The Molecular Epidemiological Survey Of Porcine Parvovirus And The Study Of Immunogenicity Of PPV3 VP2

Porcine parvovirus 1 (PPV1) was considered to be one pathogen resulting in swine reproductive failure, which had brought immense economic losses to the swine industry worldwidely. A number of novel porcine parvovirus were identified in the last few years, including porcine parvovirus 2 (PPV2), porcine parvovirus 3 (PPV3), porcine parvovirus 4 (PPV4), porcine boca-like virus (PBo-likeV), porcine bocavirus (PBoV), etc. In this study, both the prevalence status and the genetic variation of that five novel parvovirus were investigated, and the results may lay foundation for the future research of epidemiology and pathogenesis of novel parvovirus. In addition, this study used both the prokaryotic expression system and the baculovirus expression system to express the complete VP2 and two truncated protein of PPV3, and the result may establish a theoretical basis for the research of subunit vaccine of PPV3 and its immunological characteristics. Finally, this study developed an indirect ELISA with recombinant protein pET-28a-540 for detecting antibody to PPV3, and the method may serve as an efficient serological test for the detection of PPV3 clinically.1. The molecular epidemiological survey of novel porcine parvovirusThe present study was conducted to do the molecular epidemiological survey of PPV2,PPV3,PPV4, PBo-likeV and PBoV with PCR method by detecting the samples collected and stored during 2008 to 2013, and the result show that the prevalence of them was 46.4%,21.1%,6.8%,12.3% and 5.5%, respectively. In addition, the prevalence of PCV2 and PRRSV was also investigated. To analysis the co-infection of the novel parvovirus and PCV2/PRRSV generally, the result indicated that co-infection of PPV3/PRRSV and PBo-likeV/PCV2 were both prevalent in high frequency, thus it suggest that they may contribute to the infection of PCV2 and PRRSV. By comparative analysis of the nucleotide sequences and phylogenetic studies we propose that the genome of PPV3, PPV4 and PBo-likeV are highly conserved, while the variability of PPV2 and PBoV are obviously.2. The expression of PPV3 VP2 in prokaryotic expression system and the study of its immunogenicityThe prokaryotic expression system was applied to express the recombinant protein pET-28a-540, pET-28a-555 and pET-28a-VP2, and the SDS-PAGE analysis indicated that all the three proteins were exist in inclusion body. The three proteins, especially the protein pET-28a-540, were well-purified with inclusion body lotion. The immunogenicity of the three proteins was examined by mice immunization experiment, and the ELISA result showed that the titer of antibody against PPV3 induced by the vaccine based on the protein pET-28a-VP2 was significantly higher than the vaccine based on the protein pET-28a-555(P<0.05). In addition, the ELISA result showed that the immunogenicity of protein pET-28a-540 was better than pET-28a-555 but worse than pET-28a-VP2.3. The expression of PPV3 VP2 in baculovirus expression system and the study of its immunogenicityThe baculovirus expression system was also applied in the present study. Western blot analysis suggested that the recombinant protein pFastBacDual-540, pFastBacDual-555, pFastBacDual-VP2 were all successfully expressed in Sf9 cell. Develop the well-purified protein pFastBacDual-555 and pFastBacDual-VP2 into subunit vaccines and access its immune efficacy through mice experiment. The indirect ELISA showed that both of the vaccines could induce relatively high titer of antibody, and the sandwich ELISA showed that the vaccine based on the protein pFastBacDual-VP2 could significantly enhance the secretion of IL-4 and IFN-y. This study demonstrated that protein pFastBacDual-VP2 could induce both humoral immunity response and cellular immunity response.4. Development and preliminary application of indirect ELISA for detecting antibody to PPV3An indirect ELISA for detecting PPV3 specific antibody was developed based on the recombinant protein pET-28a-540 expressed in E.coli BL21. The Optimal conditions were in detailed below:the coating concentration was 0.5μg/ml, the serum dilution was 1:100, the coating antigen condition was 37℃ for 1h, the sealing solution was PBST containing 5% skim milk powder, the sealing condition was 37℃ for 2h, the reaction condition of serum was 37℃ for 1h, the dilution of HRP-SPA was 1:20000, the reaction condition of HRP-SPA was 37℃ for 30min, the reaction condition of TMB was 37℃ for 6min without light. The judging criterion for serum was:if P/N>2.1 and OD450nm>0.318, the serum was judged as positive serum; If OD450nm<0.277, the serum was judged as negative serum; If 0.277<OD450nm<0.318, the serum was suspected as positive serum. This method possessed high specification for had no cross reaction with other standard positive serums, such as CSFV, FMDV, PCV2, PRV and PRRSV. This method possessed high repeatability for the CV% of intra-batch repeatability test and inter-batch repeatability test was under 5% and 10%, respectively. The result of epidemiological survey with this method showed that PPV3 infection rate was 27.9% and was approximately with the result detected by PCR.This study was concentrated on novel porcine parvovirus, including its molecular epidemiology, serology diagnostic method and the immunogenicity of its viral protein, etc. This study laid theoretical basis for the systematic study in the future, such as the pathogenesis, biological function and immunological character.