Evaluation of Interferon Beta Protein Expression in HEK293T Cell Line Transfected by Recombinant Construction, Pbud.Ifnb 1a
Cytokines are a group of signaling glycoproteins that are expressed in respond to stimulus factors and antigens. IFNa is an important cytokine that play role in immune processes, inflammation and host defense against external factors. In this study, the expression level of IFNb protein in HEK293T cell line transfected by recombinant construction pBud.IFNb examine via Dot blot and Elisa protein tests. Sequence of IFNb gene with using of primers containing Kozak conserved sequence and enzyme restriction site of Kpnl and BglII amplified from pSVMdhfr vector. After treatment by above mentioned enzymes, IFNa gene cloned in linearized pBud.CE4.1. Structure of recombinant vector were examined via RFLP, Colony PCR and sequencing and finally transformed into competent Ecoli.TOP10 bacteria and next transfected to HEK293T cells. The results of protein tests indicate that recombinant IFNa gene expressed successfully in HEK293T cell line under eEf1a promoter of selected vector. Proteins produced in prokaryotic systems are lacking glycosylation and thus have different physicochemical properties in comparison with normal protein production in the body. This causes side effects in the patient so the production of IFNa protein in human HEK293T cells and high expression level of this protein is the benefits of this research.