Studies On The Immunogenesis In BALB/c Mice Immunized With GC Genetic Vaccine Of Duck Enteritis Virus

Duck viral enteritis (DVE), also known as duck plague, is an acute and contagious disease of ducks, geese and other animals of Anseriformes, caused by duck enteritis virus (DEV). Gene vaccine attracts study of genetic engineering vaccines, because of its unique advantage.A series of research were carried on theoretical immunogenicity of DEV gC, immunogenicity and immunoadjuvants of its gene vaccine in BALB/c mice, and so on. the contents were summarized as follows:1. Valuation of theoretical immunogenicity of gC gene of duck virus enteritis. The analysis resultes showed DEV gC had high homology and genetic relationship with MDV-1 and MDV-2. The predict results showed gC belonged to gene ontology category of immune response, had function of cell envelope and character of enzyme, but did not belong to any enzyme. Further prediction showed that the gC had abundance cell epitope,three different regions of heparan sulfate-binding domain and complement binding domain, respectively. RegionⅠof heparan sulfate-binding was located between 21-54 aa, and regionⅡand regionⅢwas 48-68 aa and 160 -200 aa, respectively. The regions of 168 -225 aa and 245-332 aa were important for C3b binding, while 29-110 aa interfereed with properdin binding to C3b. We concluded that the gC possessed good immunogenicity in BALB/c mice.2. Chitosan as deliver carrier of pcDNA-DEV-gC DNA vaccine:preparation and study of biologic character. Preparation of complexes of chitosan/DEV gC genetic vaccine was standardizde and reproducible. Entrapment rate of complexes was 95.6%±0.8. Shap of complexes was uniform and globose. Size of complexes was between 50nm and 80nm. Complexes was stable stored in 4℃, prevented plamid from degradation of nuclease effectively, had better transfection efficiency compared to plamid,and low cytotoxicity. The results we obtained showed chitosan as deliver carrier of DEV gC genetic vaccine was safe and effective.3. Cation Iiposomes as deliver carrier of pcDNA-DEV-gC DNA vaccine: preparation and study of biologic character. Preoaration of cation liposomes/DEV gC genetic vaccine complexes was standardizde and reproducible. Entrapment rate of complexes was 76.4%±0.8. Shap of complexes was uniform and globose. Size of complexes was between 400nm and 700nm. Complexes was stable stored in 4℃, prevented plamid from degradation of nuclease effectively, had better transfection efficiency compared to chitosan,and low cytotoxicity. The results we obtained showed cation liposomes as deliver carrier of gene vaccine was safe and effective.4. Established two indirect enzyme linked immunosorbent assay determined serum specific IgG and enteric IgA of mice. Concentration of covered antigen of indirect ELISA determined serum specific IgG and enteric IgA of mice were 0.5ug/ml and lug/ml respectively. Working concentration of the examined samples were 1:8 and not dilution respectively. Working concentration of enzyme labelled antibody were 1:5000 and 1:8000 respectively. Temperature and time of incubated the examined samples was all 37℃h. Temperature and time of incubated enzyme labelled antibody were 37℃1h and 37℃2h respectively. Time of substrate response was all 40min. The cut-off value were 0.259 and 0.297 respectively. Specificity and repeatability of two indirect ELISA were high. The results we obtained provided stable methods, which were applied to detect humoral immunity and mucosal immunity of DEV gC genetic vaccine.5. Studies on the immunogenesis in BALB/c mice immunized with gC genetic vaccine of duck enteritis virus. pcDNA-DEV-gC genetic vaccine induced cellular, humoral and mucosal immune responses of immuned BALB/c mice in some extent. Compared the groups which were immunized by gene gun bombardment, intramuscular injection and oral, Peripheral T lymphocyte proliferation, CD4+ and CD8+ T lymphocyte immune responses of oral groups were lower compared to gene gun bombardment and intramuscular injection groups. Humoral immune responses of gene gun bombardment groups were better compared to intramuscular injection and oral groups. Oral groups induces mucosal immune responses only in there inoculation routes. Gene gun bombardment groups showed dose-dependent character in cellular and humoral immune responses, but intramuscular injection groups prefered to cellular immune responses. Deliver carriers enhanced cellular and mucosal immune responses significantly, cation liposomes perferer to cellular immune responses, chitosan perferer to mucosal immune responses. The result showed deliver carriers were good adjuvant. Genetic vaccine induced stronger cellular immune responses compared to live attenuated vaccine of DEV, but live attenuated vaccine induced stronger humoral immune responses.