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.