Journal of Medical Microbiology and Infectious Diseases

en Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells Immune responses, deficiencies and vaccine candidates Immune responses, deficiencies and vaccine candidates Original article Original article Introduction: Developing potent therapeutic vaccines against human papillomaviruses (HPVs) is crucial for the effective management of various HPV-associated cancers. DNA-based vaccines are attractive due to their safety, stability, and capacity to elicit a targeted immune response against specific antigens. Heat shock proteins (HSPs) can enhance the efficacy of DNA vaccines when used as adjuvants. In this study, we created a recombinant DNA molecule by fusing the HPV16 e7 gene with either the hspB1 or hsp27 gene and assessed its expression in a eukaryotic cell line. Methods: Initially, we constructed a recombinant eukaryotic expression vector by inserting the hsp27-e7 fusion gene into the pcDNA3.1 (-) vector. The concentration and purity of the sample were evaluated using NanoDrop spectrophotometry. We cultured human<a name="_Hlk139703661"> embryonic kidney 293T </a> (HEK-293T) cells in RPMI 1640 medium and transfected them with the pcDNA3.1-hsp27-e7 construct using Lipofectamine 2000 transfection reagent. After 48 hours, we assessed the expression of the Hsp27-E7 fusion protein by western blotting using an anti-E7 monoclonal antibody. Results: We successfully subcloned the hsp27-e7 fusion gene into the pcDNA3.1 (-) vector, and enzymatic digestion confirmed a distinct ~975 bp band on an agarose gel. The concentration and purity of the recombinant DNA vector in a 10 mL culture were measured to be 210 ng/µL and 1.86, respectively. Furthermore, the expression of the Hsp27-E7 fusion protein in HEK-293T cells was confirmed by Western blot analysis, which detected a distinct band of approximately 38 kDa. Conclusion: Our in vitro findings demonstrate successful expression of the DNA construct encoding the hsp27-e7 gene, which can be utilized as a DNA vaccine for future in vivo investigations.   Human papillomavirus (HPV), E7, Small heat shock protein, DNA-based vaccine, Recombinant DNA construct, Eukaryotic cells 123 127 http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-441-2&slc_lang=en&sid=1 Reyhane Najafi 0009-0009-2964-6386 No Department of Biotechnology, Faculty of Advanced Science and Technology, Islamic Azad University, Tehran Medical Branch, Tehran, Iran Azam Bolhassani A_bolhasani@pasteur.ac.ir 0000-0001-7363-7406 Yes Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran Maryam Montazeri maryam104@Yahoo.com 0000-0003-2732-8551 No Department of Biotechnology, Faculty of Advanced Science and Technology, Islamic Azad University, Tehran Medical Branch, Tehran, Iran Elnaz Agi 0000-0003-3587-2952 No Iranian Comprehensive Hemophilia Care Center, Tehran, Iran