Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology

Preparation and Identification of Monoclone Antibodies Against CAP Protein of Porcine Circovirus Type 2

Author CaoYanQiong
Tutor JiangPing
School Nanjing Agricultural College
Course Preventive Veterinary Medicine
Keywords PCV2 Cap protein McAb Epitope
CLC S852.65
Type Master's thesis
Year 2011
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Porcine circovirus type2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) and can cause severe immunosuppression in swine. In recent years, PCV2 has spreaded widely in the world and impaired the swine industry. The genome of PCV2 contains two major open reading frames, ORF1 and ORF2, which encode Rep protein related with viral replication and main structure Cap protein, respectively. The Cap protein can induce protective immunity against PCV2 infection in swine. It contains several immunodominant epitopes. In this study, Balb/c mice were immunized with the recombinant Cap protein expressed with baculovirus expression system. Six McAbs specific to PCV2 Cap protein were obtained, and antigenic epitopes recognized by these six McAbs were analyzed systemically. The main contents of the research were as following:1. Balb/c mice were immunized with baculovirus-based Cap protein, and the splenocytes of the immunized mice were fused with murine myeloma cells SP2/0. After subcloning by 3 times, six hybridoma clones which produced McAbs against PCV2 steadily were screened by indirect ELISA, and named as 3H9/1H5,3H9/3C7,4A9/3E2, 4A9/3D7,6G7/7B6 and 6G7/8D3, respectively.2. The titers of the McAbs in supernatant of cell culture were 1:1024,1:1024,1:2048, 1:2048,1:1024 and 1:1024 for 3H9/1H5,3H9/3C7,4A9/3E2,4A9/3D7,6G7/7B6 and 6G7/8D3, respectively. Correspondingly, the titers in ascites were 1:204800,1:204800, 1:1310720,1:1310720,1:163840 and 1:163840. The titers of the McAbs were confirmed steadily during 20 passages of the cell clones in vitro. Moreover, the McAbs 3H9/1H5, 3H9/3C7,6G7/7B6 and 6G7/8D3 belonged to IgG1, while 4A9/3E2 and 4A9/3D7 belonged to IgG2b.All McAbs hadκchain. Western blot analysis showed that all McAbs were able to react with both recombinant Cap protein and native PCV2 virus. The results of IFA also suggested that they could react strongly with PCV2 isolated strains, which means they can recognize the native structure of PCV2.3. The six McAbs specific to PCV2 Cap were identified to recognize three different epitopes, according to the results of an ELISA assay.3H9/1H5 and 3H9/3C7,4A9/3E2 and 4A9/3D7,6G7/7B6 and 6G7/8D3 were identified to recognized three epitopes. In order to map the epitopes, five truncated fragments of PCV2 Cap(CapA:51-150aa, CapB:51-200aa, CapC:51-233aa, CapD:101-200aa, CapE:101-233aa)were expressed by E.coli system and used in Western blot. The results showed that 4A9/3E2 and 4A9/3D7 could recognize the epitope within 101-150aa, while the other four McAbs might recognize conformational epitopes or the linear epitope within 1-50aa. It should be useful for studing on the new diagnostic methods and the foundation of the Cap protein in the future.

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