title: Generation of the Fluorescent HMGB1-GFP Fusion Protein in Insect Cells and Evaluation of its Immunogenicity in Two Mice Models.
abstract: BACKGROUND
  High mobility group box 1 (HMGB1) is a highly conserved protein present in the nuclei and cytoplasm of cells which has an important role as a mediator of inflammation in the extracellular environment. HMGB1 was identified as an innate adjuvant that induces immune responses against soluble antigens in vivo.
  
  
  OBJECTIVE
  Our goal is the generation of recombinant HMGB1-GFP fusion protein in insect cells for evaluation of immune responses in mouse model.
  
  
  METHODS
  In the current study, we used a baculovirus expression system for insect cells that was based on expression of HMGB1 with target gene (GFP), and purified the recombinant HMGB1- GFP fusion protein. We then demonstrated whether immunogenicity of GFP changes in the presence or absence of recombinant HMGB1 acting as an adjuvant in C57BL/6 and BALB/c mice.
  
  
  RESULTS
  Our data showed that HMGB1 had a major influence on antibody immune responses induced by GFP in both animal models. The groups receiving HMGB1-GFP fusion protein showed total IgG and IgG2a responses significantly higher than IgG1 in BALB/c mice. Indeed, a mixed IgG1/IgG2a response was observed with high intensity toward IgG2a. In contrast, C57BL/6 mice immunized by HMGB1-GFP protein elicited the same levels of IgG1 and IgG2a. However, the levels of IgG2a and total IgG against the recombinant GFP (rGFP) in C57BL/6 mice were lower than those in BALB/c mice.
  
  
  CONCLUSIONS
  We concluded that fusion of HMGB1 with GFP was immunologically more effective than GFP alone.
authors.affiliation: Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Anvar
authors.affiliation: Department of Medical Lab Technology, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences; Tehran, Iran.
authors.forename: Rouhollah
authors.initials: R
authors.lastname: Vahabpour
authors.affiliation: Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors.forename: Iman
authors.initials: I
authors.lastname: Salahshourifar
authors.affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Azam
authors.initials: A
authors.lastname: Bolhassani
mesh_terms: D000276:Adjuvants, Immunologic; D000818:Animals; D049452:Green Fluorescent Proteins / Q000235:genetics; D024243:HMGB1 Protein / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D007114:Immunization; D007074:Immunoglobulin G / Q000276:immunology*; D007313:Insecta / Q000166:cytology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D000276:Adjuvants, Immunologic; D024243:HMGB1 Protein; C582808:HMGB1 protein, mouse; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: Baculovirus-insect cells; GFP; HMGB1; Immunogenicity; adjuvant; fusion protein