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.