Molecular Epidemiological Investigations Of Novel PPV,Immunogenicity Analysis Of PPV2 VP Protein And Its Application In An Indirect ELISA

Porcine parvovirus was a generic terms,including the viruses in the member of Parvoviridae,which could lead to miscarriage,diarrhea,and other respiratory symptoms.In recent years,a variety of new porcine parvovirus have been reported at home and abroad.As a representative of the new porcine parvovirus,PPV2 had a very high positive rate in pigs,which posed a major threat to the world's pig industry.In this study,the prevalence of the novel parvovirus in different regions of China were investigated by PCR method,and the PPV2 ORF2 genes were sequenced and analysed.VPa and VPb protein were respectively expressed through the prokaryotic and eukaryotic systems,immunogenicity of which was also detected.Indirect ELISA method was established by the use of PPV2 VPb protein obtained by the prokaryotic expression system,which provided an support for serological testing of PPV2.The details are as follow:1.Detection of the novel parvovirus,and sequence analysis of the PPV2 ORF2 genes253 collected and preserved tissue samples from 2013 to 2014 were detected by PCR method,and PPV2 ORF2 genes were amplified and sequenced in this study.The results showed that the positive rates of PPV2,PPV3,PPV4 and PBoV were 54.5%,11.1%,8.7%and 9.9%,respectively,and PPV2,PPV3 and PPV4 showed some seasonal epidemics.All the PPV2 ORF2 sequences were analysed and the phylogenetic tree was constructed,the phylogenetic tree indicated that the homology among PPV2 ORF2 genes ranged from 92.6%to 100%.PPV2 and PPV3,along with human parvovirus type 4,5 were classified into PARV4-like virus.This study laid the foundation for the further investigation of novel porcine parvovirus and the phylogenetic analysis of PPV2.2.Prokaryotic expression and the immunological properties study of the major epitope region of PPV2 VP proteinIn this study,two truncated protein containing respectively aa 172-412?nts 514-1236 of VPa gene?and aa 713-924?nts 2137-Z772 of VPb gene?were expressed with the pET-32a?+?/BL 21 E.coli classic prokaryotic expression system.The mice were vaccinated subunit vaccine prepared by the purified protein,and immunogenicity of two proteins determinated by measuring antibody level.The results showed that the two kind of proteins were successfully expressed by prokaryotic expression systems and were able to induce the production of the specific antibodies.Antibody level of group injected with VPb was higher than that of group immunized with VPa,which indicated stronger immunogenicity of VPb proteins and laid the foundation for the development of PPV2 subunit vaccine.3.Eukaryotic expression and the immunological properties study of the major epitope region of PPV2 VP proteinIn this study,the VPa and VPb genes of PPV2 ORF2 were cloned into the vectorpFastBacn^TM Dual and transferred into DHlOBac to get the recombinant Bacmid,then the recombinant Bacmid were transfected into Sf9 cells to harvest recombinant baculoviruses,VPa and VPb protein were expressed in Sf9 cells and identified by western-blot.The mice were vaccinated subunit vaccine prepared by the expressed protein,and immunogenicity of two proteins determinated by measuring antibody level.The results showed that the two kind of proteins were successfully expressed by eukaryotic expression systems.Immunized mice experiments showed that VPb protein could induce significantly higher level of antibody than VPa protein,which indicated stronger immunogenicity of VPb proteins and laid the foundation for the further study of the immunogenicity of the two proteins and the research of the subunit vaccine of PPV2 VP protein.4.Establishment and application of an indirect ELISA assay for the detection of the antibody to porcine parvovirus 2In this study,the indirect ELISA method was established using the recombinant truncated VP?VPb?protein as coating antigen obtained by prokaryotic expression system.The reaction conditions were optimized,including 0.8 ?g/mL coating antigen?purified VPb protein?,1:100 dilution of the tested serum and 1:20 000 dilution of HRP-SPA with a cut off-value of 0.4005?OD?.Analysis of specificity of the ELISA method showed that it had no reactions with the positive sera to porcine parvovirus type 1 virus,classical swine fever virus,pseudorabies virus,porcine circovirus type 2 virus and foot-and-mouth disease virus.Coefficient of variability in intra-assay and inter-assay were within 10%.480 clinical serum samples collected in Jiangsu province were detected by the established ELISA method.The results showed that the positive rate of the PPV2 antibody was 31%,which revealed that the indirect ELISA method using VPb protein as coating antigen was sensitive and specific,and could be used to detect serum antibodies in clinical.This study provided theoretical support for the further investigation of novel porcine parvovirus and the phylogenetic analysis of PPV2.Establishment of an indirect ELISA method laid an important foundation for the development of PPV2 serological detection methods.