霍乱毒素B亚单位与胰岛素原融合基因的克隆、表达及重组蛋白特性分析  

Cloning of CTB-PROIN Fusion Gene and its Expression in Escherichia coli

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

机构地区：[1]宁夏大学生命科学学院,银川,750021,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 [2]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 [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]宁夏大学生命科学学院,银川,750021

出　　处：《生物工程学报》2005年第2期204-210,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金资助项目 (No .3 0 170 818)~~

摘　　要：构建了霍乱毒素B亚单位 (CholeratoxinBsubunit,CTB)与胰岛素原 (Proinsulin)的融合基因CTB -PROIN ,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pETCPI,并将该质粒转入大肠杆菌菌株BL2 1(DE3)中 ;重组菌株经IPTG诱导后 ,其表达产物经过 15 %SDS PAGE分析表明该菌株可以表达融合蛋白 ,其分子量约为 2 1. 6kD ,且主要以包涵体形式存在 ,约占全菌蛋白的 2 5 %。含CTB- PROIN重组蛋白的包涵体经过变性和复性后 ,CTB- PROIN可以在体外自组装成五聚体结构。Westernblotting分析结果表明重组CTB PROIN蛋白可分别被霍乱毒素的抗体和胰岛素的抗体识别 ,说明该蛋白具有霍乱毒素和胰岛素的双重抗原性。同时在体外 ,CTB PROIN蛋白可与神经节苷脂GM1(monosialoganglioside)特异性结合 ,表明了该融合蛋白在体外具有生物活性。这些研究结果为利用原核生物表达系统研制廉价、高效的I型糖尿病口服疫苗奠定了基础。A fusion gene CTB-PROIN, in which Proinsulin 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 pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E.coli stain BL21(DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% 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 CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.

关 键 词：霍乱毒素B亚单位 胰岛素原 融合基因CTB-PROIN 神经节苷脂GMI 

分 类 号：R587.1[医药卫生—内分泌]