Establishment Of IELISA Method For Peste Des Petits Ruminants And Identiifcation Of CD8~+Cytotoxic T Lymphocyte Epitopes Of H And F Proteins In Bajb/c Mice

Peste des petits ruminants (PPR) is an acute and highly contagious viral disease of goats and sheepcausing high morbidity rates and sometimes high mortality rates. It is epidemic in parts of sub-SaharanAfrica, the Middle East and Asia. Owing to the high mortality rate (70–80%), PPR is notifiable toOffice International des Epizooties (OIE), the World Organisation for Animal Health.The PPRV genome consists of single stranded negative sense RNA of15,948nucleotides. Thegenome codes for six structural (N, P, M, F, H and L) and two nonstructural (C and V) proteins in theorder of3′-N-P(C/V)-M-F-H-L-5′. Among the viral proteins, F and H are considered to be veryimportant for the induction of protective host immune response against the virus, and most of theneutralizing antibodies are directed against H and F. However, during the eradication of PPR in thefuture, it will be necessary to restrict the use of the PPR live-attenuated vaccine. The subunit vaccinebased on H and F proteins will be the best choice. More fundamental immunological research isrequired for PPR to find out the immune correlates of protection. Therefore, work on cell-mediatedimmunity is an area of importance for future research.We carried out the following study.1Prokaryotic expression and purification of the extracellular domain of PPRV H fusion proteinBased on the expression vector, we expressed and purified the extracellular domain of PPRV Hprotein. SDS-PAGE revealed the recombinant protein was expressed and the molecular weight was ku.Western-blotting showed that the recombinant protein were of good immunogenicity.2Establishment of PPR indirect ELISA detection methodWe established an indirect ELISA method that incubated by the antigen expressed and purified inour lab. We showed that this ELISA method had good specificity, sensibility and repeation.3Identification of CD8+cytotoxic T lymphocyte epitopes of H and F proteinBalb/c mice were immunized with PPRV live-attenuated vaccine. We synthesized20CTL epitopesof PPRV H and F proteins bases on bioinformatics analysis. Finally, after twice immunization, micewere sacrificed and their spleen cells were separated and stimulated by the predicted CTL epitopes. Fourepitopes of H and F proteins which are capable of stimulating the IFN-γ production in mice CD8+Tcells were selected out by intracellular cytokine staining—flow cytometry and ELISPOT assay.The iELISA method we established in this paper can detect the antibody induced by PPRlive-attenuated vaccine and H protein subunit vaccine. It is the foundation of DIVA detection method.The identification of CD8+cytotoxic T lymphocyte epitopes of H and F proteins enriched the researchof cell-mediated immunity of PPR. It is an area of importance for novel vaccine and the reaction ofPPRV and host.