山羊地方性鼻内肿瘤病毒gag蛋白的原核表达、纯化及多克隆抗体的制备  

Prokaryotic expression and purification of gag protein of enzootic nasal tumor virus of goats and preparation of its polyclonal antibody

作　　者：何亚鹏[1] 张琪[1] 王景[1] 周曼[1] 付明哲[1] 许信刚[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医学报》2017年第12期2300-2303,共4页Chinese Journal of Veterinary Science

基　　金：陕西省农业科技创新与攻关资助项目(2016NY-092);陕西省重点产业创新链计划资助项目(2016KTZDNY02-06);西北农林科技大学大学生创新创业训练计划资助项目(201610712019)

摘　　要：为获得纯化的山羊地方性鼻内肿瘤病毒(enzootic nasal tumor virus of goats,ENTV)gag蛋白和抗gag蛋白的多克隆抗体,根据GenBank已登录的ENTVgag基因序列,设计合成1对特异性引物,应用PCP扩增gag基因并连接于原核表达载体pET-28a(+)中,构建重组质粒pET-gag,经鉴定正确后转化大肠杆菌Rosetta(DE3)株进行诱导表达,并进行SDS-PAGE分析。重组菌经IPTG诱导后成功表达相对分子质量约为69 000的重组蛋白,重组蛋白经镍柱亲和层析纯化、尿素梯度透析复性后免疫小鼠制备多克隆抗体。Western blot试验表明,重组蛋白能与制得的多克隆抗体反应,而与正常小鼠血清和山羊地方性鼻内肿瘤患羊血清不反应。本试验成功获得了纯化的ENTV gag蛋白和小鼠抗gag蛋白的多克隆抗体,为进一步研究gag蛋白在ENTV致病过程中的作用提供了材料。In order to obtain purified gag protein and polyclonal antibody against the gag protein, ac cording to the published sequence of gag gene of ENTV,a pair of specific primers were designed and synthesized. The gag gene was amplified by PCR from ENTV,and cloned into pET 28a vector. The prokaryotic expression plasmid pET-gag containing gag gene was successfully constructed. The induced expression of recombinant plasmid pET-gag in Rosetta （DE3） competent cell was detected by SDS-PAGE. The expressed protein was purified by Ni-NTA affinity chromatography and refolded by urea gradient dialysis,then vaccinated the mouse, polyclonal antibody against the gag protein was obtained. Western blot analysis showed that the recombinational fusion protein could react to the polyclonal antibody, and could not react to the serum of normal mice and the goat infected with ENTV. The result indicated that purified gag protein and polyclonal antibody against the gag protein were successfully obtained, which provides materials for further research.

关 键 词：山羊地方性鼻内肿瘤病毒 GAG蛋白 原核表达 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学] S853.53[农业科学—兽医学]