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


XML Print


Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

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
Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
Abstract:   (608 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.
 
Full-Text [PDF 843 kb]   (184 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

References
1. Choi S, Ismail A, Pappas-Gogos G, Boussios S. HPV, and Cervical Cancer: A Review of Epidemiology and Screening Uptake in the UK. Pathogens. 2023; 12 (2): 298. [DOI:10.3390/pathogens12020298] [PMID] [PMCID]
2. Chaberek K, Mrowiec M, Kaczmarek M, Dutsch-Wicherek M. The Creation of the Suppressive Cancer Microenvironment in Patients with HPV-Positive Cervical Cancer. Diagnostics. 2022; 12 (8): 1906. [DOI:10.3390/diagnostics12081906] [PMID] [PMCID]
3. Yousefi Z, Aria H, Ghaedrahmati F, Bakhtiari T, Azizi M, Bastan R, et al. An update on human papilloma virus vaccines: history, types, protection, and efficacy. Front Immunol. 2022; 12: 6036. [DOI:10.3389/fimmu.2021.805695] [PMID] [PMCID]
4. Haręża DA, Wilczyński JR, Paradowska E. Human papillomaviruses as infectious agents in gynecological cancers. oncogenic properties of viral proteins. Int J Mol Sci. 2022; 23 (3): 1818. [DOI:10.3390/ijms23031818] [PMID] [PMCID]
5. de Moura IA, Silva AJD, de Macêdo LS, Invenção MdCV, de Sousa MMG, de Freitas AC. Enhancing the Effect of Nucleic Acid Vaccines in the Treatment of HPV-Related Cancers: An Overview of Delivery Systems. Pathogens. 2022; 11 (12): 1444. [DOI:10.3390/pathogens11121444] [PMID] [PMCID]
6. Milani A, Basirnejad M, Bolhassani A. Heat-shock proteins in diagnosis and treatment: An overview of different biochemical and immunological functions. Immunotherapy. 2019; 11 (3): 215-39. [DOI:10.2217/imt-2018-0105] [PMID]
7. Hu C, Yang J, Qi Z, Wu H, Wang B, Zou F, et al. Heat shock proteins: Biological functions, pathological roles, and therapeutic opportunities. MedComm. 2022; 3 (3): e161. [DOI:10.1002/mco2.161] [PMID] [PMCID]
8. Ramezani A, Aghakhani A, Soleymani S, Bavand A, Bolhassani A. Significance of serum antibodies against HPV E7, Hsp27, Hsp20 and Hp91 in Iranian HPV-exposed women. BMC Infect DIS. 2019; 19: 1-8. [DOI:10.1186/s12879-019-3780-2] [PMID] [PMCID]
9. Akhatova A, Chan CK, Azizan A, Aimagambetova G. The efficacy of therapeutic DNA vaccines expressing the human papillomavirus E6 and E7 oncoproteins for treatment of cervical cancer: systematic review. Vaccines. 2022; 10 (1): 53. [DOI:10.3390/vaccines10010053] [PMID] [PMCID]
10. Yan F, Cowell LG, Tomkies A, Day AT. Therapeutic Vaccination for HPV-Mediated Cancers. Curr Otorhinolaryngol Rep. 2023; 11 (1): 44-61. [DOI:10.1007/s40136-023-00443-8] [PMID] [PMCID]
11. Milani A, Rouhollah F, Naseroleslami M, Bolhassani A. The effects of heat shock proteins on delivery of HIV-1 Nef antigen in mammalian cells. Vacres. 2020; 7 (1): 54-9. [DOI:10.29252/vacres.7.1.54]
12. Kardani K, Sadat SM, Kardani M, Bolhassani A. The next generation of HCV vaccines: a focus on novel adjuvant development. Expert Rev Vaccines. 2021; 20 (7): 839-55. [DOI:10.1080/14760584.2021.1941895] [PMID]
13. Deng H, Gao X, Peng H, Wang J, Hou X, Xu H, et al. Effect of liposome‑mediated HSP27 transfection on collagen synthesis in alveolar type II epithelial cells. Mol Med Rep. 2018; 17 (5): 7319-24. [DOI:10.3892/mmr.2018.8744] [PMID]
14. Khairkhah N, Shahhosseini F, Agi E, Milani A, Bolhassani A. Comparison of adjuvant effects of Montanide ISA-720 and heat shock protein 27 in increasing immunostimulatory properties of HIV-1 Nef-Vif fusion protein construct. Protein Pept Lett. 2023. [DOI:10.2174/0929866530666230403093538] [PMID]
15. Chong ZX, Yeap SK, Ho WY. Transfection types, methods and strategies: A technical review. PeerJ. 2021; 9: e11165. [DOI:10.7717/peerj.11165] [PMID] [PMCID]
16. Hunter M, Yuan P, Vavilala D, Fox M. Optimization of protein expression in mammalian cells. Curr Protoc Protein Sci. 2019; 95 (1): e77. [DOI:10.1002/cpps.77] [PMID]
17. Zhou J, Wang L. SAG 4 DNA and peptide vaccination provides partial protection against T. gondii infection in BALB/c mice. Front Microbiol. 2017 ; 8: 1733. [DOI:10.3389/fmicb.2017.01733] [PMID] [PMCID]
18. Khan KH, Gene Expression in Mammalian Cells and its Applications. Adv Pharm Bull. 2013; 3 (2): 257-63.
19. Talebi S, Bolhassani A, Azad TM, Arashkia A, Modaresi M. In vitro expression of HPV16 E7 linked to HMGB1 immunoadjuvant in mammalian cells. Bratislavské Lekárske Listy. 2016; 117 (10): 609-13. [DOI:10.4149/BLL_2016_118] [PMID]

Add your comments about this article : Your username or Email:
CAPTCHA

Send email to the article author


Rights and permissions
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.