传染性脾肾坏死病毒(ISKNV)ORF086基因的表达·纯化及其抗体的制备  

Expression and Purification of Infectious Spleen and Kidney Necrosis Virus(ISKNV) Gene ORF086 and Its Antibody Preparation

作　　者：彭媛媛[1,2] 付小哲[2] 石存斌[2] 余露军[1,2] 李宁求[2] 吴淑勤[2] 

机构地区：[1]上海海洋大学,上海201306 [2]中国水产科学研究院珠江水产研究所,广东广州510380

出　　处：《安徽农业科学》2009年第11期4920-4922,共3页Journal of Anhui Agricultural Sciences

基　　金：国家科技支撑计划项目(2006BAD03B05);广东省自然科学基金项目(7004728)

摘　　要：[目的]构建传染性脾肾坏死病毒(ISKNV)ORF086基因的融合表达载体,制备抗体为进一步研究ORF086蛋白的免疫保护性提供前提条件。[方法]PCR扩增目的基因,构建重组质粒,经酶切鉴定,PCR和核酸序列分析后,IPTG诱导表达,Ni-柱纯化,注射新西兰白兔获得抗血清。[结果]扩增出ORF086基因,成功构建了重组质粒pET32a-ORF086,SDS-PAGE电泳和Western-blot分析显示,获得一条36.0 kD的融合蛋白。[结论]成功构建了原核表达载体,融合蛋白主要以包涵体的形式存在,经柱层析纯化蛋白,纯度达90%以上。[Objective] The research aimed to construct the fusion expression vector of infectious spleen and kidney necrosis virus（ISKNV） ORF086 gene and prepare the antibody,in order to provide basis for future research on the protective immunity of ORF086 protein.[Method]The gene fragment was obtained by PCR to construct the recombinant rector.After enzyme digestion,PCR and gene sequencing,the recombinant vector was expressed by IPTG induction.ORF086 was purified by Ni-NTA affinity chromatograph.The purified recombinant protein was injected to New Zealand white rabbits to obtain the antiserum.[Result] ORF086 gene was amplified and recombinant plasmid pET32a-ORF086 was successfully constructed.The results of SDS-PAGE and Western blot analysis showed that one 36.0 kD fusion protein was obtained.[Conclusion] The prokaryotic expression vector was successfully constructed.The fusion protein mainly existed in the forms of inclusion body.After column chromatography purification,the purity of recombinant protein was above 90%.

关 键 词：传染性脾肾坏死病毒(ISKNV) 表达 纯化 抗体 

分 类 号：S917[农业科学—水产科学]