猪流行性腹泻病毒S蛋白和M蛋白截短表达及多克隆抗体制备  

Truncated Expressions and Polyclonal Antibody Preparations of Spike Protein and Membrane Protein of Porcine Epidemic Diarrhea Virus

作　　者：李德龙 周建方 余远迪 丁鸿飞 肖奇奇 黄庄 李悦林 罗丽娜[1] 黄涵 刘辉鑫 杨婧婧 付利芝 LI De-long;ZHOU Jian-fang;YU Yuan-di;DING Hong-fei;XIAO Qi-qi;HUANG Zhuang;LI Yue-lin;LUO Li-na;HUANG Han;LIU Hui-xin;YANG Jing-jing;FU Li-zhi(College of Veterinary Medicine,Southwest University,Chongqing 402460,China;National Center of Technology Innovation for PigsChongqing 402460,China;Chongqing Academy of Animal Sciences,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆402460 [2]国家生猪技术创新中心,重庆402460 [3]重庆市畜牧科学院,重庆402460

出　　处：《动物医学进展》2024年第8期41-44,共4页Progress In Veterinary Medicine

基　　金：国家生猪技术创新中心先导项目(NCTIP-XD/B11);重庆英才计划技术创新与应用发展项目(cstc2022ycjhbgzxm0183)。

摘　　要：为制备猪流行性腹泻病毒(PEDV)S蛋白和M蛋白的多克隆抗体,用蛋白结构分析软件对S和M基因序列进行分析,截取2个基因的相关抗原片段,依据大肠埃希氏菌偏好密码子进行密码子优化、序列合成。将合成的片段克隆至pET-32a(+),转化入大肠埃希氏菌BL21(DE3),IPTG诱导表达后进行SDS-PAGE检测。大量表达重组蛋白,免疫昆明小鼠制备多克隆抗体。结果表明,成功表达大小约为38ku和32ku的重组蛋白Sm和Mm,免疫小鼠后制备的抗Sm多克隆抗体效价超过1:25600,抗Mm多克隆抗体效价超过1:51200,且Westernblot表明多克隆抗体特异性良好。研究制备的多克隆抗体可以为建立PEDV检测方法和开发亚单位疫苗提供材料。In order to prepare polyclonal antibodies of S protein and M protein of porcine epidemic diarrhea virus,the sequences of S and M were analyzed by the protein structural analysis software.The relevant antigenic epitope fragments of the two genes were intercepted,and the codon optimization and sequence synthesis were conducted according to the E.coli preference codon.The synthesized fragments were cloned into pET-32a(+)and transformed into E.coli BL21(DE3).The expression proteins were induced by IPTG and detected by SDS-PAGE.The recombinant proteins were abundantly expressed and recovered.The Kunming mice were immunized to prepare polyclonal antibodies.The results demonstrated that Sm and Mm with the size of 38 ku and 32 ku were successfully expressed;the titers of polyclonal antibodies of Sm exceeded 1:25600;the titers of polyclonal antibodies of Mm exceeded 1:51200;the results of Western blotting showed that the polyclonal antibodies had good specificity.The polyclonal antibodies prepared in the study could provide material basis for the establishment of PEDV diagnostic methods and the development of PEDV subunit vaccines.

关 键 词：猪流行性腹泻病毒S蛋白 M蛋白 抗原表位 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]