Construction and identification of recombinant adenovirus expressing the hemagglutinin gene of H5N1 subtype avian influenza virus

In order to construct recombinant adenovirus expressing HA gene of clade 2.3.4 influenza virus,the HA gene of A/Chicken/Shandong/628/2011(H5N1) was amplified by RT-PCR and inserted into adenovirus shuttle plasmid pShuttle-CMV to construct a recombinant adenovirus shuttle plasmid pShuttle-CMV-HA.A replication-defective recombinant adenovirus expressing HA gene(rAd-HA) was generated in Ad293 cells transfected with pShuttle-CMV-HA.The adenovirus particles were observed through cell ultramicroscape.Furthermore,the transcription and expression of HA gene in Ad293 cells were also verified by RT-PCR,indiect immunofluorescence assay and western blot which had a similar immunoreactivity as influenza viral protein.This study laid a foundation for the development of new H5N1 subtype avian influenza virus live vector vaccine.