Construction And Immunogenicity Of Chimeric Peptide Antigen Composed Of M2e And NP Genes Of Avian Influenza Virus

Highly pathogenic avian influenza (HPAI) is a devastating disease of poultry leading to high mortality caused by H5 or H7 subtypes influenza A viruses. HAPI is classified as one of the list A animal disease by OIE and China Ministry of Agriculture. Matrix protein 2 (M2) of influenza A virus is one of the three integral membrane proteins, and has been remarkably conserved in all influenza A strains. Neucleoprotein (NP) of influenza A virus is an interoir structure protein which has more than three independent antgen site, and also the cytotoxic T cell recognition site. Both as a target antigen which can induce protective immunity from lethal homologous or heterologous influenza A viruses challenge. Therefore, those proteins may be an attractive candidate for the universal influenza vaccine research.Set the plasmid pET-3M2e and pBV-M2e-Fc which constructed and conserved by our lab as the template, and induct the NP antgen epitope in the prime, then got the gene sequence of 3M2e-NP1, NP2-Fc and 3M2e-NP1/2 by PCR amplification. Two promiscuous T-cell epitopes (PTCEs) from hepatitis B (HB) and tetanus toxin (TT) were artifical systhesized. The recombinant expression vector pET32α-3M2e- NP1/2-Fc,pET32α-3M2e-NP1/2,pET32α-PTCE-3M2e-NP1/2 were constructed by the cascade connection of gene sequences which involved above, and insert into the prokaryotic expression vector pET-32α(+). The recombinant vectors were transformed into the competent cells of E.coli BL21 (DE3), and the recombinant bacterium were induced by IPTG, the fusion proteins were analyzed by SDS-PAGE and Western-blotting. Resluts show that the recombinant vectors were successfully constructed and the relative molecular mass of the fusion proteins were measured to be 50kDa, 31.2kDa, 36.2kDa. The purified fusion proteins were obtained by Ni-sepharose affinity chromatography, which could be applied to study its immunogenicity.Fusion proteins 3M2e-NP1/2-Fc, 3M2e-NP1/2 and PTCE-3M2e-NP1/2was mixed with Freund's, vash oil and chitosan adjuvant then immunized 20-day-old chicken by intramuscular injection. Blood samples were cllected one week interval from priming and detected the anti-M2e antibody by ELISA. Checked the neutralizing ability of anti-serum on viral infected MDCK cell and SPF chick embryo. Detected the CD4~+ and CD8~+ T lymphocyte in peripheral blood of immunized chicken by flow cytometry. Results show that each group can be detected ELISA antibody, and the fusion protein PTCE-3M2e-NP1/2 emulsionized with Freund's adjuvant induced the highest antibody level, and the antibody reach the top at 7th week of each group. Fluorescence microscopy showed that the anti-M2e sera can bind the M2 which virus expressed on the surface of MDCK cells, but it can't neutralize them completely. Flow cytometry shows that CD4~+ and CD8~+ T lymphocyte in peripheral blood increased during immunization (P<0.05), which possess the character of cell immunization. The chimeric peptide has better immunogenicity but the immune protect efficacy must be certificated in the appointed institution.