Development And Application Of Indirect ELISA Antibody For Porcine Circovirue Type 2 And Production Of Monoclonal Antibodies Against PCV2

Postweaning multisystemic wasting syndrome (PMWS) in pigs was first identified in western Canada in 1991. Thereafter, it has been reported worldwide. The clinical signs of PMWS are fever, progressive weight loss, pallor, jaundice, respiratory and digestive disorders. The main etiological agent of PMWS was porcine circovirus type 2 (PCV2).Porcine circovirus (PCV) is a member of the family Circoviridae. According to the pathogenic and genetic constitution, PCV contains PCV1 and PCV2.PCV1 is a type of contaminant in cells, and it has no pathogenicity. PCV2 has pathogenicity, and it can depress the pig's immune system. PCV2 has increasingly been associated with various disease syndromes in pigs. So far, the infection of PCV2 has become one of the pathogens which can seriously handicap progress of pig industry worldwide.PCV2 contains eleven open reading frames. ORF1 and ORF2 are the two major open reading frames. ORF1 gene encodes replication-associated proteins (Rep) which is a protein involved in viral replication. Because of Rep protein, PCV1 and PCV2 have cross-reaction of antigenicity. The homology of them is 85%. ORF2 gene encodes the capsid protein (Cap) which is the major constitutive protein. The Cap protein was considered to be type-specific antigen, which has no cross-reaction of antigenicity in two type viruses.The research used the prokaryotic expression protein of ORF2 to establish the indirect ELISA assay, which is a sensitive, rapid, and specific method. The indirect ELISA assay can identify PCV2 efficiently. In a word, the establishment of the method has important significance for controlling the disease and early detection of the disease in pig industry.1. The prokaryotic expression of ORF2 gene fragments in E.coli. The results show as follows. The truncated fragment of PCV2 ORF2 gene was expressed correctly in E.coli BL21. The molecular weight of the expressed recombinant capsid protein was about 26.0kDa. The recombinant capsid protein could react with polyclonal antibody against PCV2.2. The recombinant Cap protein was used as the envelope antigen, and established the indirect ELISA method. We use the method to detect 80 serum samples, and contrast with the commercial kit. The total masculine ratio was 60%. The compliance rate between two methods was 93%.3. We immunized Balb/c mice with PCV2 virus antigen which was condensed and purified, and screened with the indirect ELISA method to obtain monoclonal antibodies specific for PCV. The McAbs were named 2B11-C1 and 3F2-A10. The specific experiments showed that 2B11-C1 and 3F2-A10 had no cross-reactivity with CSFV, PRV, JEV, PRRSV and PK-15 cells. This research provides a basis for setting up a PCV2-testing which has high specificity and sensibility.