Cloning And Expression Of PPRV N Protein In Baculovirus And Developing Immunological Diagnostic Methods For PPRV Infection

Peste des Petits Ruminants, a fatal infectious disease for small ruminants, caused large loss to farmers worldwidely. It spread fast in recent years, and the epidemic tendency become more critical. PPR outbroke for the first time in China Tibet, in July 2007. Therefore, it becomes more necessary for us to reinforce studies on the etiology, epidemiology, diagnosis and control of the disease.The study aims to construct the recombinant baculovirus containing PPRV N gene through Bac-to-Bac baculovirus expression system. Firstly, the recombinant baculovirus transferring vector pFastBacTM HTA-N was constructed. Then the transferring vector pFastBacTM HTA-N was used to transform E.coli DH10Bac competent cells. N gene of PPRV was inserted into bacmid in E.coli DH10Bac through Tn7 transposition system. Then the recombinant baculovirus was obtained by transfecting insect cell sf21. Finally, the recombinant baculovirus containing the PPRV N gene was constructed. After infecting sf21, the correct size of protein was expressed, and the antigenicity was identified by Western blotting. The recombinant PPRV N protein was used as the coating antigen to detect the PPRV-N antibody in goats and sheep serum by Indirect ELISA. The indirect ELISA has a high sensitivity of 100% and a high specificity of 96.2% compared with the cELISA kit supplied by CIRAD-EMVT, the coincidence rate of the two methods is 96.9%.6~8 weeks old BALB/c mice were immunized by purified bacmid-PPRV-N protein for 3 times. 3 days after the last immunization, the splenocytes of the BALB/c mice immunized were collected to fuse with the SP2/0 cells. Prokaryo-expressed pET-32a-PPRV-N protein was used as the detection antigen in indirect ELISA. 8 strains of positive hybridoma cells were filtered out after 3 times of subcloning. The inducing ascites in vivo method was employed to produce monoclonal antibodies in large amount. The titer of 1D5's culture supertant was 1∶16000, and the titer of it's ascites was 1∶106. The subtype of 1D5 monoclonal antibody is IgG2b.By employing the Bacmid-PPRV-N protein as the coating antigen, 1D5 monoclonal antibody as the competitive antibody, cELISA was established to detect the serum antibodies of animals. The method has high sensitivity and specificity. Especially, the Mab 1D5 could differentiate RP and PPR positive serum. According to the test results of 187serum samples, the cELISA established in this study has a high coincidence rate of 98.5% compared with the cELISA kit supplied by CIRAD-EMVT.