The Comparative Research Of Immunogenicity For PCV2b-1A, PCV2b-1B And PCV2b-1C Strain From Anhui

Porcine circovirus type2is the major pathogen of post-weaning multisystemic wastingsyndrome (PMWS), which divided into PCV2a, PCV2b and PCV2c genetic subtypes. Inrecent years, the herd infection mainly is PCV2b genetic subtypes.And the Anhui regionPCV2isolates were PCV2b, divided into PCV2b-1A, PCV2b-1B and PCV2b-1C genecluster, the overall amino acid sequence of PCV2b-1A and PCV2b-1B are consistent, butcompared with PCV2b-1and PCV2b-1B,the amino acid sequence of PCV2b-1C havemore obvious changes,and the changes of loci focused on two immune reaction zone ofORF2.To explore the immunogenicity differences of the different cluster but same geneticsubtype of porcine circovirus type2Anhui strains. In this study, the following contentwere studied, for PCV2vaccine research and PCV2pathogenesis of prevention and controlmeasures provide a theoretical basis.Choose the different cluster but same genetic subtype of porcine circovirus type2strains (PCV2-DY0801is PCV2b-1A, PCV2-BH0801is PCV2b-1B, PCV2-XC0801isPCV2b-1C) infect mice.To detect the IgG antibodies levels, the number of T cell subsetsand Th1/Th2cytokine levels in peripheral blood of mice by ELISA and flow cytometry.Byexpressing the PCV2ORF2which encode Cap protein and its immunological activity areidentified.Primers are designed according to the ORF2gene sequence of PCV2Anhuistrain form GenBank, ORF2gene from PCV2are amplified by PCR and clone into apET-32α expression vector, generating the pET-32α-ORF2construction. It is transformedinto the Ecoil Rosetta competent cells. Recombinant protein is expressed highly.The miceare vaccinated by intraperitoneal injections at1d and14d. And the IgG and Th1/Th2cytokines levels are detected by ELISA after immunization, observing the existencedifferences of immunogenicity of the three PCV2Anhui strains.Three PCV2Anhui strains infected mice produced highly levels of IgG, and at21dpithe BH0801group IgG levels were significantly higher than the XC0801group.Threeinfection groups CD4+, CD8+and CD4+CD8+cell number was significantly reduced thanthe control group in peripheral blood (P<0.05), at14dpi the BH0801group CD4+numberwas significantly lower than the XC0801group,but later higher than the XC0801group.Three infection groups IL-4and IL-10in serum post infection were significantlyelevated compared with the control group(P<0.05). IFN-γ increased at7dpi, and decreasedsignificantly at21dpi and28dpi than the control group (P<0.05). The BH0801group IL-2were significantly higher than the XC0801group (P<0.05). The XC0801group IL-10levels were significantly higher the BH0801and DY0801group (P<0.05).SDS-PAGEanalysis showed that the molecular weight of the protein was approximately40kD.Western blotting analysis demonstrated that the fusion protein possessed antigenic activityand could be specifically recognized by antiserum against PCV2. Three immunity groupsIgG levels in serum were significantly higher (P <0.05). Only the IL-4production of theXC0801group was significantly higher at the second immunization (P <0.05), however,IFN-γ, IL-2and IL-10all obvious increased (P>0.05).The IgG and cytokine levels of thethree immunity groups no significantly difference (P>0.05).The results show that three PCV2Anhui strains could induced high levels of humoralimmune response, but the cell immune response was suppressed, and the induction ofhumoral and cellular immune responses were some differences.The immune response ofPCV2-BH0801and PCV2-XC0801was significant difference. Three Cap protein of PCV2Anhui strains had good immunogenicity, which could induce high levels of humoralimmune response, but the induction of humoral and cellular immune response was nosignificant difference. Compared with PCV2-BH0801and PCV2-DY0801, the changes ofamino acid sequence of PCV2-XC0801’s ORF2did not result in changeing significantly inthe immunogenicity of Cap protein. So there are some differences in immunogenicity ofthe different cluster but same genetic subtype of PCV2Anhui strains, but the three Capprotein are no significant difference in immunogenicity. The reasons for differences inimmunogenicity is not only related to the changes in the amino acid sequence of ORF2,butalso other factors.