[Construction and identification of H5 subtype avian influenza vaccine candidate based on flagellin].
OBJECTIVE
To develop a novel H5 subtype avian influenza vaccine candidate based on flagellin.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Jiangsu Key Laboratory of Zoonosis, Key Laboratory of Ministry of Education for Avian Preventive Medicine, Yangzhou University, Yangzhou 225009, China.
Jing
J
Wang
Xilong
X
Kang
Yang
Y
Jiao
Dan
D
Xiong
Li
L
Song
Yun
Y
Yang
Zhiming
Z
Pan
Xinan
X
Jiao
D000818:Animals; D003001:Cloning, Molecular; D005408:Flagellin / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology*; D051379:Mice; D010957:Plasmids / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics; D051198:Toll-Like Receptor 5 / Q000378:metabolism; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
D051198:Toll-Like Receptor 5; D014765:Viral Vaccines; D005408:Flagellin