的灵敏度;在1∶120000稀释条件下,该抗体能特异且灵敏地识别天然CRP。综上所述,本研究所制备的CRP抗体不但可用于田间CWMV病株样品的精准诊断,还可用于植物体内瞬时表达CRP的检测分析,为后续CRP检测、定量分析及其亚细胞定位等研究提供了依据。Chinese wheat mosaic virus(CWMV)is one of the most important pathogens causing mosaic disease in wheat and has threatened the yield and quality of wheat for a long time.Cysteine-rich protein(CRP)of CWMV plays important and complex roles in viral infection.To further study CRP functions and CWMV infection mechanisms,the CRP coding region was amplified by reverse transcription-polymerase chain reaction(RT-PCR)from leaves of CWMV-infected wheat and cloned into the prokaryotic expression vector pET-32a.The recombinant plasmid pET-CRP was transformed into Escherichia coli BL21(DE3)for inducible expression.The recombinant CRP was purified by nickel-column affinity chromatography and used as an antigen to immunize New Zealand white rabbits for polyclonal antibody preparation.A series of immunological assays,including Western blot,indirect enzyme-linked immunosorbent assay(ELISA)and dot ELISA,showed that the purified CRP antibody had high specificity,and its titer was as high as 1∶4096000,which was four times higher than that of the unpurified antibody.The antibody could recognize 0.5 ng antigen,showing its high sensitivity.In addition,the purified CRP antibody could specifically and sensitively recognize native CRP even at a 1∶120000 dilution.In conclusion,the polyclonal antibody can be not only used for precise diagnosis of the CWMV-infected plant samples from fields,but also applied to detect CRP expressed transiently in plants,which lays a foundation for subsequent detection,quantification and subcellular localization of CRP.

关 键 词:中国小麦花叶病毒 富含半胱氨酸蛋白 原核表达 蛋白质纯化 多克隆抗体 

分 类 号:S435.121.4[农业科学—农业昆虫与害虫防治]

 

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