Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells
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 specif...
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| Format: | Article |
| Language: | English |
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Pasteur Institute of Iran
2023-09-01
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| Series: | Journal of Medical Microbiology and Infectious Diseases |
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| Online Access: | https://jommid.pasteur.ac.ir/article-1-586-en.html |
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| author | Reyhane Najafi1 Azam Bolhassani2* Maryam Montazeri1 Elnaz Agi3 |
| author_facet | Reyhane Najafi1 Azam Bolhassani2* Maryam Montazeri1 Elnaz Agi3 |
| author_sort | Reyhane Najafi1 |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-99fd3e51d08c4296be6bc40bcdcb187e |
| institution | OA Journals |
| issn | 2345-5349 2345-5330 |
| language | English |
| publishDate | 2023-09-01 |
| publisher | Pasteur Institute of Iran |
| record_format | Article |
| series | Journal of Medical Microbiology and Infectious Diseases |
| spelling | doaj-art-99fd3e51d08c4296be6bc40bcdcb187e2025-08-20T01:49:41ZengPasteur Institute of IranJournal of Medical Microbiology and Infectious Diseases2345-53492345-53302023-09-0111312313710.61186/JoMMID.11.3.123Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic CellsReyhane Najafi10https://orcid.org/0009-0009-2964-6386Azam Bolhassani2*1https://orcid.org/0000-0001-7363-7406Maryam Montazeri12https://orcid.org/0000-0003-2732-8551Elnaz Agi33https://orcid.org/0000-0003-3587-29521Department of Biotechnology, Faculty of Advanced Science and Technology, Islamic Azad University, Tehran Medical Branch, Tehran, Iran2Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran1Department of Biotechnology, Faculty of Advanced Science and Technology, Islamic Azad University, Tehran Medical Branch, Tehran, Iran3Iranian Comprehensive Hemophilia Care Center, Tehran, IranIntroduction: 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.https://jommid.pasteur.ac.ir/article-1-586-en.htmlhuman papillomavirus (hpv)e7small heat shock proteindna-based vaccinerecombinant dna constructeukaryotic cells |
| spellingShingle | Reyhane Najafi1 Azam Bolhassani2* Maryam Montazeri1 Elnaz Agi3 Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells Journal of Medical Microbiology and Infectious Diseases human papillomavirus (hpv) e7 small heat shock protein dna-based vaccine recombinant dna construct eukaryotic cells |
| title | Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells |
| title_full | Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells |
| title_fullStr | Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells |
| title_full_unstemmed | Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells |
| title_short | Successful Expression of DNA-Based Vaccine Construct Encoding Human Papillomavirus Type 16 E7 Fused to Heat Shock Protein B1 in Eukaryotic Cells |
| title_sort | successful expression of dna based vaccine construct encoding human papillomavirus type 16 e7 fused to heat shock protein b1 in eukaryotic cells |
| topic | human papillomavirus (hpv) e7 small heat shock protein dna-based vaccine recombinant dna construct eukaryotic cells |
| url | https://jommid.pasteur.ac.ir/article-1-586-en.html |
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