مجله دانشکده پزشکی اصفهان (Dec 2015)
Construction of pBud.CE4.1 IFNβ (Human Beta Interferon) and Analysis of its Expression Level in Transfected HEK293 Cell via This Construct
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.
