Expression and Characterization of Two DNA Constructs Derived from HIV-1-vif in Escherichia coli and Mammalian Cells

en Expression and Characterization of Two DNA Constructs Derived from HIV-1-vif in Escherichia coli and Mammalian Cells Background: Acquired immunodeficiency syndrome (HIV/AIDS) is still a major global concern and no effective therapeutic vaccine has been produced to prevent the problem. Among HIV-1 proteins, vif as a basic cytoplasmic protein of HIV-1 is involved in late stages of viral generation and plays important role in HIV-1 virion replication. It also increases the stability of virion cores, which probably inhibits early degradation of viral entry. Therefore, it seems rational to apply this protein as a vaccine based on its impact on HIV-1 life cycle. This study aimed at cloning, expression and production of vif protein as an HIV-1 vaccine candidate. Methods: In this study, vif sequence was amplified from pLN4-3 plasmid including HIV-1 vif gene and then cloned in pET23a to generate the recombinant plasmids of pET23a/vif with hexahistidine tags. BL21 competent cells were transformed to obtain the protein of interest. Ni-NTA column was used to purify the protein of interest and western blotting confirmed vif protein using anti-His tag antibody. In order to express the gene of interest in eukaryotic cells, vif was sub-cloned into pEGFP plasmids and HEK 293-T cells were transfected. Flow cytometry was then applied to evaluate GFP expression. Results: vif protein was expressed in BL21)DE3) strain and identified as a23 kDa band in SDS-PAGE and confirmed by anti-His antibody in western blotting. The purified protein concentration was 173.3 μg/ml using Bradford assay. HEK-293T cells were successfully transfected by recombinant pEGFP plasmids and flow cytometry confirmed the cell transfection. Conclusion: vif protein can be expressed in mammalian cells and may be a proper protein subunit vaccine candidate against HIV-1. HIV-1, Vaccines, Vif protein 131 135 https://www.ajmb.org/En/Article.aspx?id=40465 https://www.ajmb.org/PDF/En/FullText/40465.pdf FatemehZamaniDepartment of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran61746AzamBolhassaniDepartment of Hepatitis, AIDS and Blood Borne Diseases, Pasteur Institute of Iran, Tehran, Iran50SepidehShahbazi Department of Hepatitis, AIDS and Blood Borne Diseases, Pasteur Institute of Iran, Tehran, Iran61747AhmadFaghihDepartment of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran61748Seyed MehdiSadat817