猪细环病毒2型ORF1基因的原核表达及其多克隆抗体的制备  

Prokaryotic expression of ORF1 gene of torque teno sus virus 2 (TTSuV2) and preparation of its polyclonal antibody

作　　者：李淞[1] 朱玲[1,2] 周远成[1] 陈蕾[1] 徐志文[1,2] 郭万柱[1,3] 

机构地区：[1]四川农业大学 动物生物技术中心,四川雅安625014 [2]四川农业大学 动物疫病与人类健康四川省重点实验室,四川雅安625014 [3]四川农业大学动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国兽医科学》2013年第11期1162-1166,共5页Chinese Veterinary Science

基　　金：四川省科技支撑计划项目(2011FZ0065,2012NZ0001)

摘　　要：为制备猪细环病毒2型(TTSuV2)ORF1多克隆抗体,以TTSuV2阳性DNA为模板,经PCR扩增目的基因,并克隆于表达载体pET-32a(+),构建了ORF1基因的重组表达载体pET-TTSuV2-ORF1。重组质粒鉴定验证后,转化至大肠杆菌Rosetta(DE3)感受态细胞中,经IPTG诱导表达,SDS-PAGE分析结果显示重组蛋白分子质量约为45.3ku,主要以可溶性蛋白的形式存在,Western-blot分析结果显示重组蛋白能够与TTSuV2阳性血清反应。表达的蛋白经HisTrapFF组氨酸标签融合蛋白纯化柱纯化后,免疫家兔,制备多克隆抗体,Western-blot分析结果显示制备的抗体具有高度的特异性,ELISA测定抗体效价为1∶1 600。证明,成功制备了特异的TTSuV2ORF1多克隆抗体。In order to prepare polyclonal anti-recombinant TTSuV2-ORF1 antibodies, the ORF1 gene was amplified by PCR and cloned into the prokaryotic expression vector pET-32a（＋） to construct recom- binant plasmid pET-TTSuV2-ORF1, which was transformed into Escherichia coli Rosetta （DE3） and induced with IPTG for expression. SDS-PAGE analysis showed that a protein in approximately 45.3 ku was expressed to be soluble, which could react with TTSuV2 positive serum using Western-blot. The purified fusion protein was injected into rabbit to prepare for polyclonal antibodies,and the titer of the antibody was 1 ： 1 600 detected by ELISA. The protein TTSuV2-ORF1 could be specifically detected by Western-blot using the polyclonal antibodies. The results indicated that specific polyclonal antibodies against TTSuV2- ORF1 were successfully prepared.

关 键 词：猪细环病毒2型 ORF1 原核表达 多克隆抗体 

分 类 号：S852.659.2[农业科学—基础兽医学]