霍乱毒素B亚单位与胰岛素B链结构类似多肽融合蛋白的制备

为了在大肠杆菌中表达霍乱毒素B亚单位(Cholera toxin B subunit,CTB)与胰岛素B链(9-23)肽段结构类似多肽(Ins B(9-23))的融合蛋白,使用基因工程方法构建了CTB与Ins B(9-23)的融合基因CTB-Ins B-X若干,并将融合基因克隆到大肠杆菌表达载体pET28a(+)中,获得的重组质粒转化大肠杆菌BL21(DE3)。重组菌株经乳糖诱导后,其表达产物经过12%SDS-PAGE分析表明该菌株可以包含体形式表达融合蛋白,并制备得到纯度较高的重组蛋白。该重组蛋白的包含体经过变性、复性后,可以在体外自组装成五聚体结构。GM1-ELISA分析结果表明,重组蛋白在体外可以与神经节苷脂GM1(monosialoganglioside)特异性结合,表明该融合蛋白保持了CTB形成五聚体的生物活性。

Expression and Identification of the Fusion Protein of CTB-Ins B

To express the fusion protein of cholera toxin B subunit(CTB) and insulin B peptide(9-23) in E.coli,the recombinant expression plasmid pET28a-CTB-Ins B was constructed in which Ins B(9-23) genes were fused to the 3′end of CTB gene and cloned into pET28a(+) to obtain a prokaryotic expression vector pET28a-CTB-Ins B.Subsequently the recombinant plasmid was transformed into E.coli strain BL21(DE3).After being induced with 5 mmol/L lactose for 5 h,the expression products were analyzed by sodium dodecyl sulphate-polyacrylamide gel(12%) electrophoresis(SDA-PAGE),and the results indicated that the recombinant protein CTB-Ins B-X was expressed and accumulated as inclusion bodies.Purificated recombinant proteins were obtained after washing,renaturing and purifying via DEAE-52 cellulose and sephadex G-50 chromatography.The proteins were further analyzed for GM1 binding activity in a GM1-ELISA.The results of the GM1 binding assay showed(1)、(2),that the recombinant proteins could bind to monosialoganglioside specifically,although not as strong as cholera toxin B subunit,that they possessed biological activity in vitro.