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Introduction: Vaccine design is mainly considered as a therapeutic strategy to elicit HIV-specific immunity. DNA vaccines encoding an antigen and also an adjuvant can induce an effective adaptive immunity. Due to having numerous roles in viral infection, the HIV-1 Nef protein is considered as an antigen candidate for development of therapeutic vaccines. A variety of adjuvants and delivery systems have been utilized to increase the potency of DNA vaccines against viral infections, such as heat shock proteins (HSPs) which possess chaperon activity and immunostimulatory properties. Methods:  pEGFP mammalian expression vectors harboring nef, hsp27, hsp27-nef, hsp70 and hsp70-nef genes were prepared in large scale. Their concentration and purity were assessed by NanoDrop spectrophotometry. Human embryonic kidney 293T cells (HEK-293T) were grown in DMEM culture medium and transfected with these constructs using Lipofectamine 2000 transfection reagent. After 48 hours, their transfection efficiency was evaluated using fluorescent microscopy and flow cytometry. Results: The pEGFP-nef, -hsp27, -hsp27-nef, -hsp70 and -hsp70-nef constructs were successfully prepared in large scale and high purity. The results of cell transfection with each construct showed that the percentages of Nef-GFP, Hsp27-GFP, Hsp27-Nef-GFP, Hsp70-GFP, Hsp70-Nef-GFP, Hsp70-GFP + Nef-GFP and GFP expression were 53.1 ± 0.2, 64.22 ± 0.8, 57.1 ± 0.7, 68.8 ± 1.0, 61.7 ± 0.7, 77.4 ± 1.5 and 81.8 ± 1.8, respectively. Conclusion: These data showed that the potency of Hsp70 was more than Hsp27 for increasing Nef-GFP expression in HEK-293T cells. Moreover, the delivery of pEGFP-nef along with pEGFP-hsp70 increased the rate of GFP population in the cells as compared to that in pEGFP-hsp70-nef. These constructs will be used for development of HIV-1 DNA vaccine in animal model in near Future.

Keywords: HIV-1, Nef Antigen, Heat Shock Protein, Hsp27, Hsp70, DNA Vaccine

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