Preparation Of Monoclonal Antibody Against Nucleoprotein Of Peste Des Petits Ruminants Virus(PPRV) And Development Of Competitive ELISA Kit For Detection Of Antibodies Against PPRV

According to PPRV nucleotide sequences have been documented in Genbank database, we synthesized N gene ORF inserting into pBluescriptsk. The N gene fragment was obtained by regular PCR. Then the fragments were digested with designed enzymes in the primers and cloned into baculovirus donor vectors pFastBacHTA of Bac-to-Bac system. The positive recombinant plasmid was named pFastBacHTA-PPRV-N, the following sequencing result showed that the PPRV N gene was1578bp and encoded525amino acids. The recombinant plasmids pFastHTA-PPRV-N were transformed into DH10Bac host bacteria to get recombinant shuttle plasmids,Bacmid-PPRV-N. PPRV nucleoprotein was expressed by transfecting recombinant Bacmid-PPRV-N with liposomes into Sf9insect cells. Then we confirmed the recombinant virus via the SDS-PAGE and western blot,which showed that N protein was expressed in infected insect cells. The N protein had a good biological activity and was approximate61.3ku.After immunization of BALB/c mice with purified recombinant protein Bacmid-PPRV-N, two monoclonal antibodies against PPRV nucleoprotein named5B11and3H10-3B8, respectively, were produced by polyethyleneglycol (PEG)-mediated fusion of sensitized lymphocytes and myeloma cells. The specificity and biological characterization of the mAbs were identified. Isotypes and subclasses of5B11and3H10-3B8belong to IgG2b, the titres of ascites fluids of5B11was up to1:819200and3H10-3B8was1:12800. These mAbs could specifically recognize recombinant protein Bacmid-PPRV-N antigens in a serologic test. Antibodies additivity assay demonstrated that both5B11and3H10-3B8recognized the different epitopes of PPRV nucleoprotein.The number of chromosome of the hybridoma cell lines was99-104.A competitive ELISA based on the reaction between a monoclonal antibody (mAb5B11) purified by protein G and a recombinant nucleoprotein Bacmid-PPRV-N was developed. Optimum conditions were obtained by using serum samples which had positive or negative activity against PPRV. Microtitre ELISA plates were coated with N antigen diluted up to1:1000in CBS for one hour at37℃with constant agitation. After the adsorption of antigen, the sera at a final dilution of1:10were incubated simultaneously with the mAb(final dilutionl:15000) in a optimum diluent buffer (PBS, supplemented with0.5percent BSA and0.05percent Tween-20) to lead to competition for a specific epitope on the N antigen. We tested92PPRV-negative serum samples from field ruminants. The threshold of percent inhibition (PI) was determined to be50on the basis of the mean PI plus3standard deviations for serum.Three different batches of ELIS A kits to detect the antibodies against PPRV were assembled on the basis of research results in the early stage. The kits were evaluated by compared with the standard reference C-ELISA kit in their preliminary application. Clinical samples (n=977) were tested by the reference C-ELISA kits and homemade kits and the data demonstrated that the coincidence was99.38%which concluded that the homemade kits have good reliability.