霍乱毒素B亚单位与胰岛素B链融合基因的克隆及原核表达分析  

Cloning of CTB-INSB Fusion Gene and its Expression in E. coli

作　　者：陈丽[1] 欧阳凤秀[2] 钱炳俊[3] 任宏[2] 王强[4] 姜庆五[2] 王玉炯[1] 刘静波[2] 梁婉琪[5] 潘爱虎[5] 

机构地区：[1]宁夏大学生命科学学院,银川750021 [2]复旦大学公共卫生学院,上海200032 [3]复旦大学生命科学学院,上海200433 [4]南京大学生命科学学院,南京210093 [5]上海市农业科学院农业生物技术研究中心上海市农业遗传育种重点实验室,上海201106

出　　处：《中国生物工程杂志》2005年第4期56-61,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(30170818)

摘　　要：构建了霍乱毒素B亚单位(cholera toxin B subunit,CTB)与胰岛素(insulin)B链的融合基因 CTB-INSB,将该融合基因克隆到大肠杆菌表达载体pET-30a(+)中,获得重组质粒pETCIB;并将该 质粒转入大肠杆菌菌株BL21(DE3)中;重组菌株经IPTG诱导后的表达产物经15％SDS-PAGE分 析表明可以表达融合蛋白,其分子量约为15.4kDa,且主要以包涵体形式存在,约占全菌蛋白的 30％。含CTB-INSB重组蛋白的包涵体经变性和复性后,可在体外自组装成五聚体结构。Western blotting分析结果显示CTB-INSB可分别被霍乱毒素的抗体和胰岛素的抗体识别,表明该蛋白具有 霍乱毒素B亚单位与胰岛素的双重抗原性。同时GM1-ELISA分析结果表明CTB-INSB在体外可 与神经节苷脂GM1(monosialoganglioside)特异结合,进一步证实了它能够形成类似CTB五聚体的 高级结构,具有生物活性。A fusion gene CTB-INSB, in which insulin B chain gene was fused to the 3' end of CTB gene by a hinge peptide ' GPGP', was constructed and cloned into pET-30a ( + ) to obtain a prokaryotic expression vector pETCIB. Subsequently the recombinant plasmid pETCIB was transformed into E. coli strain BL21(DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and the results indicated that the recombinant protein CTB-INSB was expressed and accumulated as inclusion bodies. The recombinant CTB-INSB protein accumulated to the level of 30% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of Western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the GM1-ELISA assay showed that the protein could bind to monosialoganglioside specifically.

关 键 词：霍乱毒素B亚单位 融合基因 胰岛素 表达分析 B链 SDS-PAGE Western 原核 大肠杆菌 分析结果 神经节苷脂 表达载体 基因克隆 重组质粒 融合蛋白 表达产物 IPTG 重组菌株 重组蛋白 特异结合 高级结构 生物活性 分析表 分子量 

分 类 号：Q786[生物学—分子生物学] Q78