Volume 11, Issue 3 (9-2023)                   JoMMID 2023, 11(3): 123-127 | Back to browse issues page

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Najafi R, Bolhassani A, Montazeri M, Agi E. Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells. JoMMID 2023; 11 (3) :123-127
URL: http://jommid.pasteur.ac.ir/article-1-586-en.html
Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells
Reyhane Najafi , Azam Bolhassani * , Maryam Montazeri , Elnaz Agi
Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
Abstract:   (1288 Views)
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 embryonic kidney 293T (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.
 
Keywords: Human papillomavirus (HPV), E7, Small heat shock protein, DNA-based vaccine, Recombinant DNA construct, Eukaryotic cells
Full-Text [PDF 843 kb]   (440 Downloads)    
Type of Study: Original article | Subject: Immune responses, deficiencies and vaccine candidates
Received: 2023/08/28 | Accepted: 2023/08/28 | Published: 2023/11/11
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Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.