High Expression Of Human Bone Morphogenetic Protein-2 In E.coli

Objective: To clone integral mature peptide gene of human bone morphogenetic protein-2(BMP-2) and to express it highly in Escheria Co/i so as to obtain recombinant hBMP-2 protein which has bioactivity. Methods: ㏕wo primers were chemosynthesized according to the hBMP-2 sequence reported in Genbank. The hBMP-2 gene was gained by RT-PCR. from the total RNA which was extracted from human fetus bone tissue, and was cloned into vector pUC- 19. The recombinant plasmid pUC 1 9-hBMP-2 was extracted from E.coii , and was confirmed by sequence analysis.㏕he 5? end of hBMP-2 gene were mutated to the codon which E. Co/i likes by PCR in order to improve the expression of hBMP-2 in E. Coil .The mutated hBMP-2 gene was inserted to the exprssion vector pBV22O. The recombinant plasmid pYR(pBV22O-hBMP) was heat-induced after being transformed into E. Coil BL2 I in different conditions of bacterial activation and different times of heat-induction,so as to study the effects on expression of afromentioned both factorsX~The inclusion bodies obtained from E. Coil were purified by anion exchange chromatography DEAE and molecular sieve filter S-300. The bioactivity of rhBMP-2 protein was identifed after renaturation in vitro by mouse muscle bag model. Results: OThe result of sequence assay was in agreement with the reported hBMP-2 sequence in Genbank and mutational situs expected was changed successfully .甌he restriction endonucleases analysis of recombinant plasmid pYR shows:mutated hBMP-2 gene was inserted into pBV22O. It is optimal to express when bacteria were activated to OD髈onm 0.45 and the time of heat-induction was 4 hours ,and its exoression rate can reach 30% .瓼inally the purity of recombinanted hBMP -4- 2 after purification is greater than 95% New bone growing since rhBMP-2 embed in the muscles of mice Conclusion: The integral human BMP? mature peptide gene was cloned and its 5?end was successfully mutated.The hBMP-2 was highly expressed in E. Co/i BL2 1 and its ectopic osteoinduction activity of rhBMP-2 is ideal.