الفهرس 
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Abstract
Egyptian endemic status of Avian Influenza H5N1 accelerating the urgent need to develop potent H5N1 vaccine regimen among other control measures. In the present study, site directed mutagenesis for the full length haemagglutinin (HA) gene of a recent Egyptian strain H5N1 AIV (A/chicken/Egypt/VRLCU/2012) was carried out by deletion of multi- basic cleavage site coding sequence using PCR assays. The mutated HA gene was cloned into the baculovirus transfer plasmid (pFastBac TM) to construct the recombinant pFastBac (rpFastBac-HA). Purified rpFastBac–HA was transformed into DH10Bac competent cells, transposed with a shuttle vector (Bacmid) to construct the rBacmid-HA which was identified by PCR, restriction digestion and sequencing. Sf-9 cells were transfected with rBacmid-HA, and recombinant baculovirus was harvested. In vitro characterization of the expressed HA protein was carried out using Hemadsorption assay (HAD), hemagglutination assay (HA), hemagglutination inhibition test (HI), Immunofluorescence assay, SDS– PAGE, and western immunoblot. The expressed HA protein was identified in SDS-PAGE assay with approximately 63 KDa, which further confirmed by western blot assay using reference anti-H5N1 AIV serum. This study reports the successful expression and antigenic characterization of HA protein of recently circulating H5N1 strain among Egyptian poultry population which will provide an effective tool for production of subunit vaccine candidates and diagnostic utilities
Key words: Endemic, rBacmid-HA, Sf-9, Characterization, Expression.