大鲵虹彩病毒主要衣壳蛋白主要抗原表位区的原核表达及抗血清制备  

Prokaryotic expression and antiserum preparation for major antigenic epitope region of major capsid protein of Chinese giant salamander( Andrias davidianus) iridovirus

作　　者：周小愿[1] 张星朗[1] 贾秋红[1] 韩亚慧 高宏伟[1] ZHOU Xiaoyuan ZHANG Xinglang JIA Qiuhong HAN Yahui GAO Hongwei(Aquaculture and Fishery Engineering Laboratory, Yellow River Fisheries Institute, Chinese Academy of Fishery Sciences, Xi ＇an 710086, China)

机构地区：[1]中国水产科学院黄河水产研究所养殖与渔业工程研究室,陕西西安710086

出　　处：《细胞与分子免疫学杂志》2016年第10期1407-1411,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：陕西省科学技术发展计划(2014k01-20-02;2012K01-18)

摘　　要：目的表达大鲵虹彩病毒(CGSIV)主要衣壳蛋白(MCP)主要抗原表位区蛋白,并制备兔抗血清。方法以大鲵虹彩病毒略阳株(CGSIV-LY)基因组为模板,PCR扩增MCP基因,然后克隆到p ET-21a(+)表达载体中,转化大肠杆菌BL21(DE3)菌株,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过SDS-PAGE和Western blot法检测重组蛋白的表达和免疫活性,用镍柱亲和层析法纯化重组蛋白。以纯化的重组蛋白为免疫原免疫兔制备抗血清,采用Western blot法和ELISA检测抗血清的免疫特异性并测定效价,利用间接免疫荧光法检测鲤鱼上皮瘤(EPC)细胞CGSIV。结果表达了相对分子质量(Mr)为29 000的重组蛋白。制备的兔抗MCP血清具有良好的特异性和高滴度,间接免疫荧光法检测结果显示制备的多抗血清能识别EPC细胞中的CGSIV。结论成功表达了大鲵虹彩病毒MCP主要抗原表位区蛋白,并制备高滴度和良好特异性的兔抗血清。Objective To express the fusion protein of major antigenic epitope region of major capsid protein（ MCP） of Chinese giant salamander（ Andrias davidianus） iridovirus（ CGSIV） and prepare the rabbit antiserum. Methods Using the genomic DNA of CGSIV Lueyang strain（ CGSIV-LY） as a template,the gene fragment of major antigenic epitope region of MCP was amplified by PCR and cloned into the prokaryotic vector p ET-21a（ ＋） to construct the prokaryotic expression recombinant plasmid p ET-21a-MCP. The recombinant plasmid was transformed into Escherichia coli BL21（ DE3）. His-tagged fusion protein was induced by IPTG. After identified by SDS-PAGE and Western blot analysis,the recombinant protein was purified by nitrilotriacetic acid（ Ni-NTA） agarose resin. New Zealand rabbits were immunized with the purified recombinant protein to generate antiserum. Specificity and titer of the antiserum were determined by Western blotting and indirect ELISA,and then the antiserum was used to detect the CGSIV in the infected EPC cells by indirect immunofluorescence assay.Results The recombinant protein with the relative molecular mass of 29 000 was expressed. The prepared rabbit antiserum had a good specificity and a high titer. Indirect immunofluorescence assay showed that the antiserum could recognize CGSIV in the infected EPC cells. Conclusion The fusion protein of major antigenic epitope region of MCP of CGSIV is successfully expressed and the rabbit antiserum with a high titer and a good specificity been prepared.

关 键 词：大鲵虹彩病毒 主要衣壳蛋白 主要抗原表位区 原核表达 抗血清 

分 类 号：S947.3[农业科学—水产养殖] S966.6[农业科学—水产科学]