Cloning and Expression of Recombinant Immunotoxin using Diphtheria Toxin and Granulocyte Colony Stimulating Factor (G-CSF)

Immunotoxin (IT) is a directed toxin containing two distinct sections (immune and toxin parts) covalently bond using specific chemical or peptide linkers. The aim of this study is to produce a recombinant and hybrid protein containing diphtheria toxin (DT) and granulocyte colony stimulating factor (G-CSF).
According to the structure of the first commercial recombinant immunotoxin (Ontak, hybrid protein containing DT fused interlukine2), gene encoding of DT and G-CSF was amplified using specific primers and plasmid template of pET-IDZ3 (pET21 harboring the gene encoding ontak immunotoxine) and pET-GCSF (pET23 harboring G-CSF), respectively. The DT-GCSF fusion protein produced using soeing PCR and specific primers. Finally, pET-DT-GCSF construction prepared using subcloning of DT-GCSF into pET21a() and confirmed by sequencing, SDS-PAGE and western blot technique.
Gene encoding of DT-GCSF inserted into NdeI/EcoRI site of pET21 and the construction of strain producing DT-GCSF recombinant immunotoxin was confirmed using customary methods.
The cytokine fusion protein, DT-GCSF, could be used for the inhibition of G-CSF receptor bearing cancer cells.