重组早孕因子的原核表达及多克隆抗体制备  

Prokaryotic expression of recombinant early pregnancy factor and preparation of polyclonal antibody

作　　者：王聪 王菡 张海玲[2] 肖毅然 郭禹汐 刘梦迪 李浩菘 吴宗澄 段芙春 卢士英 WANG Cong;WANG Han;ZHANG Hai-ling;XIAO Yi-ran;GUO Yu-xi;LIU Meng-di;LI Hao-song;WU Zong-cheng;DUAN Fu-chun;LU Shi-ying(College of Veterinary Medicine/Key Laboratory of Zoonosis Research,Ministry of Education,Jilin University,Changchun130112,China;Institute of Specialty,Chinese Academy of Agricultural Sciences,Changchun130112,China)

机构地区：[1]吉林大学动物医学学院/人兽共患病研究教育部重点实验室,长春130112 [2]中国农业科学院特产研究所,长春130112

出　　处：《湖北农业科学》2023年第5期129-134,共6页Hubei Agricultural Sciences

基　　金：国家自然科学基金面上项目(32072943)。

摘　　要：对牛源早孕因子进行生物信息学分析,构建了含有促溶标签的原核表达载体pET-28a-srEPF,经IPTG成功诱导表达,亲和层析纯化早孕因子,再以重组蛋白为免疫原,免疫动物制备多克隆抗体,Western blotting对表达蛋白及其多抗进行检测。结果表明,成功制备早孕因子重组蛋白,分子质量为26 ku,纯化后BCA法测定其浓度为1.1 mg/mL,早孕因子重组蛋白和去除抑制区的EPF蛋白均可与制备的多抗特异性结合,间接ELISA测定其效价为1∶256000。Bioinformatics analysis of bovine early pregnancy factor was conducted,and prokaryotic expression vector pET-28a-srEPF containing pro-soluble labels was constructed.The prokaryotic expression vector was successfully induced by IPTG,and the early pregnancy factor was purified by affinity chromatography.Then,recombinant protein was used as immunogen to immunized animals to prepare polyclonal antibodies.The expressed proteins and their polyantibodies were detected by Western blotting.The results showed that the recombinant protein of early pregnancy factor had been successfully prepared,and its molecular weight was about 26 ku.After purification,the concentration was determined by BCA method to be 1.1 mg/mL.Both the recombinant protein of early pregnancy factor and EPF protein after enzyme digestion could be specifically bound to the prepared polyclonal antibody,and the titer determined by indirect ELISA was 1∶256000.

关 键 词：早孕因子 原核表达 多克隆抗体 

分 类 号：S85[农业科学—兽医学]