猪流行性腹泻病毒S1蛋白的表达纯化及多克隆抗体制备  

Expression and purification of S1 protein and preparation of polyclonal antibody for porcine epidemic diarrhea virus

作　　者：李佳晋 杨克礼[2] 朱啟超 任曼[1] 江昌盛 李升和[1] LI Jiajin;YANG Keli;ZHU Qichao;REN Man;JIANG Changsheng;LI Shenghe(College of Animal Science,Anhui Science and Technology University,Fengyang 233100,China;Institute of Animal Husbandry and Veterinary Medicine,Hubei Academy of Agricultural Sciences,Wuhan 430064,China)

机构地区：[1]安徽科技学院动物科学学院,安徽凤阳233100 [2]湖北省农业科学院畜牧兽医研究所,湖北武汉430064

出　　处：《安徽科技学院学报》2025年第1期9-16,共8页Journal of Anhui Science and Technology University

基　　金：湖北省农业科技创新中心项目(2021-620-000-001-17);湖北省科技重大专项(2022ABA002)。

摘　　要：以猪流行性腹泻病毒(PEDV)的S1蛋白为研究对象,构建PEDV S1的杆状病毒系统并稳定表达高纯度的S1蛋白,制备高特异性的S1蛋白多克隆抗体。基于多种基因型PEDV S1基因序列信息,使用生物信息学技术分析其理化特性、跨膜结构域、抗原表位和二级结构等性质。进一步对S1基因序列进行适用于昆虫细胞表达的密码子优化,通过基因合成、载体构建、杆粒转化、细胞培养等步骤,构建适用于表达PEDV S1重组蛋白的Bac-to-Bac杆状病毒表达系统。通过镍柱亲和层析和超滤技术纯化S1重组蛋白,将其与弗氏佐剂混合乳化后免疫BALB/c小鼠,制备鼠多克隆抗血清(多抗血清)。通过间接免疫荧光(IFA)、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫印迹(Western Blot)等方法,成功证明了本研究所建立的杆状病毒表达系统能够有效地表达S1重组蛋白。ELISA和IFA验证了所得抗体在特异识别PEDV感染Vero细胞方面的有效性。本文成功构建了PEDV S1的杆状病毒表达系统,稳定表达了高纯度的S1蛋白,并制备了具有高特异性的S1蛋白多克隆抗体,为未来猪流行性腹泻单克隆抗体的制备及检测试剂的研发奠定了基础。The S1 protein of porcine epidemic diarrhea virus(PEDV)was employed as the research object.The baculovirus system of PEDV S1 was constructed,enabling the stable expression of high-purity S1 protein,and the polyclonal antibody of high-specific S1 protein was prepared.Based on the sequence information of PEDV S1 gene of multiple genotypes,bioinformatics technology was utilized to analyze its physicochemical properties,transmembrane domains,epitopes,and secondary structures.Furthermore,the codon of the S1 gene sequence suitable for the expression of insect cells was optimized,and a Bac-to-Bac baculovirus expression system suitable for the expression of PEDV S1 recombinant protein was constructed through steps such as gene synthesis,vector construction,bacmid transformation,and cell culture.The S1 recombinant protein was purified by nickel column affinity chromatography and ultrafiltration technology,mixed with Freund's adjuvant and emulsified with BALB/c mice,and murine polyclonal antiserum(polyserum)was prepared.Indirect immunofluorescence(IFA),sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and Western Blot(Western Blot)were used to successfully confirm that the S1 recombinant protein could be effectively expressed by the baculovirus expression system established in this study.ELISA and IFA verified the effectiveness of the obtained antibodies in specifically recognizing PEDV-infected Vero cells.In this study,a baculovirus expression system for PEDV S1 was successfully constructed,which stably expressed high-purity S1 protein,and a polyclonal antibody to S1 protein with high specificity was prepared,laying a foundation for the preparation of monoclonal antibodies to porcine epidemic diarrhea and the research and development of detection reagents in the future.

关 键 词：猪流行性腹泻病毒 S1蛋白 生物信息学 杆状病毒表达 多克隆抗体 

分 类 号：S855.3[农业科学—临床兽医学]