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Res Mol Med (RMM) 2021, 9(1): 1-10 |
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Badiee Kheirabadi S E, Mashayekhi K, Moghadam M, Mousavi M J, Sankian M. Cloning, Expression, and Purification of Recombinant Mouse Interferon-γ. Res Mol Med (RMM) 2021; 9 (1) :1-10
URL: http://rmm.mazums.ac.ir/article-1-398-en.html
URL: http://rmm.mazums.ac.ir/article-1-398-en.html
Seyedeh Elham Badiee Kheirabadi1 
, Kazem Mashayekhi2 , Malihe Moghadam3 , Mohammad Javad Mousavi 4, Mojtaba Sankian5
, Kazem Mashayekhi2 , Malihe Moghadam3 , Mohammad Javad Mousavi 4, Mojtaba Sankian5
1- Department of Laboratory Sciences, School of Paramedical Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydarieh, Iran.
2- Department of Immunology, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
3- Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
4- Department of Hematology, Faculty of Allied Medicine, Bushehr University of Medical Sciences, Bushehr, Iran. ,sm-mousavi@razi.tums.ac.ir
5- Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
2- Department of Immunology, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
3- Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
4- Department of Hematology, Faculty of Allied Medicine, Bushehr University of Medical Sciences, Bushehr, Iran. ,
5- Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Abstract: (3024 Views)
Background: Interferon-gamma [IFN-γ) is the most important cytokine in the immune system. This protein has been expressed in bacterial cells. However, bacterial cloning is not an easy task. We aimed to clone, express, and purify recombinant mouse IFN-γ and overcome problems in favor of commercial purposes.
Materials and Methods: To amplify the gene product for cloning, we primarily designed two specific primers for the target gene. Following PCR amplification, the amplicon was inserted into the pET-21b[+) vector. The E. coli BL21 [DE3) CodonPlus strain was chosen for the expression of the target gene. Finally, the expressed recombinant mouse IFN-γ was assessed through the western blotting method.
Results: We performed a cloning process and produced recombinant mouse IFN-γ in an optimal condition. We also noticed that monomeric protein could be transformed to a homodimeric structure which can be observed using the SDS PAGE [SDS-polyacrylamide gel electrophoresis) and western blotting.
Conclusion: Experimental conditions strongly affect the large-scale cloning procedures required to be optimized in each laboratory. The expressed recombinant mouse IFN-γ described here is appropriate for commercial purposes.
Materials and Methods: To amplify the gene product for cloning, we primarily designed two specific primers for the target gene. Following PCR amplification, the amplicon was inserted into the pET-21b[+) vector. The E. coli BL21 [DE3) CodonPlus strain was chosen for the expression of the target gene. Finally, the expressed recombinant mouse IFN-γ was assessed through the western blotting method.
Results: We performed a cloning process and produced recombinant mouse IFN-γ in an optimal condition. We also noticed that monomeric protein could be transformed to a homodimeric structure which can be observed using the SDS PAGE [SDS-polyacrylamide gel electrophoresis) and western blotting.
Conclusion: Experimental conditions strongly affect the large-scale cloning procedures required to be optimized in each laboratory. The expressed recombinant mouse IFN-γ described here is appropriate for commercial purposes.
Keywords: Cloning, E. coli BL21 (DE3) Codon-Plus, pET21-b (+) vector, Interferon gamma, Recombinant protein
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