title: In Vitro Evaluation of Influenza M2 and Leishmania major HSP70 (221-604) Chimer Protein.
abstract: BACKGROUND
  Permanent antigenic variation of influenza viruses causes a major concern to develop an effective human influenza vaccine. Conserved antigens are new vaccine candidates because it is not necessary to match the prepared vaccine with circulating strains. Ion channel M2 protein is conserved among all influenza A viruses, allowing the virus to enter host cells.
  
  
  OBJECTIVE
  To prepare an effective vaccine against influenza A viruses, a chimerical DNA plasmid encoding Influenza virus M2 protein and Leishmania major HSP70 was constructed.
  
  
  METHODS
  Influenza A/New Caledonia/20/99 (H1N1) was inoculated into MDCK cell line and total RNA was extracted. The full length M2 gene was amplified by RT-PCR using designed specific primers, cloned into pGEM-T Easy cloning vector and completely sequenced. The M2 gene was then subcloned into the pcDNA upstream of HSP70 gene. Recombinant plasmids were transfected into COS-7 cells to evaluate protein expression.
  
  
  RESULTS
  The recombinant plasmids were confirmed by PCR, restriction enzyme analysis and sequencing. Three dimensional structure of chimer protein was assessed using specific software. Transient protein expression in eukaryotic cells was confirmed by specific mRNA detection, indirect Immunofluorescence test and western blotting.
  
  
  CONCLUSIONS
  M2-HSP70 chimer protein was successfully expressed in eukaryotic cells. Computational studies of chimer peptide sequence revealed that fusing HSP to the C-terminal of M2 protein does not mask the predominant epitope of M2. HSP70 is a molecular chaperon and immunostimulatory component. Genetically fusing antigens to HSPs leads to the enrichment of DNA vaccine potency. The immunogenicity of this construct with different formulation would be evaluated in further investigations.
authors.affiliation: Influenza Research Lab, Department of Virology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Fotouhi
authors.affiliation: Influenza Research Lab, Department of Virology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Behrokh
authors.initials: B
authors.lastname: Farahmand
authors.affiliation: Influenza Research Lab, Department of Virology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Behnaz
authors.initials: B
authors.lastname: Heidarchi
authors.affiliation: Virology Department, Iran University of Medical Sciences, Tehran, IR Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Esghaei
authors.affiliation: Molecular Immunology and Vaccine Research Lab, Department of Immunology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Sima
authors.initials: S
authors.lastname: Rafati
authors.affiliation: Influenza Research Lab, Department of Virology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Masoumeh
authors.initials: M
authors.lastname: Tavassoti Kheiri
keywords: Heat Shock Protein; Influenza Virus; M2 Protein; Molecular Chaperone; Vaccine