Partial Characterization Of Duck Plague Virus GC Gene Product And Studies On The Immunogenesis In Ducks Immunized With GC Genetic Vaccine Of Duck Plague Virus

Transcriptional analysis of gC gene in DPV infected duck embryo fibroblasts (DEF) In this study real-time fluorescent quantitative PCR (FQ-PCR) was performed to determine the transcription kinetics of DPV gC gene during viral infection. The result showed that DPV gC gene transcripts were increasing steadily since 4 hpi, reached a peak between 28 and 36 hpi, and declined slowly thereafter but maintained at a relative high level at 72 hpi. The result indicated that DPV gC gene may be a late viral gene.Time course expression of gC protein in DPV infected DEF Western blot was further performed to determine the expression kinetics of DPV gC gene during viral infection. A 45kDa protein was readily detected between 4 and 72 hpi, and the maximum gC expression was achieved by 48 hpi. Here, from the aggregate analysis of time course transcription and expression of DPV gC, it can be presume that the expression of this gene occurred at the late stage of infection, suggesting that DPV gC gene belongs to a late gene.Subcellular localization analysis of DPV gC gene The subcellular localization of gC protein was examined by indirect immunofluorescence staining. The result showed that a small amount of specific fluorescences in the cytoplasm of infected cells became detectable as early as 4 hpi. At later times of infection, DPV gC protein localized predominantly to the perinuclear region of the cytoplasm. The distribution characteristics indicated that DPV gC protein was synthetized in cytoplasm, and gC protein may play an important role in the process of virus assembly and mature.Studies on cell-mediated immune response in ducks immunized with gC genetic vaccine of duck plague virus gC genetic vaccine was administrated to ducks with the dose of 6,3, lμg per duck respectively by route of gene gun bombardment and the dose of 200,100,50μg per duck respectively by route of intramuscular injection(IM). And the control groups, empty vector, DPV attenuated vaccine and blank were included. T lymphocyte proliferation tests (MTT) and fluorescence activated cell sorter (FACS) were used to detect the proliferation of T lymphocytes and the number of CD4+and CDg+T lymphocytes of duck peripheral blood (PBL) respectively. gC DNA immunized groups were different (P＜0.05) from control groups. At 3 days post inoculation, the reaction of T lymphocyts to ConA and the numbers of CD4+and CD8+T lymphocyte in duck PBL were obviously higher than control groups, and achieved peak level at 5～7 days post inoculation. Comparing the groups which were immunized by gene gun bombardment and intramuscular injection, the proliferation of T lymphocytes and the numbers of CD4+ and CD8+T lymphocytes of IM groups were lower compared to gene gun bombardment. Comparing the different routes of DNA vaccine administration, both routes showed dose-dependent character in cell-mediated immune responses.6μg per duck administrated via gene gun bombardment and 200μg per duck of pcDNA-DPV-gC administrated via IM were better in the induction of cell-mediated immune responses.Studies on humoral immune response in ducks immunized with gC genetic vaccine of duck plague virus Indirect ELISA and virus neutralization test were used to detect the changes of anti-DPV antibody and neutralizing antibody titer with different inoculation routes and different doses in ducks respectively. The results showed that all ducks (except blank control group) produced specific IgG antibody and neutralizating antibody against DPV. Gene gun bombardment and IM groups had no significant difference. Neutralizing antibody titers of DPV attenuated vaccine group were significantly higher than that of the DNA vaccine immunized groups at each sampling times. The evidence was that pcDNA-DPV-gC genetic vaccine was able to elicit humoral and cell-mediated immune responses in ducks, and gene gun bombardment delivery could induce a more potent cell-mediated immune responses compared to the IM route. Compare genetic vaccine with DPV attenuated vaccine, the former induced stronger cell-mediated immune responses, and the latter induced stronger humoral immune responses.