The Cap Protein Expression And Monoclonal Antibody Preparation Of Porcine Circovirus Type2and The Study Of Viral Infeciton Of Monocyte-deirved Cells In Vitro

Porcine circovirus disease (PCVAD) caused by porcine circovirus type2(PCV2) is widespread allover the world, causing serious economic losses. PCV2is hazards to the immune function of infectedpigs, result in host immune suppression, and produce secondary infection. PCV2infection is often in theform of subclinical infection that easily overlooked. Many uncertainty factors made it difficult to studyon PCV2and its related diseases. Therefore, it plays a groundbreaking role to establish a series of studiesmeans for research, diagnosis and prevention of the PCV2.PCV2has a single strand covalently closed DNA genome about1766～1769bp.The viral particle hasa diameter about17nm without capsule,and strong resistance to the external environment.The are twolarge open reading frame, encoding replication-related protein (Rep) and the nuclear capsid protein (Cap).Capsid protein has good immunogenicity, therefore carried out Cap protein research are extraordinarysignificance for diagnostic methods and vaccine development of PCV2.In this study, PCR detect45disease material collected suspected PCV2infection, and13samples arePCV2-positive. And from Beijing, Jilin suspected PCV2infected pigs isolated two strains of viruses, theidentified PCV2were named as PCV2-BJ and PCV2-JL. Six porcine circovirus type2(PCV2) completegenome sequences of isolates and positive disease materials were got, and construct the phylogenetic treeof the PCV2.To study on PCV virus infection and the media may cause the PCV spread, as well as the viral loadand distribution characteristics in the host body,we established quantitative PCR of two virus types.Inthis study PCV1and PCV2sequences accepted on Genbank and sequenced were compared,andquantitative PCR of each type of virus were established.The results show that the quantitative PCRmethod of two virus types are specific, sensitive, and good reproducibility, and provide the necessarymeans for virus detection and quantification.According to the genome sequence of PCV2separated, combined with the characteristics ofBac-to-Bac expression system, we designed a pair of PCV2gene-specific primers to amplify ORF2, andthe introduction of appropriate restriction sites at5’ ends of the primers. Cloning the PCV2target geneinto the shuttle plasmid. Recombinant shuttle plasmid was transform to Bacmid DH10Bac competentscreening positive recombinant, and transfected Sf9cells getting recombinant baculovirus, named rBac-Cap.Western-Blot experiments and indirect immunofluorescence fluorescence confirmed rCap proteinexpression, the results show that the success to build a Baculovirus system expressing PCV2Cap protein.Immune Balb/c mice with purified porcine circovirus type2, after several times of immunization,took mouse spleen cells fusion with mouse myeloma cell line SP2/0, then use the baculovirus expressionCap protein as screening antigen to filter hybridization-positive tumor cells by indirect ELISAmethod.And with limited diluted subclone, two hybridoma was screened.After hybridoma preparation、 subtype identification, titer determination and indirect immunofluorescence analysis, the results showthat the successful preparation of monoclonal antibodies against PCV2Cap protein, which laid thefoundation for PCV2research.As the advantages of pig peripheral blood mononuclear cells (pPBMC) was easy to get, experimentalconditions stable, this study uses in vitro infection pPBMC method, detection the chemokine mRNAexpression of pPBMC in the affected experiments after infected with PCV2.The results confirmed thatpPBMC chemotaxis induced by LPS was inhibited after PCV2infection, and provide a good model of invitro study on PCV2infection.PCV2infection maybe influence the chemotaxis of dendritic cells derived from pigs, in vitroprepared monocyte-derived dendritic cells was infected with PCV2.The cytokine mRNA expression wasdetected by the established quantitative PCR methods.The results show that dendritic cell inducedchemokine receptor7expression was suppressed by PCV2virus infection, providing a theoreticalreference for further elucidate PCV2immune suppression mechanism.