Expression And Immunoassay Of Avian Influenza Virus H9N2,H5N1 HA1 And Chicken IgY Fc Fragment Fusion Protein In Insect Cells

The widespread of the H5N1 highly pathogenic avian influenza(HPAI)and H9N2low-pathogenic avian influenza(LPAI)has accelerated the occurrence of new influenza and the risk of influenza pandemic.H5N1 and H9N2 avian influenza virus(AIV)have demonstrated significance to public health as it could not only directly infect mankind,but also donate partial or even whole cassette of internal genes to generate novel human-lethal reassortants like H7N9,H10N8 viruses.Therefore it is imperative to prevent the H5N1 and H9N2 avian influenza.AIV mainly infects animals through the respiratory tract and so on.Subunit vaccines introduced by intranasal administration can block an infection at its primary site by inducing mucosal immune responses.A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers.However,effective transport of antigens is restricted due to the complexity of the mucosal immune system.In avian species,the chicken yolk sac Ig Y receptor(Fc Ry)can mediate Ig Y transport.In our laboratory previous studies,it was demonstrated that Fc Ry can transcytose chicken Ig Y Fc-fused subunit vaccine across the mucosal barrier.In this study,the HA1,a major protective antigen of the H9N2 and H5N1 AIV,was fused to chicken Ig Y Fc fragments and expressed in the Bac-to-Bac baculovirus system.The recombinant protein combined with Cp G ODN2007 adjuvant was used as a bivalent subunit vaccine to inoculate chickens by intranasal immunization.The results show that the recombinant protein can be transported through respiratory mucosal barrier and induce the body to produce effective mucosal and systemic immune responses against H9N2,H5N1 avian influenza virus.The main research results are as follows:1.Expression of H9N2 AIV HA1 and preparation of mouse anti-chicken H9N2 HA1 polyclonal antibodyThe H9N2 HA1 gene fragment was amplified by PCR to construct the p GEX-KG-HA19 prokaryotic expression vector and expressed in E.coli BL21.The GST-HA19 fusion protein was purified by inclusion body purification.The purified protein HA1 with an adjuvant was immuned the mouse as an immunogen,succeeded to prepare the mouse anti-chicken HA1 serum.Using the indirect ELISA to detect serum antibody titer,the titer was up to 1:256000.The mouse-anti chicken HA1 polyclonal antibody had good specificity by Western Blot.2.Construction of recombinant plasmids p Fast Bac HTb-sHA19-HA15-Fc and p FastBac HTb-sHA19-Fc and expression and purification of fusion proteinsWe selected H9N2 avian influenza sHA1,H5N1 avian influenza HA1 and chicken Ig Y Fc fragment,the three fragments were connected and cloned into the eukaryotic expression vector p Fast Bac HTb,the combinant eukaryotic expression plasmid p Fast Bac HTb-sHA19-HA15-Fc was constructed.In addition,fused H9N2 avian influenza sHA1 and chicken Ig Y Fc fragment and cloned into the eukaryotic expression vector p Fast Bac HTb,the combinant eukaryotic expression plasmid p Fast Bac HTb-sHA19-Fc was constructed.Using Bac-to-Bac expression system,sHA19-HA15-Fc and sHA19-Fc fusion protein was expressed and purified.The fusion protein is about 140 KDa and 105 KDa by SDS-PAGE and Western Blot analysis,respectively.In non-reduced condition,the size of protein is about 280 KDa and 210 KDa,respectively.The result showed that the fusion protein appeared as a dimer under non-reducing condition and was similar to the structure of Ig Y.3.Evaluation of immune effect of bivalent subunit vaccine of sHA19-HA15-Fc fusion protein in chickensThe sHA19-HA15-Fc fusion protein was supplemented with the adjuvant CpG-ODN2007 intranasally to immunize chickens,and an effective local mucosal immune response and systemic immune response response could be induced at the same time.(1)On the10 th day after booster immunization,the trachea and lungs washings of sHA19-HA15-Fc intranasal group,anti-H9N2 or H5N1 HA1-specific s Ig A and Ig Y titers were the highest,the difference was extremely significant compared with other immunized groups(P<0.01),except sHA19-Fc and sHA15-Fc intranasal immunization groups.(2)On the10 th day after booster immunization,the serum IFN-? and IL-2 levels of sHA19-HA15-Fc intranasal group were extremely significant(P<0.01).The differences were extremely significant(P<0.01)compared to sHA19-HA15 intranasal immunization group and inactivated vaccine group.(3)On the 10 th day after booster immunization,the chicken spleen lymphocyte proliferation index was significantly higher than that of sHA19-HA15-Fc subcutaneous immunization group and sHA19-HA15 intranasal immunization group(P<0.01).(4)On the 14 th day after booster immunization,The anti-H9N2 or H5N1 HA1 specific Ig Y and HI in sHA19-HA15-Fc intranasal immunization group were the highest,it was extremely higher than sHA19-HA15 intranasal immunization group(P<0.01).(5)On the 3th day after H9N2 virus challenge,sHA19-HA15-Fc and sHA19-Fc intranasal immunization group were not detected the hemagglutination activity of the H9N2 virus indicate both groups were 100% protected against the intranasal viral challenge,sHA19-HA15-Fc subcutaneous immunization group and Inactivated vaccine immunization group were 75%and 87.5% protected respectively,while the sHA19-HA15 was 25%.sHA19-HA15-Fc fusion protein,together with the adjuvant CpG,across respiratory mucosa epithelial cells by Fc Ry mediated Ig Y transport pathway.This immunization could induce efficient mucosal and systemic immune responses,completely protected sHA19-HA15-Fc immunized chickens after challenge with virulent H9N2.