| Rabbit heamorrhagic disease(RHD)and rabbit coccidiosis cause sever damage to the sound development of rabbit industry.There are unsolved issues in prevention and control of RHD including biosafety risks of inactivated virus vaccine,short duration of immunity by such vaccine in sucklings,and prolonged virus shedding in chronical infections.On the other hand,rabbit coccidia are of excellent immunogenicity that single vaccination can induce sufficient mucosal immunity.For many years,our research group have been studying transgenic Eimeria spp.as live vaccine vectors in order to provide alternative immunization strategies which are safer and more convenient in practical use.Thus,in this dissertation,we explored to construct transgenic rabbit Eimeria spp.expressing P2 subdomain of capsid protein VP60 of RHDV.By investigation of the specific immunity,we evaluated the potential of transgenic rabbit coccidia as vaccine vectors,offering novel prospectives for vaccine development of RHD and rabbit coccidiosis.Fist,we obtaind the regulatory sequences of E.magna profilin gene,and confirmed its capacity to drive expression of exogenous proteins in in vitro transfections,yet we failed in stable transfection.Therefore,we adapted the regulatory sequences from Histone 4(His4)of E.tenella,and established a transgenic E.magna line expressing enhanced yellow fluorescent protein(EYFP)and red fluorescent protein(RFP)(EmagER).We observed the exogenous proteins were expressed throughout the parasites'whole life cycle.The fecundity and immunogenicity of EmagER was similar to those of the wild-type parasites.In order to evaluate the immune response elicited by transgenic rabbit coccidia,we established an immunodetection platform of rabbit cell-mediated immunity due to the lack of reliable tools for rabbit immunology and vaccinology.We detected the relative transcriptional level of immunity-related cytokines by quantitative real-time PCR(qPCR)in EmagER infected rabbits and found that immunization with EmagER stimulated Th-1 immunity in mesenteric lymph nodes(MLN).Additionaly,we tested custom ordered mAbs against rabbit CD3,CD4,CD8,IFN-y,TNF-a by Western-blot and flow cytometry.We identified mAbs clones of CD4 and TNF-a that can be applied for flow cytometry providing useful tools for studies on rabbit immunology.Next,we constructed a transgenic.magatline expressing P2 subdoain of VP60 of RHDV(EmagE-VP60).We found that VP60-P2 was expressed in the cytoplasma and on the membrane of the merozoites during schizogony stage.Immunization assay demonstrated that humoral immunity and percentage of VP60-P2 specific-CD8+TNF-a+ cells in peripheral blood mononuclear cells(PBMC)was slightly elevated in rabbits immunized with EmagE-VP60 compared to those with the wild-type parasites.Relative transcriptional level of Th-1 related cytokines in MLN and payer's patches(PP)of rabbits immunized with EmagE-VP60 were up-regulated after stimulation with VP60-P2 protein compared with rabbits immunized with the wild type parasites.All the results suggested that VP60-P2 expressed by EmagE-VP60 elicited specific intestinal immunity in rabbits.In conclusion,we established a platform for rabbit coccidia transfection and constructed transgenic E.magna expressing viral antigen of RHDV.Immunization with the transgenic E.magna induced exogenous antigen-specific intestinal immunity in rabbits.Our study laied the groundwork for subsequent studies on immunologica mechanism and application of transgenic rabbit as recombinant vaccine vectors. |
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