tetano
Editor, Senior Moderator
Adv Pharm Bull. 2015 Dec;5(Suppl 1):673-81. doi: 10.15171/apb.2015.092. Epub 2015 Dec 31.
[h=1]Generation of New M2e-HA2 Fusion Chimeric Peptide to Development of a Recombinant Fusion Protein Vaccine.[/h] Ameghi A[SUP]1[/SUP], Baradaran B[SUP]2[/SUP], Aghaiypour K[SUP]3[/SUP], Barzegar A[SUP]4[/SUP], Pilehvar-Soltanahmadi Y[SUP]5[/SUP], Moghadampour M[SUP]6[/SUP], Taghizadeh M[SUP]6[/SUP], Zarghami N[SUP]7[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]PURPOSE:[/h] 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.
[h=4]METHODS:[/h] 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.
[h=4]RESULTS:[/h] 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.
[h=4]CONCLUSION:[/h] 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.
[h=4]KEYWORDS:[/h] Cloning; Expression; Fusion PCR; HA2; Influenza; M2e
PMID: 26793615 [PubMed] PMCID: PMC4708040 Free PMC Article
[h=1]Generation of New M2e-HA2 Fusion Chimeric Peptide to Development of a Recombinant Fusion Protein Vaccine.[/h] Ameghi A[SUP]1[/SUP], Baradaran B[SUP]2[/SUP], Aghaiypour K[SUP]3[/SUP], Barzegar A[SUP]4[/SUP], Pilehvar-Soltanahmadi Y[SUP]5[/SUP], Moghadampour M[SUP]6[/SUP], Taghizadeh M[SUP]6[/SUP], Zarghami N[SUP]7[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]PURPOSE:[/h] 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.
[h=4]METHODS:[/h] 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.
[h=4]RESULTS:[/h] 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.
[h=4]CONCLUSION:[/h] 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.
[h=4]KEYWORDS:[/h] Cloning; Expression; Fusion PCR; HA2; Influenza; M2e
PMID: 26793615 [PubMed] PMCID: PMC4708040 Free PMC Article