Construction And Immune Responses Of DNA Vaccines Based On The Capsid Gene Of Duck Circovirus

Duck circovirus(DuCV)is the smallest virus infecting water birds,the infected ducks mainly show some clinical characteristics like poor body condition,feathering disorders and immunosuppressive.The DuCV has been spreading globally,damaging the ducks industry in many countries.However,studies about vaccines against DuCV were limited due to the lack of a suitable cell lines for its propagation.In this study,based on the DuCV capsid(Cap)gene and plasmid pcDNA3.1(+),some recombinant plasmids for eukaryotic expression were constructed and the ducks’ specific immune responses and protective rates against DuCV separately induced by these plasmids were evaluated initially.1.Construction and expression of recombinant plasmids based on DuCV Cap gene.The nuclear localization signal regions of DuCV Cap and the secretory signal peptides of tPA were predicted,the Cap gene and Cap△NLS gene(the NLS region of Cap was deleted)were amplified from the template of DuCV GH01,and the fusion gene tPA’-Cap△NLS was obtained using overlapping PCR.Results of sequencing and restriction digestion showed that the length of the fragments of Cap,△NLS and tPA’-△NLS were 774 bp,666 bp and 735 bp separately,and they were the same as their theoretical values.The gene fragments of Cap,Cap△NLS and tPA ’-CapANLS were cloned into pcDNA3.1(＋)to construct the recombinant plasmids of pcDNA3,1-Cap,pcDNA3.1-CapANLS and pcDNA3.1-tPA’-Cap△NLS.After that,these plasmids would be transfected into COS7 cells,the cells would be detected by IFA and their supernatants would be detected by Western blot after 48 h of the transfection.The results showed that all of the plasmids pcDNA3.1-Cap,pcDNA3.1-Cap△NLS and pcDNA3.1-tPA’-Cap△NLS were successfully expressed in COS 7 cells.The plasmid of pcDNA3.1-tPA ’-Cap△NLS had the highest expression level,and its target protein could be partially secreted outside the cells.2.Evaluation of immunogenicity of the recombinant plasmids of pcDNA3.1-Cap,pcDNA3.1-Cap△NLS and pcDNA3.1-tPA’-Cap△NLSThe viral supernatants containing DuCV GH01 were produced,with a median infective dose(ID50)of 5.93 ×103.5 copies/mL.Ninety cherry valley ducks with ages about 4 days were divided into 5 groups and separately injected with the recombinant plasmids of pcDNA3.1-Cap,pcDNA3.1-Cap△NLS,pcDNA3.1-tPA’-CapANLS,pcDNA3.1(+)and PBS.The dosage was 200 μg every duck and the boost immunization was carried out 2 weeks later than the first injection.The sera were collected at the 0 w,1st w,2th w,3 th w,4 th w,5th w,6th w and 8th w after the first immunization,and the peripheral blood(PB)were collected at the 0 w,1st w,2 th w,and 4th w after the second immunization.Meanwhile,ten ducks of each group were selected casually,every duck were challenged by DuCV GH01 with a dosage of 105.5ID50 after 4 weeks of the first immunization,and then the ducks’spleens,brursa of fabridus,thymus and hardenrian gland were collected 4 weeks later.The results obtained were listed below.① the Cap specific antibodies of every immunization group showed positive after 3 weeks of the first immunization and had increased towards a high level since then,compared with the controls(P<0.01);the specific serum antibody level of group pcDNA3.1-tPA ’-Cap△NLS was significantly higher than that of group pcDNA3.1-Cap and group pcDNA3.1-Cap△NLS(P<0.05),even showing a trend of slight growth after 8 weeks of the first immunization,and the serum antibody level of group pcDNA3.1-Cap was significantly than that of group pcDNA3.1-Cap△NLS(P<0.05)at the 3 th w,5th w,6th w after the first immunization.② the levels of CD4+,CD8+ IL-4,IL-10,IL-12 and IFN-γ from the groups of pcDNA3.1-Cap,pcDNA3.1-Cap△NLS and pcDNA3.1-tPA’-Cap△NLS were significantly higher than that from the negative groups,which indicates that the ducks’ cells immune response could be activated by the three recombinant plasmids,as well as the ducks’ Thl and Th2 immune responses.There were no obvious gaps existed among the three groups when the quantities of CD4+,CD8+,IL-4,and IL-12 were analysised(P>0.05),but the secretory levels of IL-10 and IFN-y from the group of pcDNA3.1-Cap were markedly higher than that from the groups of pcDNA3.1-Cap△NLS and pcDNA3.1-tPA’-Cap△NLS(P<0.05).③ After 4 weeks of the virus challenge,the spleen,brursa of fabridus,thymus and hardenrian gland from the controls were markedly bigger and heavier than that from the negative group(P<0.05),with an infection rate of 100%(10/10),and the copies of DuCV in ducks’ spleen was 104.5copies/mg;while these clinical signs did not happen to the immunized groups,the infection rates of group pcDNA3.1-Cap,group pcDNA3.1-Cap△NLS and group pcDNA3.1-tPA ’-Cap△NLS were 20%(2/10),40%(4/10)and 10%(1/10)respectively,with a low viral titer of 101.5copies/mg in the DuCV-infected ducks’ spleen.