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南芥菜花叶病毒衣壳蛋白的克隆、原核表达和多克隆抗体制备
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引用本文:苏学思,张玉宝,王亚军,郭志鸿,邱 阳,唐国亮.南芥菜花叶病毒衣壳蛋白的克隆、原核表达和多克隆抗体制备[J].西北农业学报,2021,30(6):894~900
DOI:10.7606/j.issn.1004-1389.2021.06.011
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作者单位
苏学思,张玉宝,王亚军,郭志鸿,邱 阳,唐国亮 (1.中国科学院 西北生态环境资源研究院兰州 7300002.中国科学院大学北京 1000493.甘肃省生态与农业综合试验野外科学观测研究站兰州 730000) 
基金项目:陇原青年创新创业人才(团队)项目(2020-339);兰州市人才创新创业项目(2019-HLJC-9);兰州市城关区科技计划(2019-6-2)。
中文摘要:为开发百合中南芥菜花叶病毒(Arabis mosaic virus,ArMV)的血清学检测技术及研究ArMV的致病机理,原核表达ArMV衣壳蛋白(coat protein,CP),纯化并制备其多克隆抗体。以侵染东方百合杂交品种‘木门’(‘Conca D’or’)的ArMV基因组为模板,克隆ArMV CP基因全长,构建ArMV CP基因原核表达载体,转化E.coli BL21(DE3)原核表达ArMV CP蛋白,并以纯化ArMV CP蛋白为抗原制备兔抗多克隆抗体,Western blot鉴定多克隆抗体的特异性。结果表明:克隆获得百合ArMV CP序列(GenBank登录号:MT323117)全长,其长度为1 515 bp,与已知的ArMV CP基因核苷酸序列相似性最高达到93.6%,氨基酸序列相似性最高达到98.2%;SDS-PAGE表明CP蛋白在E.coli BL21(DE3)中大量表达,蛋白相对分子质量为 56 ku;Western blot结果显示抗体能够特异性结合ArMV CP蛋白,具有良好的特异性。
中文关键词:百合  南芥菜花叶病毒  衣壳蛋白  原核表达  多克隆抗体
 
Preparation of Polyclonal Antibody against Arabis mosaic virusby Cloning and Prokaryotic Expression of Coat Proteins
Abstract:In order to improve serological detection of Arabis mosaic virus(ArMV), and to facilitate further study of its pathogenic mechanism, the coat protein (CP) was expressed by prokaryote cells, and the polyclonal antibody was prepared. In this study, the genomic RNA of ArMV infecting oriental hybrid lily (Lilium oriental cv.‘Conca D’or’) was used as a template to amplify the full-length CP gene. The CP gene of 1 515 bp was constructed into the prokaryotic expression vector pET-28a (+) to express the fusion CP protein. Further CP protein was purified by using a Ni-NTA Gravity Column and this antigen was used to prepare rabbit anti-polyclonal antibodies to ArMV.A Western blot assay was then developed to identify the specificity of these polyclonal antibodies. BLAST results showed that the similarity of nucleotide and amino acid sequences was up to 93.6% and 98.2%, respectively, between the cloned ArMV CP sequence (GenBank Accession:MT323117) and the known ArMV CP sequence (GenBank Accession:MH316599). SDS-PAGE indicated that CP protein was highly expressed in E.coli BL21 (DE3), and had a protein relative molecular mass of 56 ku. Western blot results demonstrated that the prepared polyclonal antibody successfully bound CP protein and had high specificity in detecting ArMV.
keywords:Lily  Arabis mosaic virus  Coat protein  Prokaryotic expression  Polyclonal antibody
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