Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type ۱۶ E۷ Fused to Heat Shock Protein B۱ in Eukaryotic Cells

Publish Year: 1402
نوع سند: مقاله ژورنالی
زبان: English
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JR_JMMI-11-3_001

تاریخ نمایه سازی: 29 بهمن 1402

Abstract:

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 HPV۱۶ e۷ gene with either the hspB۱ or hsp۲۷ gene and assessed its expression in a eukaryotic cell line. Methods: Initially, we constructed a recombinant eukaryotic expression vector by inserting the hsp۲۷-e۷ fusion gene into the pcDNA۳.۱ (-) vector. The concentration and purity of the sample were evaluated using NanoDrop spectrophotometry. We cultured human embryonic kidney ۲۹۳T (HEK-۲۹۳T) cells in RPMI ۱۶۴۰ medium and transfected them with the pcDNA۳.۱-hsp۲۷-e۷ construct using Lipofectamine ۲۰۰۰ transfection reagent. After ۴۸ hours, we assessed the expression of the Hsp۲۷-E۷ fusion protein by western blotting using an anti-E۷ monoclonal antibody. Results: We successfully subcloned the hsp۲۷-e۷ fusion gene into the pcDNA۳.۱ (-) vector, and enzymatic digestion confirmed a distinct ~۹۷۵ bp band on an agarose gel. The concentration and purity of the recombinant DNA vector in a ۱۰ mL culture were measured to be ۲۱۰ ng/µL and ۱.۸۶, respectively. Furthermore, the expression of the Hsp۲۷-E۷ fusion protein in HEK-۲۹۳T cells was confirmed by Western blot analysis, which detected a distinct band of approximately ۳۸ kDa. Conclusion: Our in vitro findings demonstrate successful expression of the DNA construct encoding the hsp۲۷-e۷ gene, which can be utilized as a DNA vaccine for future in vivo investigations.

Authors

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

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  • Choi S, Ismail A, Pappas-Gogos G, Boussios S. HPV, and ...
  • Chaberek K, Mrowiec M, Kaczmarek M, Dutsch-Wicherek M. The Creation ...
  • Yousefi Z, Aria H, Ghaedrahmati F, Bakhtiari T, Azizi M, ...
  • Haręża DA, Wilczyński JR, Paradowska E. Human papillomaviruses as infectious ...
  • de Moura IA, Silva AJD, de Macêdo LS, Invenção MdCV, ...
  • Milani A, Basirnejad M, Bolhassani A. Heat-shock proteins in diagnosis ...
  • Hu C, Yang J, Qi Z, Wu H, Wang B, ...
  • Ramezani A, Aghakhani A, Soleymani S, Bavand A, Bolhassani A. ...
  • Akhatova A, Chan CK, Azizan A, Aimagambetova G. The efficacy ...
  • Yan F, Cowell LG, Tomkies A, Day AT. Therapeutic Vaccination ...
  • Milani A, Rouhollah F, Naseroleslami M, Bolhassani A. The effects ...
  • Kardani K, Sadat SM, Kardani M, Bolhassani A. The next ...
  • Deng H, Gao X, Peng H, Wang J, Hou X, ...
  • Khairkhah N, Shahhosseini F, Agi E, Milani A, Bolhassani A. ...
  • Chong ZX, Yeap SK, Ho WY. Transfection types, methods and ...
  • Hunter M, Yuan P, Vavilala D, Fox M. Optimization of ...
  • Zhou J, Wang L. SAG ۴ DNA and peptide vaccination ...
  • Khan KH, Gene Expression in Mammalian Cells and its Applications. ...
  • Talebi S, Bolhassani A, Azad TM, Arashkia A, Modaresi M. ...
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