Alireza Milani, Azam Bolhassani, Masoumeh Heshmati: Small heat shock protein 27 increases the expression of HIV-1 nef gene in human embryonic kidney 293T cells. The Second International and 10th National Biotechnology Congress, Tehran-Iran; 08/2017. (Oral)
Abstract: Practical consideration of DNA vaccines as an alternative to other therapeutic methods against HIV depends on the improved delivery systems and different adjuvants. Hsp27 belongs to small heat shock proteins family that has similarity in a-crystalline and C-terminal domain with other small members. Recent studies demonstrated that Hsp27 can modulate immune system through interaction with receptors such as TLR-2 and TLR-4. In this study, at first, HIV-1 pNL4.3 Nef, Hsp27 and Hsp27-Nef fusion constructs were designed in pEGFP mammalian expression vector. Human Embryonic Kidney 293T cells (HEK-293T) were grown in DMEM and transfected with these constructs using TurboFect and Lipofectamine2000 transfection reagents. After 48 hours, their transfection efficiency was evaluated using fluorescent microscopy, flow cytometry, and western blotting. Our results indicated a ~ 648 bp, ~ 720 bp, and ~ 1368 bp band for Nef, Hsp27, and Hsp-Nef, respectively on agarose gel electrophoresis. The DNA transfection efficiency was determined by the percentage of proteins expression using GFP reporter marker. The percentage of Nef-GFP, Hsp27-GFP, Hsp27-Nef-GFP, GFP proteins expression was 50.16, 75.19, 69.26, 88.13 for TurboFect system, and 41.36, 64.72, 56.20, 81.39 for lipofectamine, respectively. Western blot analysis showed the clear bands of ~ 53 kDa, ~ 54 kDa, ~ 80 kDa, and ~ 27 kDa for Nef-GFP, Hsp27-GFP, Hsp27-Nef-GFP, and GFP, respectively. Generally, the efficiency of TurboFect transfection reagent was significantly higher than lipofectamine in in vitro gene delivery. Moreover, Hsp27 could increase HIV-1 Nef expression in HEK-293T cells. These constructs will be used for development of DNA vaccine in animal model in near Future.
Keywords: HIV-1-Nef, Small Heat Shock Protein, Hsp27, Turbofect, Lipofectamine
