پزشکی بالینی ابن سینا (Dec 2014)
Cloning of BMP-2 Gene and Its Expression Study in E. coli in Order to Produce a Recombinant Drug
Abstract
Introduction & Objective: Bone morphogenetic proteins are a group of cytokines that belongs to superfamily TGFβ. These proteins play an important role in evolution of many of organs and tissues through germinal period followed by amending and rebuilding of bone tissue and car-tilage. The aim of this study was to clone and expression analysis of BMP-2 gene in E. coli bacteria. Materials & Methods: In this experimental study the sequence of cDNA related to the mature peptide of human morphogenetic protein-2 (BMP-2) in E.coli was synthesized and cloned in a PET system. After sequencing, recombinant plasmid pET28a/BMP-2 was transformed into the expression host, E.coli BL21 (DE3). The transformed bacteria were cultured in LB me-dium containing kanamaycin antibiotic at 37° C for O/N. Then, induction with IPTG took place. The expression was evaluated by reverse transcriptase PCR and SDS-PAGE followed by western blotting to confirm its identity. The observed band on SDS-PSGE showed the presence of the expressed protein at the 14k Dalton segment which was confirmed by west-ern blotting technique. Results: The gene sequence was amplified by PCR. After gene and plasmid preparation, lega-tion was performed. Sequencing confirmed accuracy of cloning. Protein expression was demonstrated by RT-PCR and SDS-PAGE. Results were confirmed by western blotting. Conclusion: In this study over-expression of this recombinant protein was achieved in a pro-karyotic system. Different concentrations of inducer were applied and harvesting was per-formed in different times after induction. The best expression was detected in 4 hours after induction with a concentration of 1mM IPTG.
