家蚕核型多角体病毒多角体蛋白的多克隆抗体制备及应用试验  

Preparation and Application Test of Polyclonal Antibody Against Bombyx mori Nucleopolyhedrovirus Polyhedrin

作　　者：胡志刚 董战旗 蒋亚明 董非凡 潘敏慧 Hu Zhigang;Dong Zhanqi;Jiang Yaming;Dong Feifan;Pan Minhui(State Key Laboratory of Silkworm Genome Biology,Chongqing 400716,China;Key Laboratory for SericuItural Biology and Genetic Breeding of Agricultural Ministry,Chongqing 400716,China;College of Biotechnology,Southwest University,Chongqing 400716,China)

机构地区：[1]家蚕基因组生物学国家重点实验室,重庆400716 [2]农业部蚕桑生物学与遗传育种重点实验室,重庆400716 [3]西南大学生物技术学院,重庆400716

出　　处：《蚕业科学》2018年第3期398-403,共6页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.31472152);现代农业产业技术体系建设专项(No.CARS-18)

摘　　要：杆状病毒（baculovirus）多角体蛋白（polyhedrin）是晚期高水平表达的蛋白质,对于维持病毒粒子在自然环境下的稳定性和在宿主之间的传播至关重要。PCR扩增家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus,Bm NPV）多角体蛋白基因polh,亚克隆到原核表达载体p ET32,获得重组质粒p ET32-polh,并转化至大肠埃希菌（Escherichia coli）BL21（DE3）表达菌株进行原核表达,诱导表达的融合蛋白带His标签,经镍柱纯化后用于免疫新西兰大白兔,制备抗多角体蛋白的多克隆抗体。Western blot检测该多克隆抗体能够特异识别Bm NPV的多角体蛋白。利用该多克隆抗体进行免疫荧光和Western blot分析多角体蛋白的亚细胞定位及表达时相,结果显示多角体蛋白定位于细胞质,且在病毒感染后48 h才可以被检测到。该多克隆抗体可用于Bm NPV多角体蛋白的功能研究。The polyhedrin highly expressed in late phase of baculovirus life cycle is crucial for the stability of virions under natural environment and the horizontal transfer between the hosts. The polh gene of Bombyx mori nucleopolyhedrovirus（ Bm NPV） was amplified by PCR and subcloned into the prokaryotic expression vector pET32 to obtain the recombinant plasmid pET32-polh and then transformed into Escherichia coli expression strain BL21（ DE3） for prokaryotic expression.The induced His-tagged fusion protein was purified by nickel column and then used to immunize New Zealand white rabbits to prepare the polyclonal antibody against polyhedrin. Western blot analysis showed that the polyclonal antibody could specifically recognize polyhedrin of Bm NPV. This antibody was used to study the subcellular localization and the expression phase of polyhedrin in immunofluorescence and Western blot assay. The results showed that the polyhedrin localized in the cytoplasm of Bm N cells and could be detected at 48 h post infection. This polyclonal antibody can be used in the function study of Bm NPV polyhedrin.

关 键 词：家蚕核型多角体病毒 多角体蛋白 多克隆抗体 亚细胞定位 表达时相 

分 类 号：Q523.3[生物学—生物化学] Q786