猪丁型冠状病毒NSP14蛋白截短表达及其间接ELISA抗体检测方法的建立  

Truncated expression of porcine delta coronavirus protein NSP14 and establishment of indirect ELISA antibody detection method

作　　者：刘思雨 何颖[2] 卢冰霞[2] 赵武[2] 赵硕 许心婷 全琛宇 秦毅斌[2] 欧阳康[1] LIU Si-yu;HE Ying;LU Bing-xia;ZHAO Wu;ZHAO Shuo;XU Xin-ting;QUAN Chen-yu;QIN Yi-bin;OUYANG Kang(College of Animal Science and Technology,Guangxi University/Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease/Guangxi Engineering Research Center of Veterinary Biologics,Nanning,Guangxi 530004,China;Guangxi Veterinary Research Institute Guangxi/Key Laboratory of Veterinary Biotechnology Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of China,Nanning,Guangxi 530001,China)

机构地区：[1]广西大学动物科学技术学院/广西高校动物疫病预防与控制重点实验室/广西兽用生物制品工程研究中心,广西南宁530004 [2]广西兽医研究所/广西兽医生物技术重点实验室/农业农村部中国(广西)-东盟跨境动物疫病防控重点实验室,广西南宁530001

出　　处：《南方农业学报》2024年第4期1238-1248,共11页Journal of Southern Agriculture

基　　金：广西创新驱动发展专项(桂科AA17204057-1);国家现代农业产业技术体系广西创新团队建设专项(nycytxgxcxtd-2023-15-02);南宁市科学研究与技术开发计划项目(20232039);广西农业科技自筹经费项目(Z202221)。

摘　　要：【目的】进行猪丁型冠状病毒(PDCoV)NSP14蛋白截短原核表达,制备PDCoV NSP14蛋白的多克隆抗体,并建立检测PDCoV抗体的间接酶联免疫吸附试验(ELISA),为探究PDCoV NSP14蛋白的生物学功能及进行PDCoV监测和诊断提供参考依据。【方法】以PDCoV 1468B毒株为模板,构建NSP14截短基因原核重组质粒pET32a-NSP14,经双酶切和测序鉴定后,通过原核表达系统获得NSP14融合蛋白,将其纯化后免疫新西兰大白兔获得NSP14蛋白的多克隆抗体,进行Western blotting验证,并用间接ELISA测定其效价。以NSP14融合蛋白为抗原包被酶标板,采用方阵滴定法优化一系列反应条件,构建检测PDCoV抗体的间接ELISA。【结果】在构建的pET32a-NSP14重组质粒中,NSP14蛋白在大肠杆菌BL21(DE3)中以包涵体形式表达,其大小约40.2 kD;制备的NSP14蛋白多克隆抗体效价在1∶512000以上,经Western blotting验证,能与纯化的NSP14融合蛋白发生特异性反应。经过筛选,确定所构建检测PDCoV抗体间接ELISA的最佳反应条件:NSP14融合蛋白最佳包被浓度为4.0μg/mL,包被条件为37℃、2.0 h;封闭条件为5%脱脂奶粉封闭1.0 h;待检血清按1∶400稀释,孵育2.0 h;酶标二抗1∶2500稀释,孵育60.0 min;TMB底物显色时间为30.0 min。特异性试验结果表明,猪病阳性血清PRRSV、JEV、PCV2、PPV、PEDV和CSFV的OD450均小于0.235,说明优化的间接ELISA检测的病原与其他猪病毒阳性血清无反应,特异性良好。重复性试验结果表明,优化的间接ELISA检测猪病阳性血清的批内和批间变异系数均小于10.000%,说明优化的ELISA具有较好的重复性。【结论】成功制备具有高效价和良好免疫反应性的PDCoV NSP14蛋白多克隆抗体,并建立检测该抗体的特异性、敏感性和重复性良好的间接ELISA,可供进一步研究PDCoV NSP14蛋白的生物学功能及进行PDCoV监测和诊断参考应用。【Objective】The truncated NSP14 protein of porcine delta coronavirus(PDCoV)was expressed in prokaryo-tic,and the polyclonal antibody of PDCoV NSP14 protein was prepared,and an indirect enzyme-linked immunosorbent assay(ELISA)method for detecting PDCoV antibody was established.It provided a reference for exploring the biological function of PDCoV NSP14 protein and conducting PDCoV monitoring and diagnosis.【Method】In this study,the prokaryo-tic recombinant plasmid pET32a-NSP14 of NSP14 truncated gene was constructed using PDCoV 1468B strain as a template,and after identified by double digestion and sequencing,the recombinant protein pET32a-NSP14 was obtained by prokaryotic expression system.They were purified and then reimmunized to New Zealand white rabbits to obtain polyclonal antibodies against NSP14 protein,the Western blotting was performed and and its titer was determined by indirect ELISA method.NSP14 recombinant protein was used as antigen coated enzyme-linked immunosorbent assay plate,and a series of reaction conditions were optimized by square matrix titration method to construct an indirect ELISA method for detecting PDCoV antibody.【Result】The recombinant plasmid pET32a-NSP14 was constructed successfully,and the NSP14 protein was expressed as an inclusion body in Escherichia coli BL21(DE3)competent cells with a size of approximately 40.2 kD.The NSP14 protein polyclonal antibody was produced successfully,with an antibody titer above 1∶512000,and it could specifically react with the purified recombinant NSP14 protein.After screening,the optimal reaction conditions of the indirect ELISA antibody detection method were determined:the optimal coating concentration of NSP14 recombinant protein was 4.0μg/mL,and the coating condition was 37℃for 2.0 h;the blocking condition was 5%skimmed milk powder for 1.0 h;the serum to be tested was diluted 1∶400 and incubated for 2.0 h;the enzyme labeled secondary antibody was diluted 1∶2500 and incubated for 60.0 min;the TMB substrate reaction time was 30.

关 键 词：猪丁型冠状病毒 NSP14蛋白 原核表达 多克隆抗体制备 间接ELISA 

分 类 号：S855.3[农业科学—临床兽医学]