Adv Pharm Bull. 2015;5(5):673-681.
doi: 10.15171/apb.2015.092
PMID: 26793615
PMCID: PMC4708040
Scopus id: 84954120716
  Abstract View: 455
  PDF Download: 193

Original Research

Generation of New M2e-HA2 Fusion Chimeric Peptide to Development of a Recombinant Fusion Protein Vaccine

Ali Ameghi 1,2, Behzad Baradaran 3, Khosrow Aghaiypour 4 * , Abolfazl Barzegar 5, Yones Pilehvar-Soltanahmadi 3,6, Masood Moghadampour 2, Morteza Taghizadeh 2, Nosratollah Zarghami 3,1,6 *

1 Department of Clinical Biochemistry, Tabriz University of Medical Sciences, Tabriz, Iran.
2 Department of Influenza, Razi Vaccine and Serum Research Institute, Alborz, Iran.
3 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
4 Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute, Alborz, Iran.
5 Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran.
6 Department of Medical Biotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran.

Abstract

Purpose: 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. Conclusion: 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.
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