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

构建了霍乱毒素B亚单位(choleratoxinBsubunit,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。Westernblotting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。

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

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.