CONSTRUCTION AND CLONING OF A SYNTHETIC GENE FOR EXPRESSION OF β-INTERFERON (IFN-β) IN E. coli UNDER THE COTROL OF lac AND tac PROMOTERS

Human interferon-β (IFN-β) is an antiproliferative and antiviral glycoprotein that is typically produced by fibroblasts as a natural glycoprotein, and it has already been used to treat viral hepatitis, glyoma and melanoma. Its beneficial effects on patients suffering from relapsing –remitting MS have been also demonstrated. In this study, a synthetic gene encoding 166 residues of interferon-β was constructed. For an efficient expression of IFN- β in E. coli, the synthetic gene was designed according to the E. coli codon usage. Cystein 17 has been replaced with serine to avoid multimer formation and random intramolecular disulfide bridges as well. The sequence of the designed gene was analyzed with Web Cutter and RNA structure softwares to make ensure about the cloning and expression of the recombinant protein in the proposed cloning and expression vectors. Twelve overlapping primers ranging from 55 to 83 nucleotides in length were designed. A step-wise assembly polymerase chain reaction was used for construction of the synthetic gene. The synthetic gene was constructed through three steps PCR programs using overlapping adjacent primers as well as PCR products of the previous steps. The final PCR product was further amplified and cloned into T/A cloning vector. Dideoxy Termination Method of sequencing revealed that the synthetic gene codes for IFN-β. The gene was sub-cloned into pQE30 and pkk-223 expression vectors.

Keywords:

synthetic gene , Interferon ? , step-wise PCR , codon usage