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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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Pasteur Institute of Iran
2023-09-01
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| Series: | Journal of Medical Microbiology and Infectious Diseases |
| Subjects: | |
| Online Access: | https://jommid.pasteur.ac.ir/article-1-586-en.html |
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| Summary: | 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. |
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| ISSN: | 2345-5349 2345-5330 |