Construction of a pAED4-M2 vector for expressing avian influenza A (H9N2) virus M2 gene as a universal recombinant vaccine model

We expressed the M2 gene in prokaryotic cells using the pAED4 expression vector system to produce native and purified M2 protein as a candidate for universal recombinant vaccine against influenza A subtypes. The open reading frame (ORF) of avian influenza A/chicken/Iran/101/1998 (H9N2) M2 gene was amplified by 2-step RT-PCR using specific primers and pfu DNA polymerase. pAED4 was used as expression vector, purified PCR product digested by Nde I and EcoR I restriction enzymes was ligated to the same digested site in the vector using T4 ligase to form pAED4-M2. The cloned M2 gene was confirmed by PCR and restriction enzymes pattern. M2 polypeptide was produced through the expression of this recombinant expression vector (pAED4+M2) in Escherichia coli BL21 (DE3) strain. The expressed M2 polypeptide was analyzed on SDS-PAGE and confirmed by western blotting assay. The level of 100% homology between the N-terminal domain of H5 and H9 isolates was considerable. It seems that recombinant vaccine based on Iranian isolate A/chicken/Iran/101/1998(H9N2) M2 protein might cover all H5 and H9 circulating in Iran and neighboring regions significantly. Further research will be needed to evaluate the immunity of the expressed M2 protein in the lab animal model to check the native conformational structure of this expressed protein by challenging with other influenza isolates.