Expression Of PCV2-ORF2 In Prokaryoti Cells And Its Preparation And Identificat Of Monoclonal Antibodies Against Cap Protein Of PCV2

Porcine circovirus (PCV) was divided into the family porcine circovirus, genus porcine circovirus, which included two serotypes of PCV1 and PCV2. PCV1 had no pathogenicity of swine, but PCV2 was main pathogen of postweaning multisystemic wasting syndrome (PMWS) and was closely related to many diseases. PCV2 can in propagate in the hugely bite the cell, and appeared Immature granulocytes of low density, which can reduce the quantity of the immunity cells, lacking the valid immunity answer ability. The method establishing of PCV2 pathogen and infection serum antibody had very important significance for preventing PCV.In this study, we used PCR method to amplify gene fragment about 576bp removing sighal sequence of nuclear location .The gene fragment we obtained was connected with expressing vector pPRO-HTb, and the products were transformed into competent cell BL21-ΔE3. Analysis identification IPTG induction and SDS-PAGE ,The result showed that recombinant fusion protein was expressed, molecular weight was 27ku.After western-blot identification, positive antiserum of PCV2 can specifically combine with recombinant protein. Therefore, we can know that recombinant protein had a very good reactionogenicity, which can be used establishing to detection method for screening antibody of PCV2 serum.Immuning BALB/c mice with purified and renatured recombinant protein, we fused SP2/0 myeloma cell fuse with spleen cell of immuning mice,prepared hybridoma. Through limiting dilution and there or four screening and cloning, we obtained three hybridoma strains of secreting stable monoclonal antibody resisting PCV2-Cap protein after identificationand name them 1C2,3C11and 3E11. Titers of culture supernatant of hybridoma cells were1:l600,1:12800 and 1:l600. Identification of subclass Ig of three strains monoclonal antibody are IgM. Analysis results of western-blot and Dot-ELISA and indirect immunofluorescence showed that 1C2,3C11and3E11 response with protein PCV2-Cap,but they did not response with PrV,PRRSV,PPV,PEDV and TGEV.The result showed there monoclonal antibodies have strongly specificity.Obtaining three monoclonal antibody provided a powerful tool for establishing the method for diagnosing PCV2 .