| Avian influenza virus(Avian influenza virus, AIV) is one of the main hazards pathogenic in poultry industry, and brought serious economic losses to poultry industry. AIV have a number of serotype, and genome is highly variable, therefore, we need to constantly change vaccine strain according to the variation of influenza virus. Because of frequency large, protracted and painstaking, so the development of the vaccine cannot keep up with the speed of mutation rate of the virus.NP of AIV in different subtypes of influenza viruses are highly conserved(Saha S et al, 2006; Ohba K et al, 2007; Altstein A D et al, 2006). NP posesses both stocks and type-specific proteins, and it is highly conserved between different subtypes of the virus. NP is the major protein antigen-induced cytotoxic T lymphocyte(CTL) response, and includes three independent antigenic sites at least.Cell immunity mediated by MHC molecules plays an important role in antiviral infection.The important part of immune cells blongs to T cell epitopes and restricted by MHC, which different MHC molecules bind to different antigen peptides. After CTL epitopes of NP bind to MHC I molecules which can be recognized by CD8 + T cell, and can cause T cell proliferation, and the ability to activate the secretion of IFN-?. Therefore, by detecting the content of cells to secrete IFN-? and IFN-? expression of mRNA to validate the T cell epitope is correct.In order to identify the T cell epitope in avian influenza virus nucleoprotein(NP), four T cell epitope candidate peptides(NP120-128?NP163-172?NP85-92?NP67-74)of the avian influenza virus nucleoprotein(NP) were predicted with the protein structure prediction technology, and synthesize the corresponding candidate peptides. To verify the immunogenicity of these peptides, the recombinant plasmid pCAGGS-NP was constructed and the expression of NP was confirmed by indirect immunofluorescence and western blot in transfected 293 T cell. The antibodies against NP were also detected by ELISA in pCAGGS-NP inoculated SPF chickens. The splenic cells were collected from inoculated chickens and stimulated with the peptides NP120-128, NP163-172, NP85-92, NP 67-74 or negative control, respectively. The results of SBRY Green qPCR show that spleen lymphocytes stimulated with the peptide NP67-74 can significantly increase the secretion of IFN-?, which indicated that the peptide NP67-74 may be AIV NP CTL epitope. The results possess significance to mechanism of protective immunity against influenza virus and the research towards universal flu vaccine.To explore whether T cell epitope peptide NP protein NP67-74 have a good effect on 4M2 e immune effect, this study will add freund's adjuvant in polypeptide NP67-74- KLH and 4 M2e- MAP, and immune mice with the preparation of synthetic peptide, and then evaluate its immune effect. Then the immune protection of the synthetic peptide was evaluated by mice challenged detection?viral titers and histopathological slices changes in lung after infection. Monitoring weight of mice after mice challenged,the result of changing curve showed that the weight change trend of jointing immune polypeptide NP67-74- KLH and 4 M2e- MAP is not much clear than immuning the pepitide 4M2 e, and the weight of the former regained quickly. Q-PCR and histopathological slices results showed jointing immune polypeptide NP67-74- KLH and 4 M2e- MAP can inhibit virus replication in the lungs and reduce the pathological damage of the lung and have protection force against the attack on AIV, so that it can produce the better immune effect. This provides a good reference for the combined use of peptide vaccine against avian influenza research. |
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