Molecular Cloning of Bone Morphogenetic Protein-2 (BMP- 2) in pGEM-b1 Vector and Transformation into E.coli Rosetta Strain

  • Zahra Sadri Najaf Abadi 1 1 Dept. of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
  • Farnaz Fatemi 1
  • Masoud Khosravani 2 2 Dept. of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
  • Majid Rahmati 3 3 Dept. of Medical Biotechnology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran.
  • Esmaeil Sadroddiny 1*

Abstract

Background: Bone Morphogenetic Protein 2 (BMP- 2) belongs to the TGF-β superfamily of proteins and plays an important role in the development of bone and cartilage. BMP- 2 is also associated with maintenance and repair of damaged bone. Recombinant human bone morphogenetic protein 2 (rhBMP- 2) 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- 2 gene from human osteoblast cell line MG-63, insert the amplified BMP- 2 gene into pGEM-b1 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- 2 expression.Methods: After culturing MG-63 cells, approximately 5 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- 2 gene was amplified by specific primers and the PCR product was cloned in the pGEM-b1 vector. Chemically competent E.coli cells were prepared using CaCl2 0.1 M and transformed with recombinant pGEM-b1 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- 2 gene in pGEM- b1 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 1100 bp fragment was obtained from PCR amplification with specific primers, indicating that the BMP- 2 gene was inserted into pGEM-b1 vector.Conclusions: The pGEM-b1 vector and E.coli Rosetta strain were not used for BMP- 2 cloning in previous investigations. Therefore, this method may be a useful approach to reduce the challenges ahead of the optimization of BMP- 2 production. 
Published
Jan 30, 2016
How to Cite
SADRI NAJAF ABADI 1, Zahra et al. Molecular Cloning of Bone Morphogenetic Protein-2 (BMP- 2) in pGEM-b1 Vector and Transformation into E.coli Rosetta Strain. International Journal of Health Studies, [S.l.], p. page:20-23, jan. 2016. ISSN 2423-6594. Available at: <http://ijhs.shmu.ac.ir/index.php/ijhs/article/view/79>. Date accessed: 25 may 2020. doi: http://dx.doi.org/10.22100/ijhs.v1i3.79.
Section
Articles

Keywords

Bone morphogenetic protein 2, Molecular cloning, Genetic vectors, Bacterial transformation