title: Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Neda
authors.initials: N
authors.lastname: Mousavi Niri
authors.affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Arash
authors.initials: A
authors.lastname: Memarnejadian
authors.affiliation: Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Jamshid
authors.initials: J
authors.lastname: Hadjati
authors.affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Aghasadeghi
authors.affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Shokri
authors.affiliation: Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Yones
authors.initials: Y
authors.lastname: Pilehvar-Soltanahmadi
authors.affiliation: Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Abolfazl
authors.initials: A
authors.lastname: Akbarzadeh
authors.affiliation: Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Clinical Biochemistry, Radiopharmacy Lab, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Nosratollah
authors.initials: N
authors.lastname: Zarghami
keywords: FOXP3 protein; Fusion protein; Immunoglobulin G (IgG)