猪巨细胞病毒gB优势抗原表位区的原核表达及其多克隆抗体的制备  

Prokaryotic expression of the gB epitope region of porcine cytomegalovirus and preparation of the polyclonal antibodies

作　　者：江一帆[1] 刘骁[1] 廖珊[1] 朱玲[1,2] 周远成[1] 

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

出　　处：《中国兽医科学》2013年第9期936-940,共5页Chinese Veterinary Science

基　　金：四川省青年基金项目(200930421);教育部新世纪优秀人才支撑计划项目(1NCET 11-1059);四川省科技支撑计划项目(2010NZ0112;2012NZ0001)

摘　　要：为制备猪巨细胞病毒(PCMV)gB优势抗原表位区重组蛋白的多克隆抗体,进一步分析PCMV gB蛋白的生物学特性,建立PCMV抗体血清学检测方法。对PCMV gB基因优势抗原表位区进行了PCR扩增,将回收的目的片段连接入pET32a(+)表达载体,转化入E.coli Rosetta(DE3),构建了重组表达菌E.coli Rosetta-pET32a(+)-gB,经IPTG诱导表达了gB优势抗原表位区重组蛋白。对重组蛋白进行纯化后,通过Western-blot试验验证了重组蛋白的反应原性,并免疫家兔制备了多克隆抗体。结果表明,该重组蛋白大量可溶性表达,且具有良好的反应原性。琼脂扩散试验及Western-blot检测表明,多克隆抗体效价达到1∶8,且能与gB优势抗原表位区重组蛋白特异性结合。证实,成功制备了PCMV gB优势抗原表位区重组蛋白多克隆抗体。This study aimed to prepare polyclonal antibodies of the epitope region of gB gene of porcine cytomegalovirus（PCMV）, further to analyze biological properties of PCMV gB protein,and to establish the serological methods for the detection of PCMV antibodies. The fragment of gB epitope region was amplified and cloned into pET32a（＋） expression vector,transformed into Escherichia coli Rosetta（DE3） to obtain the expression bacteria E. coli Rosetta-pET32a（＋）-gB. The recombinant protein was expressed after in- duction by IPTG. After the recombinant protein was purified, the reactinogenicity of the recombinant pro- tein was confirmed by Western-blot, and polyclonal antibodies were prepared by immunizing rabbits. The results showed that the recombinant protein was expressed in a soluble state with good reactinogenicity. Agar-gel diffusion test and Western-blot analysis showed that the polyclonal antibodies＇titer was 1 ： 8 and the polyclonal antibodies could specifically bind to the gB epitope region of recombinant protein. The poly- clonal antibodies of the PCMV gB epitope region of recombinant protein was prepared.

关 键 词：猪巨细胞病毒 gB优势抗原表位区 多克隆抗体 

分 类 号：S852.659.6[农业科学—基础兽医学] Q503[农业科学—兽医学]