Construction Of Expression Vector Of Capsid Proteins From Goose Parvovirus And Investigation Of The Immunogenicity

Gosling plague is an acute and septicaemic infectious disease induced by goose parvovirus (GPV) which can cause low spirits, lack of appetite, diarrhea and sometimes neurological symptoms. At present, it has no effective drugs against goose parvovirus and mainly through immune inoculating of traditional vaccines or hyperimmune serum for breeding gooses for prevention. China is the world's largest waterfowl breeding country. The requirement on poultry meat and eggs are becoming higher along with the living standard improves. GPV has the character of rapid transmission and high mortality. It has put a threat to the goose cultural industry so that we should give insight into its pathogenic mechanism and molecular biology characteristics. This thesis concluded the following contents:1. We constructed three recombinant plasmids pcDNA3.1-VPl, pcDNA3.1-VP2, and pcDNA3.1-VP3, which were further transfected into in 293T cells and goose embryo fibroblast (GEF). The expression level of recombinant proteins was detected by western blot. The results revealed that the recombinant protein could be recognized by specific anti his-tag antibody and anti-GPV antiserum in around 80 kDa,70 kDa and 60 kDa respectively by western blot which indicated the successful expression of targeted protein in this system.2. The cellular immunogenicity of VPs was compared on GEF and goose peripheral blood mononuclear cells (PBMCs). GEF were transfected with pcDNA3.1-VP1, pcDNA3.1-VP2, pcDNA3.1-VP3, and pcDNA3.1 for 60 h and dissolved in trizol. Real time PCR (RT-PCR) was used to detect the transcript of IL-1?,IL-6, and IFNs. Then we transfected GEF with pcDNA3.1-VP1, pcDNA3.1-VP2, pcDNA.1-VP3, and pcDNA3.1 for 48h, which were further used in treating goose PBMCs for 6 h. The mRNA expression level of IL-6, IL-1? and IFNs were detected. The recombinant protein VP2 which was expressed in GEF and purified by sucrose density-gradient centrifugation then stained with 1% phosphotungstic acid was finally examined under a transmission electron microscope (TEM). The result indicated that both VP2 and VP3 can up-regulate IL-6 and IL-1? expression. While in PBMCs model, all genes barely had any changes by treating VP1. However, the expression level of IFN? and IFN? upregulated significantly by treating VP2. The above data suggested that the immunogenicity of VP2 and VP3 were superior than that of VP1. The VP2 protein of GPV is able to self assemble and consequently capable of forming VLPs when expressed in GEF.3. Finally we chose VP2 gene as a DNA vaccine candidate. In the animal experiment, MPLA or ODN2006 were co-immunized with VP2 DNA vaccine via i.m route. RT-PCR and indirect ELISA was applied to detect the expression level of CD4, CD8a, IL-1?, IL-18, and IFNs, as well as the specific antibody in sample serum respectively. The result indicated that, during the 28 d after immunization, CD4 and CD8a expression saw a steady increase. CD8a mRNA expression level were increased significantly at 7 d and 14 d after immunized of pcDNA3.1-VP2. Moreover, IL-1? and IL-18 were significantly up-regulated at 7 d and 21 d respectively after immunized of pcDNA3.1-VP2 with ODN2006. The ELISA result indicated that after post-immunization of 14 d, the specific antibody produced. The IgY antibody level became highest level at 28 d and all the three experimental groups up-regulated significantly. Besides, pcDNA3.1-VP2 group up-regulated significantly at 21 d.