Express Of Avian Influenza Virus H5N1 HA1 And Chicken IgY Fc Fragment Fusion Protein And Mucosal Immunity

The H5N1 highly pathogenic avian influenza (HPAI) not only causes great economic loss to poultry, and also poses a great threat to the health of human beings in the past two decades. In addition, the H5N1 avian influenza virus often recombines with other subtypes of influenza virus, leading to highly pathogenic and pandemic avian influenza. Therefore it is imperative to prevent the H5N1 avian influenza. Avian influenza virus mainly infects animals through the respiratory tract or the digestive tract and so on. Preventing pathogens from invading the body, the better immune way is intranasal or oral vaccination way compared with other ways which induces the body to produce mucosal and systemic immune responses to resist early virus infection. Some literatures reported that FcRn can efficiently transport IgG across mucosal epithelium. FcRy that transport IgY is similar to FcRn. We selected H5N1 avian influenza sHA1 and chicken IgY Fc fragment to be fused, chickens were inoculated this fusion protein with mucosal adjuvants by intranasal immunization, it can be transported through respiratory mucosal barrier and induce the body to produce mucosal and systemic immune responses. The main research results are as follows:1. Preparaion of rabbit anti-chicken HA1 polyclonal antibodyPurified protein HA1 with an adjuvant was immuned the rabbit as an immunogen, succeeded to prepare the rabbit anti-chicken HA1serum. Using the indirect ELISA to detect serum antibody titer, the titer was up to 1:16000. The rabbit-anti chicken HA1 polyclonal antibody had good specificity by Western Blot.2. Construction of recombinant plasmid pFAST HTb-sHAl-Fc and expression and purifications sHA1-Fc fusion proteinWe selected H5N1 avian influenza sHA1 and chicken IgY Fc fragment, the two fragments were connected and cloned into the eukaryotic expression vector pFAST HTb, the combinant eukaryotic expression plasmid pFAST HTb-sHAl-Fc was constructed. Using Bac-to-Bac expression system, sHA1-Fc fusion protein was expressed and purified. The fusion protein is about 82KD by SDS-PAGE and Western Blot analysis. In not-reduced condition, the size of protein is about 160 KD. The result showed that the fusion protein appeared as a dimer under non-reducing condition and was similar to the structure of IgY.3. Detection of the H5N1 HA1 fusion protein across respiratory tract mucous membrane barrierBiotin labeled sHA1-Fc, GST-HAl and chicken IgY were immuned chickens by intranasal immunization. After 8 hours, we detected the content of biotin labeled protein in chicken serum. The results showed that sHAl-Fc fusion protein (OD450=0.384) can effectively go across the respiratory epithelium compared with GST-HA1 fusion protein (OD450=0.104).The difference were extremely significant (P<0.01).4. The mucosal immune research of H5N1 HA1 fusion proteinChickens were immuned by sHA1-Fc and GST-HA1 fusion protein with CpG-ODN 2006 through intranasal or subcutaneous immunization. Results showed the immune groups can induce specific mucosal and systemic immune responses, sHA1-Fc fusion protein induced stronger immune responses:(1) On the 14th day after booster immunization, in sHA1-Fc intranasal group, IgY antibody titers were the highest. It was extremely higher than GST-HA1 intranasal group by ELISA (P<0.01), it was significant compared to intranasal GST-HA1 intranasal group by HI (P<0.05). (2) On the 10th day after booster immunization, the trachea and lungs washings of sHA1-Fc intranasal group, specific IgY titers and SIgA titers were the highest. The differences were extremely significant (P<0.01) compared to other groups and significant to sHA1-Fc subcutaneous and inactivated vaccine groups (P<0.05). (3) On the 10th day after booster immunization, the serum IFN-y and IL-2 levels of sHA1-Fc intranasal group were extremely significant (P<0.01). The differences were extremely significant (P<0.01) compared to the level of IL-2 in other groups. (4) On the 10th day after the boost, the chicken spleen cell stimulation index in sHA1-Fc intranasal group was 2.92, the results of statistical analysis showed the differences were extremely higher than GST-HA1 intranasal and sHA1-Fc subcutaneous groups (P<0.01).We determined FcRy can mediate mucosal polarized epithelial cells to transport IgY by binding IgY Fc fragments, effectively stimulated local mucosal immunity and systemic immune system to make the body get local specificity and systemic immunity against avian influenza virus. This will have a good foundation and provide a new strategy for development of new rasorial type of mucosal vaccines.