Fusion and sequence analysis of the influenza A (H9N2) virus M2e and C-terminal fragment of Mycobacterium tuberculosis HSP70 (H37Rv)

Moradi Bidhendi, S.; Toghyani, H.; Varshovi, H.R.; Paykari, H.; Mirjalili, A.; Tebianian, M.; Ebrahimi, S.M.; Attaran, H.R.

doi:10.22092/ari.2009.103835

Fusion and sequence analysis of the influenza A (H9N2) virus M2e and C-terminal fragment of Mycobacterium tuberculosis HSP70 (H37Rv)

Authors

10.22092/ari.2009.103835

Abstract

The present study was aimed to construct a fusion plasmid harboring the extracellular domain of the influenza A M2-protein (M2e), which was fused to the N-terminus of the truncated HSP70 (HSP70359–610) molecule as a new approach for future vaccine research against influenza A. The amplified fragments, M2e and HSP70359-610 genes, were gel-purified. The products were then single digested with BamHI restriction enzyme separately. The digested products were again gel-purified and ligated by T4 DNA ligase to form M2e- HSP359-610 gene. The PCR product containing both M2e and HSP359-610 genes as a single open reading frame (ORF) was gel-purified and double digested with EcoRI and XbaI restriction enzymes and then ligated into the EcoRI / XbaI double digested pPICZαA expression vector to form recombinant expression vector. Finally, the fused gene was sequenced, and then confirmed according to the related deposited gene in Genbank. The extracellular domain of the M2 protein, M2e, which consists of N-terminal 24 residues, showed to be remarkably conserved, and the N-terminal epitope SLLTEVET (residues 2-9) was conserved among all subtypes of influenza A viruses. Because of M2e limited potency hence, low immunogenicity, it seems by linking this M2e-peptide to an appropriate carrier such as mycobacterium tuberculosis C-terminal 28-kDa domain of HSP70 (hsp70 359–610) we can render it very immunogenic, but further study needs to express it in both prokaryotic and eukaryotic systems and then evaluate this fusion protein in animal model.

Keywords