Preliminary Establishment Of Diagnostic Methods For Antibody And Antigen Of Peste Des Petits Ruminants

Peste des petits ruminants(PPR), caused by the Peste des petits ruminants virus(PPRV), is an highly contagious acute infectious disease of goats and sheep. After the first report of PPR in Africa in 1942, the disease has been quickly spread to most countries around the world. In 2007, the disease was first reported in China. Since then there have also been frequent outbreak of PPR in more than 20 provinces of our country. Currently, there is no effective treatment for PPR. The control and prevention of PRR still depends on vaccine. Therefore establishing a fast PPR antigen and antibody diagnostic methods will be of great importance in diagnosis and antibody screening of PRR. This study initially established two diagnostic methods and explored their application in practical :(1) This study successfully expressed H protein and N protein of PPRV by prokaryotic expression system. The target genes were amplified and cloned into the prokaryotic expression vector pET-30 a. The recombinant plasmid pET-30a-H and pET-30a-N were successfully transformed into E.coli BL21(DE3) competent cells and induced by IPTG to expression. The SDS-PAGE analysis showed that the two recombinant proteins have been expressed in inclusion bodies. The PPRV-H recombinant protein failed to link to nickel column. Therefore, the purification of the protein was achieved by extraction from the SDS-PAGE Gel. The PPRV-N recombinant protein was purified by a nickel affinity chromatography. Imidazole concentration of 300 mM was used to elution. The purified recombinant protein was identified by Western blot. Results showed that the recombinant protein has a good reactogenicity.The purified PPRV-H was used as a coating antigen to establish diagnostic method of ELISA. And the coating antigen concentration, serum dilution, the work concentration of HRP-labeled rabbit anti-goat IgG, antigen-antibody reaction time and the incubation time of the rabbit anti-goat IgG-HRP were optimized. The CV of intro-batch duplicability test and inter-batch duplicability test were all less than 10%, indicating that the method had a good reproducibility. The results were all negative by detecting the positive serums for contagious ecthyma, sheep pox and foot-and-mouth disease, indicated that the method had a good specificity. Comparing of the i-ELISA with the c-ELISA kit(United Kingdom Pirbright Laboratory) by detecting 80 clinical serums, the coincidence rate was 93.75%. Therefore, this method can be used for clinical detection of antibody serums and epidemiological investigation of PPR.(2) The hyperimmune serum were collected from rabbits immunized by purified PPRV-H protein and PPRV-N protein and sheep immunized by PPRV vaccine. The serum IgG is purified by protein G affinity chromatography. The corresponding immunomagnetic beads(IMB) were prepared using the purified IgG to label nano-beads. The IMB combinding with RT-PCR initially established a rapid, sensitive method for the diagnosis of PPR antigen. Comparing the ability to enrich PPRV of three IMB, the results showed that the sensitivity of three IMB is from high to low in order to the sheep anti-PPRV-IMB, the rabbit anti-PPRV-H-IMB, the rabbit anti-PPRV-N-IMB.