title: Generation of New M2e-HA2 Fusion Chimeric Peptide to Development of a Recombinant Fusion Protein Vaccine.
abstract: OBJECTIVE
  The purpose was to design a new construction containing influenza virus (H1N1) M2e gene and HA2 gene by bioinformatics approach, cloning the construct in to Escherichia coli and produce M2e-HA2 peptide.
  
  
  METHODS
  The procedure was done by virus cultivation in SPF eggs, hemagglutination assay (HA), RNA isolation, RT-PCR, primers designed (DNAMAN 4 and Oligo7), virtual fusion construction translation (ExPASy), N-Glycosylated sites prediction (Ensemblegly-Iowa), complete open reading frame (ORF), stop codon studied (NCBI ORF Finder), rare codon determination (GenScript), Solvent accessibility of epitopes (Swiss-PdbViewer), antigenic sites prediction (Protean), fusion PCR of M2e-HA2 gene, sequence analysis, nested PCR, gel electrophoresis, double digestion of pET22b(+) plasmid and the fusion construct, ligation of them, transformation of the ligated vector (pET22b-M2e-HA2) to E.coli (BL21), mass culture the cloned bacterium ,induction the expression by isopropyl-beta-D-thiogalactopyranoside (IPTG), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), purification the fusion peptide by Ni-NTA column, western blot to verify the purification.
  
  
  RESULTS
  In this study we developed a new approach for fusion of Influenza virus M2e (96 nucleotides) and HA2 (663 nucleotides) genes based on fusion PCR strategy and produced a fused fragment with 793 nucleotides. The construct was successfully cloned and expressed.
  
  
  CONCLUSIONS
  This construct is a 261 amino acid chimeric fusion peptide with about 30 KD molecular weight. According on the latest information; this is the first case of expression and purification M2e-HA2 fusion chimeric peptide, which could be used for development of a recombinant M2e-HA2 fusion protein vaccine.
authors.affiliation: Department of Clinical Biochemistry, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Influenza, Razi Vaccine and Serum Research Institute, Alborz, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Ameghi
authors.affiliation: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Behzad
authors.initials: B
authors.lastname: Baradaran
authors.affiliation: Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute, Alborz, Iran.
authors.forename: Khosrow
authors.initials: K
authors.lastname: Aghaiypour
authors.affiliation: Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran.
authors.forename: Abolfazl
authors.initials: A
authors.lastname: Barzegar
authors.affiliation: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Medical Biotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Yones
authors.initials: Y
authors.lastname: Pilehvar-Soltanahmadi
authors.affiliation: Department of Influenza, Razi Vaccine and Serum Research Institute, Alborz, Iran.
authors.forename: Masood
authors.initials: M
authors.lastname: Moghadampour
authors.affiliation: Department of Influenza, Razi Vaccine and Serum Research Institute, Alborz, Iran.
authors.forename: Morteza
authors.initials: M
authors.lastname: Taghizadeh
authors.affiliation: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Clinical Biochemistry, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Medical Biotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Nosratollah
authors.initials: N
authors.lastname: Zarghami
keywords: Cloning; Expression; Fusion PCR; HA2; Influenza; M2e