Abstract
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. 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. 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. 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.
Highlights
Influenza virus is an enveloped single-stranded negative-sense RNA virus, belongs to the Orthomyxoviridae family
The procedure was done by virus cultivation in specific pathogen free (SPF) eggs, hemagglutination assay (HA), RNA isolation, RT-Polymerase chain reactions (PCRs), primers designed (DNAMAN 4 and Oligo7), virtual fusion construction translation (ExPASy), N-Glycosylated sites prediction (EnsembleglyIowa), 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-hemagglutination 2 (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 to E.coli (BL21), mass culture the cloned bacterium,induction the expression by isopropylbeta-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
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
Summary
Influenza virus is an enveloped single-stranded negative-sense RNA virus, belongs to the Orthomyxoviridae family This virus causes an acute and highly contagious respiratory disease. Seasonal influenza each year: affects 5 to 15 percent of the world population and causes 3 to 5 million serious infections. It is responsible for the death of up to 500 thousand patients worldwide.[1,2] Pandemic is the other face of this disease. Two major characterization of influenza virus; the error-prone polymerase and segmented genome cause antigenic drift and shift respectively. Antigenic shift causes genome segments dealing between the different subtypes of the virus and makes new genetic combinations. Antigenic shift could lead to terrible pandemics by emerging new viruses that can be transferable between different species.[4]
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