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Journal of Medical Microbiology and Infectious Diseases، جلد ۱۱، شماره ۳، صفحات ۱۲۳-۱۲۷
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عنوان انگلیسی Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells
چکیده انگلیسی مقاله 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.
 
کلیدواژه‌های انگلیسی مقاله Human papillomavirus (HPV), E7, Small heat shock protein, DNA-based vaccine, Recombinant DNA construct, Eukaryotic cells
نویسندگان مقاله | Reyhane Najafi
Department of Biotechnology, Faculty of Advanced Science and Technology, Islamic Azad University, Tehran Medical Branch, Tehran, Iran


| Azam Bolhassani
Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran


| Maryam Montazeri
Department of Biotechnology, Faculty of Advanced Science and Technology, Islamic Azad University, Tehran Medical Branch, Tehran, Iran


| Elnaz Agi
Iranian Comprehensive Hemophilia Care Center, Tehran, Iran
نشانی اینترنتی http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-441-2&slc_lang=en&sid=1
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کد مقاله (doi)
زبان مقاله منتشر شده en
موضوعات مقاله منتشر شده Immune responses, deficiencies and vaccine candidates
نوع مقاله منتشر شده Original article
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