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Article History
Submitted: 10 Oct 2015
Revised: 22 Dec 2015
Accepted: 23 Dec 2015
First published online: 31 Dec 2015

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Adv Pharm Bull. 2015;5(5):673-681 doi: 10.15171/apb.2015.092
PMID:26793615        PMCID:PMC4708040

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

Original Research

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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Articles by Ameghi A
Articles by Baradaran B
Articles by Aghaiypour K
Articles by Barzegar A
Articles by Pilehvar-Soltanahmadi Y
Articles by Moghadampour M
Articles by Taghizadeh M
Articles by Zarghami N

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Articles by Ameghi A
Articles by Baradaran B
Articles by Aghaiypour K
Articles by Barzegar A
Articles by Pilehvar-Soltanahmadi Y
Articles by Moghadampour M
Articles by Taghizadeh M
Articles by Zarghami N

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