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.