DNA Vaccine Which Co-Expression HA Gene And NA Gene Of H5N1 Subtype Hpaiv Against Hpai In Chinken

A series of studies have demonstrated that the protective efficacy of AIV genetic engineering vaccines, in which hemagglutinin is a major component. It is high against an epidemic of homologous virus but is low against an epidemic of heterogonous virus. However , because of highly frequent changing of antigenic variation of gene of avian influenza virus , especially HA gene, to develop a more effective vaccine providing cross protection against different subtype of AIV remains an important means to control AIV. On the other hand, NA , which is one of the surface proteins of AIV, elicited specific immune responses and provide protection against AIV, as a result, a DNA vaccine which contains NA gene and HA gene maybe an ideal strategy for high protective efficacy because of the relatively slower antigenic evolution of NA, than HA .In order to study HA gene and NA gene DNA vaccine, the NA gene of H5N1 subtype HPAIV,derived from plasmid pBDNA by PCR ,was subcloned into eukaryotic expressing vectors pCI which contains the CMV promoter and enhancer and pCAGGS which contains the chicken's β -acting promoter, and were designated as pCINA and pCANA, respectively. Then, we used the recombinant plasmids pCINA pCANA and plasmids pCIoptiHA pCAoptiHA to construct double-gene plasmids pCIoptiH5N1 pCAoptiH5Nl. in double-gene vaccine, both NA gene and HA gene were promoted by CMV/ β -actin promoter , and were followed by Poly(A) sequence.The mammal monolayer 293T cells were transected by these constructions respectively and the HA protein and NA protein expression were confirmed by western-blotting analysis. Indirect immunofluorescence assay indicated the optimized HA gene and NA gene were expressed respectively without obvious influence each other. This study lays the foundation for developing immune test on chickens and the assessment of immune efficacy.To evaluate the vaccine efficacy, groups of 3-week-old SPF chickens were intramuscular inoculated with two doses of 10μg of pCIoptiHA pCAoptiHA pCIoptiHA+pCANA pCAoptiHA+pCANA pCIoptiH5N1 pCAoptiH5N1 pCANA, respectively, in a 3 weeks interval.'A group of chickens were injected with 200μl PBS as control. Sera were collected every week after vaccination for detecting of the HI antibody. Two-weeks after the second dose, all chickens were challenged with 100LD₅₀ of highly pathogenic GD/1/96(H5N1) or A/FPV/Rostock/34(H7Nl), Oropharyngeal and cloacal swab specimens were collected from all chickens 3, 5 and 7 days after inoculation for titration of virus in eggs, and chickens were observed daily for disease signs and deaths for 2 weeks.In research of all chickens against 100LD₅₀ of highly pathogenic GD/1/96(H5N1):except of pCANA and control, all chickens generated high HI...