Molecular Cloning of Bone Morphogenetic Protein-۲ (BMP- ۲) in pGEM-b۱ Vector and Transformation into E.coli Rosetta Strain

Background: Bone Morphogenetic Protein ۲ (BMP- ۲) belongs to the TGF-β superfamily of proteins and plays an important role in the development of bone and cartilage. BMP- ۲ is also associated with maintenance and repair of damaged bone. Recombinant human bone morphogenetic protein ۲ (rhBMP- ۲) is now produced by genetic engineering techniques and used in treatment of thin bone fractures in the jaw and spine. In this study we aimed to extract and amplify the BMP- ۲ gene from human osteoblast cell line MG-۶۳, insert the amplified BMP- ۲ gene into pGEM-b۱ cloning vector, and then transform the recombinant vector into the E.coli strain of Rosetta. This technique can be used in future research and BMP- ۲ expression.Methods: After culturing MG-۶۳ cells, approximately ۵ million viable cells were used for extraction of Total RNA. The extracted RNA was used for cDNA synthesis in RT-PCR reaction. Then, the BMP- ۲ gene was amplified by specific primers and the PCR product was cloned in the pGEM-b۱ vector. Chemically competent E.coli cells were prepared using CaCl۲ ۰.۱ M and transformed with recombinant pGEM-b۱ vector under heat shock. The transformed E.coli Rosetta bacteria were inoculated on LB agar medium containing Ampicillin. Bacterial colony containing recombinant vector was isolated and used for plasmid extraction. The extracted plasmid was used for specific PCR to confirm the presence of BMP- ۲ gene in pGEM- b۱ vector.Results: After transformation, the E.coli Rosetta had the ability to become resistant to ampicillin and could grow on ampicillin- containing medium. While non-transformed E.coli Rosetta could not grow on LB agar containing ampicillin. The ۱۱۰۰ bp fragment was obtained from PCR amplification with specific primers, indicating that the BMP- ۲ gene was inserted into pGEM-b۱ vector.Conclusions: The pGEM-b۱ vector and E.coli Rosetta strain were not used for BMP- ۲ cloning in previous investigations. Therefore, this method may be a useful approach to reduce the challenges ahead of the optimization of BMP- ۲ production.Background: Bone Morphogenetic Protein ۲ (BMP- ۲) belongs to the TGF-β superfamily of proteins and plays an important role in the development of bone and cartilage. BMP- ۲ is also associated with maintenance and repair of damaged bone. Recombinant human bone morphogenetic protein ۲ (rhBMP- ۲) is now produced by genetic engineering techniques and used in treatment of thin bone fractures in the jaw and spine. In this study we aimed to extract and amplify the BMP- ۲ gene from human osteoblast cell line MG-۶۳, insert the amplified BMP- ۲ gene into pGEM-b۱ cloning vector, and then transform the recombinant vector into the E.coli strain of Rosetta. This technique can be used in future research and BMP- ۲ expression. Methods: After culturing MG-۶۳ cells, approximately ۵ million viable cells were used for extraction of Total RNA. The extracted RNA was used for cDNA synthesis in RT-PCR reaction. Then, the BMP- ۲ gene was amplified by specific primers and the PCR product was cloned in the pGEM-b۱ vector. Chemically competent E.coli cells were prepared using CaCl۲ ۰.۱ M and transformed with recombinant pGEM-b۱ vector under heat shock. The transformed E.coli Rosetta bacteria were inoculated on LB agar medium containing Ampicillin. Bacterial colony containing recombinant vector was isolated and used for plasmid extraction. The extracted plasmid was used for specific PCR to confirm the presence of BMP- ۲ gene in pGEM- b۱ vector. Results: After transformation, the E.coli Rosetta had the ability to become resistant to ampicillin and could grow on ampicillin- containing medium. While non-transformed E.coli Rosetta could not grow on LB agar containing ampicillin. The ۱۱۰۰ bp fragment was obtained from PCR amplification with specific primers, indicating that the BMP- ۲ gene was inserted into pGEM-b۱ vector. Conclusions: The pGEM-b۱ vector and E.coli Rosetta strain were not used for BMP- ۲ cloning in previous investigations. Therefore, this method may be a useful approach to reduce the challenges ahead of the optimization of BMP- ۲ production.

کلیدواژه ها:

Bone morphogenetic protein ۲ ، Molecular cloning ، Genetic vectors ، Bacterial transformation