Construction of Expression Vector for Recombinant DT/bFGF Fusion Protein and Purification of Expressed Product

Objective To construct the expression vector for DT/bFGF fusion protein and prepare highly purified DLF fusion toxin.Methods The DNA sequences encoding DT389 and bFGF were amplified by PCR and linked together by restriction endonuclease digestion and ligation,then cloned into expression vector pET-30a.The constructed plasmid pET-30a/DLF was transformed to E.coli BL21(DE3),and the positive clones were screened.The fusion protein was expressed by IPTG induction and purified by nickel ion affinity chromatography,then identified by Western blot.Results Sequence analysis showed that the inserted DNA fragment encoded fusion protein DLF consisting of 545 amino acid residues.SDS-PAGE showed that the fusion protein in a form of inclusion body was expressed in E.coli and contained about 20% of total somatic protein.The purity of the fusion protein reached more than 90% after purification and showed antigenicities of both DT and bFGF as proved by Western blot.Conclusion The expression vector of DT/bFGF fusion protein was successfully constructed,and highly purified DLF fusion toxin was obtained.