IBRV gD蛋白的原核表达及其间接ELISA检测方法的建立  

Prokaryotic expression for gD protein of infectious bovine rhinotracheitis virus and establishment of indirect ELISA-linked testing method

作　　者：魏鑫 张建华[1] 郭婷 吕天星[1] 周雅坪 张新竹 吴倩 陈新迪 王艳芳 白帆[1] 郝永清[1] WEI Xin;ZHANG Jian-hua;GUO Ting;LYU Tian-xing;ZHOU Ya-ping;ZHANG Xin-zhu;WU Qian;CHEN Xin-di;WANG Yan-fang;BAI Fan;HAO Yong-qing(Laboratory of Microbiology and Im munology,College of Veterimary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China)

机构地区：[1]内蒙古农业大学兽医学院

出　　处：《中国兽医学报》2019年第11期2129-2134,共6页Chinese Journal of Veterinary Science

基　　金：内蒙古科技厅应用技术研发资助项目(201802066)

摘　　要：利用生物学软件分析牛传染性鼻气管炎病毒(IBRV)gD蛋白的抗原决定簇,发现相应的核酸序列,设计特异性引物用PCR扩增IBRVgD基因片段并克隆到原核表达载体pET-32a中,通过双酶切鉴定正确和测序正确后进行诱导表达。利用间接ELISA方法和Western blot鉴定gD重组蛋白的免疫原性和反应原性。最后将gD重组蛋白用作包被抗原,并通过优化相应的反应条件建立检测IBRV抗体的间接ELISA方法。结果显示,表达的gD重组蛋白具有良好的免疫原性和反应原性,间接ELISA阴阳性临界值为:D450nm值=0.23。重组抗原gD与口蹄疫、牛病毒性腹泻、赤羽病、副结核病、布鲁菌病、结核病等阳性血清无交叉反应,具有比较好的特异性。组内和组间变异系数均小于5%,重复性较好。建立的ELISA方法结果显示,当测试相同的200个临床血清样本时,与IDEXX牛传染性鼻气管炎病毒gB X3抗体检测试剂盒相比,两者符合率为96%。本试验为IBRV抗体间接ELISA检测试剂盒的开发奠定了基础。In present research,we aimed to learn immunological characteristics of gD protein originated from infectious bovine rhinotracheitis virus(IBRV)and utilize it to establish a method for rapid testing of specific antibody against IBRV.The epitopes of gD protein and corresponding nucleic acid sequences were anchored and screened.Afterwards,specific primer pair was designed to amplify gene segments of gD protein,and the segments were cloned into vector pET-32afor prokaryotic expressions of gD proteins under optical induction conditions.Immunogenicity and reactionogenicity of gD recombinant protein were analyzed with ELISA and Western blot utilizing anti-IBRV-NMHS-1rabbit serum as the primary antobody and HRP-conjugated goat anti-rabbit lgG as the secondary antobody.Finally,gD recombinant proteins were enveloped as antigen to establish indirect ELISA detecting method under orthogonally screened reaction conditions.The results showed that gD proteins has satisfactory immunogenicity and reactionogenicity.Established ELISA method showed a D450nmvalue of 0.23for each diagnostic positivity threshold,and excellent specificity without cross reactions to antisera from dairy cows infected respectively with FMD,BVD,akabane disease,paratuberculosis,brucella,and tuberculosis.Coefficient variations within groups and among groups were both less than 5%and repetitiveness was great.Our established ELISA method showed a 96%consistency with IDEXX ELISA testing kits for antobody against gB X3of IBR when testing the same 200clinical antisera samples.The present research lays essential foundation for development of ELISA-linked testing for IBR.

关 键 词：牛传染性鼻气管炎病毒 gD蛋白 原核表达 ELISA 

分 类 号：S852.65[农业科学—基础兽医学]