Preparation And Characterization Of Monoclonal Antibodies Against PCV2-Cap Protein

As a member of the family Circoviridae, Porcine Circovirus (PCV) contains two types genome: PCV1 and PCV2. PCV1 has no pathogenicity, PCV type2 (PCV2) is associated with Postweaning multisystemic wasting and relation to many dieases, including CT, PDNS, PRDS. So far, the mainly diagnosis of PCV2 is serological diagnosis methods and pathogen detection method. However, as PCV1 and PCV2 cross-reaction between the antigen, serological diagnosis is not very reliable and pathogen detection depend on strict laboratory operations and is hard to be used widely, it is very important to establish a simple diagnosis way which is rapid and accurate. The purpose of this reserch was to prepare monoclonal antibody of PCV2-Cap protein and prepare for the new diagnosision way of PCV2.In this research, PCV2-ORF2 recombiant protein was expressed in E.coli and purified through His-bind affinity chromatography. The purified PCV2-ORF2 recombiant protein was used to immune BALB/C mice. The immunitied mice spleen cells and SP2/0 myelomas cells were fused and the positive clone was screened by limiting dilution method. Then the McAbs were prepared and identificated. The results indicated that:1. The recombinant fusion proteins were highly expressed after IPTG inducement and the molecular weight was about 45 ku as expected. The recombinant protein was purified through Ni-chelating affinity chromatography. The UV absorption determination indicated that the recombinant protein concentration was about 5 mg/mL.2. Splenic cells of Balb/c injected were hybridized with SP2/0 by cell fusion and the hybridoma cells were screened by ELISA and subcloned approach. Finally three hybridoma cells (1C7A4, 4F3F5 and 4G8F7) stably secreting anti-PCV2-Cap protein monoclonal antibodies were obtained, and ascites were induced to produce monoclonal antibodies. Hybridoma cells and the monoclonal antibodies were identified ultimately. The results indicated that hybridoma cells which have normal chromosome were stably secreting antibodies after freezing and thawing and passage culture.3. The McAbs we made after a series of biological nature experiment showed that they belonged to IgGl, IgG1 and IgG2b subgroup and belonged to two antigen epitopes. Western-blot indicated that three strain McAbs presented high specificity to the recombinant PCV2-Cap protein expressed in the prokaryotic cells. The 4G8F7 strain McAb could bind to PCV2-Cap protein expressed in eukaryotic cells highly specifically. In the end, we produced anti- PCV2-Cap protein monoclonal antibodies successfully, which were foundation for the further study on PCV2.