Cloning and expression of pdgf-bb gene in eschrishia coli

Background and Aims: Platelet-derived growth factor (PDGF) is an important mediator in wound healing. PDGF is a potent mitogen for fibroblasts and other cells of mesenchymal origin . It is chemotactic for neutrophils, monocytes, fibroblasts, and smooth muscle cells . PDGF stimulates production of several connective tissue molecules and supports angiogenesis. A gel formulation of recombinant human PDGF (rhPDGF) is currently approved to treat full-thickness ulcers in diabetic patients. Methods: coding sequence of PDGF-BB gene after optimization was synthesized, PDGF-BB gene and PET15b vector were digested with restriction enzymes. The gel-purified digested PDGF-B gene was cloned into pET15b. Successful construction of the pET15bPDGF-B clone was confirmed by restriction digestion and PCR amplification. For protein production, host E. coli strain BL21(DE3) was transformed by insertion of a recombinant PDGF-B plasmid. After Growth and harvesting of E. coli BL21(DE3)/pET15-hPDGF-B in LuriaBertani (LB) medium containing 100 μg ampicillin/ml at 37oC until the OD600 reached 0.6, after which 1 mM IPTG was added and culture continued for another 4 h. Analysis of the expression samples was carried out using SDS-PAGE method. Results: After cell culture and induction of expression with IPTG , SDS-PAGE was done & The gel shows increasing expression of gene product following induction with IPTG. Conclusions: The construction of recombinant prokaryotic expression vector pET-PDGF-BB and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB Protein.