| Peste des Petits Ruminants is a fatal disease caused by Peste Des Petits Ruminants Virus (PPRV), which mainly occurred in goats and sheep. Nowadays, more and more countries had attached great importance to this disease. Therefore, it is very necessary to do some studies on the virology, epidemiology, clinical characters, pathology, diagnose and vaccine development of the disease.On the base of bioinformatical therory, the physical-chemical properties, secondary structure and antigenic properties of N protein of Peste des Petits Ruminants virus (PPRv) were analyzed on the internet software. In the results, we concluded 9 antigenic epitopes in PPRv-N protein. Three peptides among them were synthesized on PS3 peptide synthesizer according to Fmoc solid phase synthesis principle. From the indirect ELISA results, we learned that the 3 antigenic epitopic peptides can react with PPRv positive sera differentially, especially the Pep2 peptide reacts only with the PPRv positive serum, but not with the RPv positive serum. These results show that the 3 peptides are epitopes of N protein and Pep2 peptide is an identical epitope.Coupled with KLH, Pep2 was injected differently into 6-8 weeks old Balb/c mice in three times. Three days later of the last immunization, the B cells were collected from spleens and amalgamated with SP2/0 cells in a proper proportion. After cultured and selected in HAT-DMEM medium, 15 hybridoma clones were selected, which can secrete positive antibodies. Among them, 4F11 monoclonal hybridoma was further cloned which can secrete monoantibody steadily. Results show that the 4F11 has 96 chromosomes, and the monoclonal antibody is an isotype of IgG2a. The antibody can differentially identify PPRv positive sera and the other paramyxoviridae virus positive sera by c-ELISA method.To establish a peptide c-ELISA, Pep2 was coupled to BSA by glutaraldehyde and 4F11 monoantibody was chosen as a competitively antibody. Compared with the standard cELISA kit bought from OIE, the established peptide cELISA method has a coincidence rate of 96.85% according to the test results of 381 serum samples. |
