鸭坦布苏病毒全长E蛋白可溶性表达及免疫原性分析  

Soluble Expression and Immunogenicity Analysis of Duck Tembusu Virus Full-length E Protein

作　　者：龚凤平 冯晓声 王伟[1] 罗梦萍 马海彬 王贵平[1] 袁建丰 GONG Feng-ping;FENG Xiao-sheng;WANG Wei;LUO Meng-ping;MA Hai-bin;WANG Gui-ping;YUAN Jian-feng(Guangdong Haid Institute of Animal Husbandry&Veterinary Medicine,Guangzhou,Guangdong,511400,China)

机构地区：[1]广东海洋大学畜牧兽医研究院

出　　处：《动物医学进展》2019年第11期14-17,共4页Progress In Veterinary Medicine

基　　金：国家重点研发计划项目(2017YFD0500800);广州市科技计划项目(201704020083);广东省科技计划项目(2014A020208052);广东省科技发展专项资金项目(2016B020234006,2017B090904029)

摘　　要：鸭坦布苏病毒病是由鸭坦布苏病毒(Duck tembusu virus,DTMUV)引起的一种以产蛋量和采食量严重下降为主要特征的传染病。应用RT-PCR方法扩增DTMUV GDHD2014-3毒株的囊膜(E)蛋白全基因序列,并将其克隆至原核表达载体pMAL-c5x,构建重组表达质粒E-pMAL。将其转化E.coli BL21感受态细胞,经诱导表达条件优化,在30℃条件下IPTG诱导,可溶性表达重组的全长E蛋白(rf E),大小约110 ku。用Amylose Resin对目的蛋白进行纯化,并将纯化的rf E蛋白免疫Balb/c小鼠,制备多克隆血清抗体。经Nu-PAGE以及Western blot分析,成功实现rf E蛋白可溶性表达,并能与DTMUV灭活疫苗免疫鸭血清多抗产生特异性反应。间接免疫荧光试验结果表明,rf E蛋白免疫小鼠获取的多克隆血清抗体能与DTMUV反应。实现了rf E蛋白的可溶性表达及纯化,并验证rf E蛋白具有良好的免疫原性,为DTMUV的亚单位疫苗的研制以及相关诊断试剂的开发奠定了基础。Duck Tembusu virus(DTMUV)disease is an infectious disease caused by duck Tembusu virus(DTMUV),which is mainly characterized by severe decline of egg production and feed intake.In this study,the full-length sequence of envelope(E)protein of DTMUV GDHD2014-3 strain was amplified by RT-PCR and cloned into prokaryotic expression vector pMAL-c5x to construct recombinant expression plasmid E-pMAL.The expression vector was transformed into E.coli BL21 competent cells and the induced expression conditions were optimized.The soluble recombinant full-length E(rf E)protein was successfully expressed at 30℃,which is the size of about 110 ku.The rf E protein was purified using Amylose Resin,and the purified rf E protein was immunized to Balb/c mice to prepare polyclonal serum antibodies.The Nu-PAGE analysis showed that the soluble rf E protein was successfully expressed,the Western blot analysis indicated that the soluble rf E protein could react specifically with the duck polyclonal serum antibodies against DTMUV inactivated vaccine.The indirect immunofluorescence assay showed that the polyclonal serum against rf E protein could react with DTMUV.In this study,the soluble rf E protein was successfully expressed and purified,the results verified that rf E protein had good immunogenicity,which laid a foundation for the development of DTMUV subunit vaccine and the development of related diagnostic reagents.

关 键 词：鸭坦布苏病毒 囊膜(E)蛋白 可溶性表达 免疫原性分析 

分 类 号：S852.65[农业科学—基础兽医学]