Expression Analysis of Interferon Beta Level in HEK۲۹۳T Cells Using Real-Time PCR and Protein Tests

Background: Interferons are some kind of natural cytokines which express in response to a variety of antigens including viral RNA, bacterial products, and tumor proteins. Interferon beta is used in the treatment of autoimmune diseases such as multiple sclerosis. Moreover, this drug inhibits cellular proliferation as well as angiogenesis and as a result, helps to cure cancer. In this research, in addition to cloning the interferon-beta gene along with conserved kozak sequence after the strong eEf۱a promoter in the pBud.CE۴.۱ vector, the expression of this recombinant gene was compared to basal expression in HEK۲۹۳T cell line using real-time PCR, SDS PAGE and western blot tests. Materials and Methods: In the beginning, the interferon-beta gene was amplified from the pSVM dhfr vector containing the gene, using primers including BglII and KpnI restriction sites as well as conserved kozak sequence. Then the duplicated gene was digested and inserted in the linear pBud.CE۴.۱ vector. After ensuring entry of the gene using RFLP, colony PCR and sequencing, the recombinant vector was transfected into E. coli TOP۱۰ competent bacteria. After that, amplified recombinant vector was extracted and transfected into HEK۲۹۳ cell line. Results: The expression of interferon-beta cloned in pBud.CE۴.۱ vector showed a ۷۹.۹-fold increase, in comparison with the basal expression in HEK۲۹۳T cell line. Moreover, non-recombinant vector transfection has increased the expression of interferon-beta up to ۲.۸۷ times in the cell line that is probably due to the existence of the viral promoter in the vector. Conclusion: Real-time PCR and protein test results showed that recombinant beta interferon gene had been successfully expressed in HEK۲۹۳T cell line. In order to produce more of this protein, optimization of various conditions are required for the HEK۲۹۳T cell line.

Keywords:

HEK۲۹۳T cells , Interferon Beta ۱a , Real-time PCR , SDS PAGE , Western blot