Ali Ameghi1, Behzad Baradaran2, Khosrow Aghaiypour3, Abolfazl Barzegar4, Yones Pilehvar-Soltanahmadi5, Masood Moghadampour6, Morteza Taghizadeh6, Nosratollah Zarghami7. 1. Department of Clinical Biochemistry, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Influenza, Razi Vaccine and Serum Research Institute, Alborz, Iran. 2. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 3. Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute, Alborz, Iran. 4. Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran. 5. 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. 6. Department of Influenza, Razi Vaccine and Serum Research Institute, Alborz, Iran. 7. 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.
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.
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.
Authors: Liudmila A Stepanova; Eugenia S Mardanova; Marina A Shuklina; Elena A Blokhina; Roman Y Kotlyarov; Marina V Potapchuk; Anna A Kovaleva; Inna G Vidyaeva; Alexandr V Korotkov; Elizaveta I Eletskaya; Nikolai V Ravin; Liudmila M Tsybalova Journal: J Biomed Sci Date: 2018-04-09 Impact factor: 8.410