A型塞内卡病毒VP3蛋白的原核表达及多克隆抗体制备  

Prokaryotic Expression of the VP3 Protein of Senecavirus A and Preparation of Its Polyclonal Antibodies

作　　者：林铱婷 姬康 谭姗姗 晋怡 宋若琪 马瑞一 王颖[1] 牛胜 赵宇军[1] 田文霞[1] 任建乐 Lin Yiting;Ji Kang;Tan Shanshan;Jin Yi;Song Ruoqi;Ma Ruiyi;Wang Ying;Niu Sheng;Zhao Yujun;Tian Wenxia;Ren Jianle(College of Veterinary Medicine,Shanxi Agricultural University,Jinzhong 030801,Shanxi,China)

机构地区：[1]山西农业大学动物医学学院,山西晋中030801

出　　处：《中国动物检疫》2024年第4期96-102,共7页China Animal Health Inspection

基　　金：山西省科技创新人才团队专项(202204051001022);山西省优秀博士来晋奖励基金项目(SXBYKY2021036);山西农业大学博士科研启动基金项目(2020BQ59)。

摘　　要：为制备A型塞内卡病毒(SVA)VP3蛋白多克隆抗体,采用同源重组技术将SVA VP3基因连接至原核表达载体pET-28a中,构建了重组表达质粒pET-SVA-VP3。将重组质粒转化至大肠杆菌感受态细胞BL21(DE3),以1 mmol/L的IPTG进行诱导表达并纯化表达产物,随后将纯化的VP3重组蛋白皮下多点注射新西兰白兔制备多克隆抗体,利用间接ELISA、间接免疫荧光试验(IFA)和Western blot分别对抗体效价、反应性和特异性进行鉴定。结果显示:重组VP3蛋白以包涵体形式表达,相对分子质量约36 kDa;制备的VP3蛋白多克隆抗体能与VP3重组蛋白和SVA发生特异性反应,其ELISA效价达1:10000以上,Western blot和IFA效价达1:5000。综上,本研究成功制备了SVA VP3多克隆抗体,其具有较高的效价和特异性,可为后续SVA致病机制及检测方法等研究奠定基础。In order to prepare polyclonal antibodies against VP3 protein of Senecavirus A(SVA),a recombinant expression plasmid named as pET-SVA-VP3 was constructed through cloning SVA VP3 gene into a prokaryotic expression vector known as pET-28a using homologous recombination technology,and transformed into Escherichia coli competent cell BL21(DE3)to express VP3 protein by IPTG induction at a concentration of 1 mmol/L,and the expressed protein was purified,then the purified VP3 recombinant protein was injected into New Zealand white rabbits via subcutaneous multi-point to prepare polyclonal antibodies,followed by evaluation on its titer,reactivity and specificity by indirect ELISA,indirect immunofluorescence assay(IFA)and Western blot.The results showed that the VP3 recombinant protein was expressed in a form of inclusion body with a molecular mass of about 36 kDa;the prepared antibodies could specifically react with VP3 recombinant protein and SVA,and the titer evaluated by ELISA reached up to above 1:10000,and those by Western blot and IFA were both 1:5000.In conclusion,SVA VP3 polyclonal antibodies were successfully prepared,which were with high titer and specificity.The study laid a foundation for future researches on the pathogenic mechanism and detection of SVA.

关 键 词：A型塞内卡病毒 VP3蛋白 原核表达 多克隆抗体 效价 特异性 

分 类 号：S852.65[农业科学—基础兽医学]