1
Department of Biology, School of Science, University of Isfahan, Isfahan, Iran
2
Associate Professor, Department of Biology, School of Science, University of Isfahan, Isfahan, Iran
Abstract
Background: Interferon beta (IFNβ) is one of the important cytokines expressed in response to stimulating factors such as antigens and plays roles in immunity and inflammatory process. In present study, the expression level of IFNβ-1a was examined in HEK293 cell line using real-time polymerase chain reaction (Real-Time PCR).Methods: IFNβ gene sequence was amplified using specific primers contained KpnI and BglII restriction site from pSVMdhfr-IFNβ plasmid as template. It was cloned in similarly digested pBud.CE4.1 linear vector. Construction of recombinant plasmid was verified via restriction fragment length polymorphism (RFLP) analysis, colony PCR and gene sequencing. Recombinant plasmid was transformed into competent Escherichia coli Top10 cells finally. After amplification, recombinant plasmid was purified and transfected into HEK293. At last, RNA extraction, cDNA synthesis and analysis of expression level of gene were performed using Real-Time PCR method.Findings: IFNβ gene was expressed under eEf1a promoter in HEK293 successfully. The expression level of target gene was increased 79.9 times in comparison with the control via transfection. Transfection of null vector showed 2.87 times elevation of target gene expression in response to the alien genome entered into the cell.Conclusion: The proteins produced in prokaryotic systems were non-glycosylated thus they had different physicochemical properties in comparison with the natural form. So, the production of IFNβ protein in human cell line under strong promoter of selected vector is one of the advantages of this research. Protein studies in this field are targeted for the future studies.
Chelbi-Alix MK, Wietzerbin J. Interferon, a growing cytokine family: 50 years of interferon research. Biochimie 2007; 89(6-7): 713-8.
Pestka S, Krause CD, Walter MR. Interferons, interferon-like cytokines, and their receptors. Immunol Rev 2004; 202: 8-32.
Pang KR, Wu JJ, Huang DB, Tyring SK, Baron S. Biological and clinical basis for molecular studies of interferons. Methods Mol Med 2005; 116: 1-23.
Zago P, Baralle M, Ayala YM, Skoko N, Zacchigna S, Buratti E, et al. Improving human interferon-beta production in mammalian cell lines by insertion of an intronic sequence within its naturally uninterrupted gene. Biotechnol Appl Biochem 2009; 52(Pt 3): 191-8.
Mark DF, Lin LS, Lu SDY. Human recombinant cysteine depleted interferon-β muteins [Google Patent: US4588585A]. 1986.
Runkel L, Meier W, Pepinsky RB, Karpusas M, Whitty A, Kimball K, et al. Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-beta (IFN-beta). Pharm Res 1998; 15(4): 641-9.
Hossler P, Khattak SF, Li ZJ. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 2009; 19(9): 936-49.
Morris A, Zvetkova I. Cytokine research: the interferon paradigm. J Clin Pathol 1997; 50(8): 635-9.
Pestka S, Baron S. Definition and classification of the interferons. Methods Enzymol 1981; 78(Pt A): 3-14.
Meyer O. Interferons and autoimmune disorders. Joint Bone Spine 2009; 76(5): 464-73.
Bertolotto A, Deisenhammer F, Gallo P, Solberg SP. Immunogenicity of interferon beta: differences among products. J Neurol 2004; 251(Suppl 2): II15-II24.
Giovannoni G, Munschauer FE, III, Deisenhammer F. Neutralising antibodies to interferon beta during the treatment of multiple sclerosis. J Neurol Neurosurg Psychiatry 2002; 73(5): 465-9.
Skoko N, Argamante B, Grujicic NK, Tisminetzky SG, Glisin V, Ljubijankic G. Expression and characterization of human interferon-beta1 in the methylotrophic yeast Pichia pastoris. Biotechnol Appl Biochem 2003; 38(Pt 3): 257-65.
Smith GE, Summers MD, Fraser MJ. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol 1983; 3(12): 2156-65.
Squires CH. Human interferon beta-1b production in Pseudomonas fluorescens. BioProcess International 2010; 8(7): 132.
Amir-Kalvangh P, Ebtekar M, Azadmanesh K, Hartoonian C, Mahdavi M. Cloning of human Ifnλ-1 (IL-29) from monocyte derived dcs and its expression in Hek 293 T. J Arak Univ Med Sci 2011; 14(57): 69-78. [In Persian].
Thomas P, Smart TG. HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods 2005; 51(3): 187-200.
Pfaffl MV. Quantification strategies in real-time PCR. In: Bustin SA, editor. A-Z of quantitative PCR. La Jolla CA: International University Line; 2004. p. 87-112.
Norouzi, R., & Hojati, Z. (2015). Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct. Journal of Isfahan Medical School, 33(354), 1691-1700.
MLA
Raheleh Norouzi; Zohreh Hojati. "Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct". Journal of Isfahan Medical School, 33, 354, 2015, 1691-1700.
HARVARD
Norouzi, R., Hojati, Z. (2015). 'Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct', Journal of Isfahan Medical School, 33(354), pp. 1691-1700.
VANCOUVER
Norouzi, R., Hojati, Z. Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct. Journal of Isfahan Medical School, 2015; 33(354): 1691-1700.
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