西伯利亚鲟(Acipenser baerii)IFN-γ基因克隆、原核表达和单克隆抗体制备  

Cloning,prokaryotic expression and monclone antibody prepartion of IFN-γof Acipenser baerii

作　　者：田照辉 TIAN Zhaohui(Beijing Academy of Agriculture and Forestry Sciences Fisheries Research Institute and National Engineering Research Center for Freshwaters(Beijing),Beijing 100068,China)

机构地区：[1]北京市农林科学院水产科学研究所暨国家淡水渔业工程技术研究中心(北京),北京100068

出　　处：《生物学杂志》2024年第6期69-73,110,共6页Journal of Biology

基　　金：北京市农业农村局种业联合攻关项目(G20220628008)。

摘　　要：利用转录组信息克隆西伯利亚鲟IFN-γ的可读编码框(ORF)528 bp,编码176个氨基酸,具有IFN-γ的特征序列和核定位序列,参照此序列构建N端含有6His(组氨酸)的原核表达载体Pet30α-IFN-γ,转化至大肠杆菌BL21(DE3),经IPTG(异丙基硫代半乳糖苷)诱导表达,SDS-PAGE蛋白电泳和免疫印迹Western Blot鉴定,重组蛋白为22.8 ku,主要以包涵体形式存在,温度37℃时的最佳表达条件为0.75 mmol/L的IPTG诱导6 h,利用镍柱层析得到纯化的重组蛋白,重组蛋白免疫小鼠,进行细胞融合获得8株阳性细胞株,利用protein G亲和层析纯化的单克隆抗体效价为2×105,为深入研究西伯利亚鲟IFN-γ免疫学功能奠定基础。Using transcriptome information,the readable coding frame(ORF)528 bp encoding 176 amino acids of IFN-γof Acipenser baerii was cloned with the characteristic sequence and nuclear localization sequence of IFN-γ.According to this sequence,a prokaryotic expression vector Pet30α-IFN-γwas constructed containing 6His(histidine)at the n-terminal.Then the Pet30α-IFN-γplasmid was transformed into Escherichia coli BL21(DE3),which was induced by IPTG(isopropyl-β-d-thiogalactoside),and the expressed recombinant protein was identified by SDS-PAGE electrophoresis and Western Blot.The recombinant protein was 22.8 ku,mainly existing in the form of inclusion bodies,and the optimal expression condition was induced at 37℃with 0.75 mmol/L IPTG for 6 h.The purified recombinant protein was obtained by nickel column chromatography.The recombinant protein was immunized to mice,8 positive cell lines were obtained by cell fusion,monoclonal antibody purified by protein G affinity chromatography,the valence of antibody was 2×105,which laid a foundation for further study of the immunological function of IFN-γof Acipenser baerii.

关 键 词：西伯利亚鲟 IFN-Γ基因 原核表达 重组蛋白 单克隆抗体 

分 类 号：Q78[生物学—分子生物学] Q959.463