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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。
The fusion gene CTB-INSB of cholera toxin B subunit (CTB) and insulin B chain was constructed, and the fusion gene was cloned into E. coli expression vector pET-30a (+) to obtain a recombinant plasmid pETCIB. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The expressed product induced by IPTG showed that the fusion protein was expressed by 15% SDS-PAGE and its molecular weight was about 15.4 kDa. Body form exists, accounting for about 30% of the whole protein. Inclusion bodies containing CTB-INSB recombinant proteins can be self-assembled into pentameric structures in vitro after degeneration and renaturation. Western blotting analysis showed that CTB-INSB was recognized by cholera toxin antibody and insulin antibody respectively, indicating that the protein has the dual antigenicity of cholera toxin B subunit and insulin. At the same time, the results of GM1-ELISA analysis showed that CTB-INSB could specifically bind with ganglioside GM1 (monosialoganglioside) in vitro, further confirming that it can form a higher-order structure resembling CTB pentamer with biological activity.