[Construction and identification of H5 subtype avian influenza vaccine candidate based on flagellin]

To develop a novel H5 subtype avian influenza vaccine candidate based on flagellin.

HA1-2-fljB was amplified through splicing hemagglutinin gene fragment HA1-2 of A/Goose/Jiangsu/1/2000 H5N1 and Salmonella typhimurium II phase flagellin fljB gene by overlap PCR, and then the gene was inserted to pET32a(+) to construct the recombinant plasmid pET32a-HA1-2-fljB. The recombinant plasmid was transformed into E.coli BL21 (DE3) and was induced with IPTG. The expression of the fusion protein was identified by SDS-PAGE and Western blotting. The biological activity of the fusion protein was evaluated by stimulating HEK293-TLR5 cells.

The recombinant plasmid pET32a-HA1-2-fljB was constructed. The fusion protein, whose relative molecule weight was about 100 000, had a good immunoreactivity. TLR5 bioassay demonstrated the flagellin part of the fusion protein could be recognized by TLR5 of HEK293-TLR5 cell line to secrete a higher level of IL-8 when compared with HA1-2 protein (P<0.01).

The flagellin-based fusion protein HA1-2-fljB with TLR5 bioactivity was expressed correctly, and the results would establish a basis for the further research on avian influenza virus H5 vaccine.