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Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein
BACKGROUND: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell (DC) based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Avicenna Research Institute
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842243/ https://www.ncbi.nlm.nih.gov/pubmed/27141264 |
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author | Mousavi Niri, Neda Memarnejadian, Arash Hadjati, Jamshid Aghasadeghi, Mohammad Reza Shokri, Mehdi Pilehvar-soltanahmadi, Yones Akbarzadeh, Abolfazl Zarghami, Nosratollah |
author_facet | Mousavi Niri, Neda Memarnejadian, Arash Hadjati, Jamshid Aghasadeghi, Mohammad Reza Shokri, Mehdi Pilehvar-soltanahmadi, Yones Akbarzadeh, Abolfazl Zarghami, Nosratollah |
author_sort | Mousavi Niri, Neda |
collection | PubMed |
description | BACKGROUND: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell (DC) based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc(IgG) can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I (cross priming). METHODS: DNA construct containing fragment C (Fc) portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli (E. coli) strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining. RESULTS: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells. Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification. CONCLUSION: Due to successful expression of Foxp3-Fc (IgG), it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means. |
format | Online Article Text |
id | pubmed-4842243 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Avicenna Research Institute |
record_format | MEDLINE/PubMed |
spelling | pubmed-48422432016-05-02 Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein Mousavi Niri, Neda Memarnejadian, Arash Hadjati, Jamshid Aghasadeghi, Mohammad Reza Shokri, Mehdi Pilehvar-soltanahmadi, Yones Akbarzadeh, Abolfazl Zarghami, Nosratollah Avicenna J Med Biotechnol Original Article BACKGROUND: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell (DC) based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc(IgG) can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I (cross priming). METHODS: DNA construct containing fragment C (Fc) portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli (E. coli) strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining. RESULTS: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells. Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification. CONCLUSION: Due to successful expression of Foxp3-Fc (IgG), it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means. Avicenna Research Institute 2016 /pmc/articles/PMC4842243/ /pubmed/27141264 Text en Copyright© 2016 Avicenna Research Institute This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. |
spellingShingle | Original Article Mousavi Niri, Neda Memarnejadian, Arash Hadjati, Jamshid Aghasadeghi, Mohammad Reza Shokri, Mehdi Pilehvar-soltanahmadi, Yones Akbarzadeh, Abolfazl Zarghami, Nosratollah Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein |
title | Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein |
title_full | Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein |
title_fullStr | Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein |
title_full_unstemmed | Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein |
title_short | Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein |
title_sort | construction and production of foxp3-fc (igg) dna vaccine/fusion protein |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842243/ https://www.ncbi.nlm.nih.gov/pubmed/27141264 |
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