抗人红细胞/抗猪瘟病毒双功能单链抗体的制备及功能鉴定  

Expression and functional identification of bifunctional single-chain antibody of anti-human red blood cell and anti-classical swine fever virus

作　　者：刘金凤 覃绍敏 石胜 林俊 杜毅超 马玲 秦树英 白安斌 陈凤莲 张振江 吴健敏 LIU Jinfeng;QIN Shaomin;SHI Sheng;LIN Jun;DU Yichao;MA Ling;QIN Shuying;BAI Anbin;CHEN Fenglian;ZHANG Zhenjiang;WU Jianmin(Guangxi Key Laboratory of Veterinary Biotechnology/Guangxi Veterinary Research Institute,Nanning 530001,China;Shangai HILE Bio-pharmaceutical Company,Shanghai 201403,China)

机构地区：[1]广西壮族自治区兽医研究所/广西兽医生物技术重点实验室,广西南宁530001 [2]上海海利生物技术股份有限公司,上海201403

出　　处：《畜牧与兽医》2021年第4期107-112,共6页Animal Husbandry & Veterinary Medicine

基　　金：广西自然科学基金(2016GXNSFBA380078);国家现代农业产业技术体系广西生猪创新团队建设项目(nycytxgxcxtd-15-01);广西创新驱动发展专项资金项目(桂科AA18118051)。

摘　　要：旨在构建检测猪瘟病毒(CSFV)的双功能单链抗体。通过连接肽将抗人红细胞膜H抗原单链抗体基因2E8-ScFv和抗猪瘟病毒单链抗体基因CSFV-ScFv拼接成融合基因,大肠杆菌密码子优化及人工合成后,构建原核表达载体pCzn1-2E8-CSFV,转化BL21(Plyss)进行重组蛋白IPTG的诱导表达,利用SDS-PAGE对表达产物进行鉴定,通过间接免疫荧光(IFA)、ELISA和血凝试验,验证重组蛋白双功能活性。结果表明,重组蛋白主要以包涵体形式在大肠杆菌中表达,分子质量约为55 kDa。IFA和ELISA结果表明,纯化复性后的重组蛋白与猪瘟病毒具有良好的结合活性;红细胞凝集试验表明,重组蛋白能与人红细胞特异性结合,同时也能与猪瘟病毒发生特异性结合,具有双功能特性。本研究成功表达了抗人红细胞膜H抗原/抗猪瘟病毒双功能单链抗体,为进一步构建猪瘟病毒红细胞凝集试验快速检测方法奠定基础。To construct a recombinant bifunctional single-chain antibody for detection of classical swine fever virus,the single-chain antibody 2 E8-ScFv was spliced against a human erythrocyte membrane H antigen and the single-chain antibody CSFV-ScFv against porcine scorpion into a fusion gene by a linker peptide.fusion gene sequence underwent codon optimization and artificial synthesis,and was then inserted into prokaryotic expression vector called pCzn1-2 E8-CSFV,which was transformed into BL21(Plyss)and expression of the recombinant protein was induced by IPTG.The expressed product was identified by SDS-PAGE.The results showed that the recombinant protein was expressed in E.coli in the form of inclusion bodies with a molecular mass of about 55 kDa.The results of indirect immunofluorescence and ELISA showed that the purified recombinant protein had a good activity in binding to classical swine fever virus.The red blood cell agglutination test showed that the recombinant protein could specifically bind to human red blood cells and also specifically combined to the classical swine fever virus,suggesting that it had a dual function.In this study an anti-swine fever virus bifunctional single-chain antibody was successfully expressed,which laid a foundation for further establishing a rapid method for swine fever pathogens by red blood cell agglutination test.

关 键 词：猪瘟病毒 双功能单链抗体 原核表达 功能鉴定 

分 类 号：S855.3[农业科学—临床兽医学]