| Peste des petits ruminants (PPR) is an infectious viral disease of sheep and goats. Owing to fastspreading and high mortality, PPR is one of the OIE list of notifiable terrestrial animal diseases. Anattenuated vaccine against PPR is an effective means of controlling the disease, but its thermolabilityand biological safety are concerns, which often cause immune failure. Therefore PPR vaccine's designfocuses on how to improve the safety and stability. In this research, on the basis of homology analysis ofthe whole genome sequences between PPRV and other member of Morbillivirus family, the tertiarystructure of PPRV six structural proteins were predicted, the P, N and L gene cloned and expressed, thefull-length cDNA constructed, the DNA vaccine prepared and immunology research conducted. Theresults were as the following:1. The homology of whole genome sequences between PPRV and other member of Morbillivirusfamily was analysed by biology software. The results showed the homology were more than64%, andthe sequences of the5' end and3' end of the whole genomes were highly homologous, almost100%.Phylogenetic analysis showed that the genetic relationship of the CDV and DMV was relatively close,located at the same clade, and that MV and RPV was grouped into the same branch, while PPRV formeda distinct one. In addition, the tertiary structures of the PPRV structure proteins were predicted usingSwiss-Model and I-TASSER.2. Prokaryotic expression vector pET30/P harboring phosphoprotein gene was successfullyconstructed. Optimum conditions of expression were determined. The soluble recombinant protein waspurified by Ni+Sepharose affinity chromatograph method immunized mice and used to preparepolyclonal antibodies, of which the titerwas up to1:25600.3. Eukaryotic expression vectors expressing P, N and L proteins, named as pcDNA3.1/P,pcDNA3.1/N and pcDNA3.1/L, were successfully constructed. These recombinant plasmids weresuccessfully transfected into Vero cells, respectively, as confirmed by indirect immunofluorescenceassay, SDS-PAGE and Western blottingting.4. Prokaryotic fractional expression vector expressing large protein was successfully constructed.The soluble recombinant protein was purified by Ni+Sepharose affinity chromatograph and used toimmunize mice and also to prepare polyclonal antibodies, of which the titer was up to1:25600.5. Using RT-PCR, five over-lapping cDNA fragments of a PPRV Nigeria75/1strainwere amplified.Using pOK12as a plasmid vector, the full-length cDNA clone, pok12-PPRV of Nigeria75/1, wasassembled by connecting the five cDNA fragments via unique restriction endonuclase sites of the PPRVgenome. Ribozyme sequences and eukaryotic promoter sequence were introduced immediately at theupstream of5′end, while HDV sequence and terminator sequence downstream of3′end. The sequencing and sequence alignment analysis showed the Nigeria75/1strain of full-length cDNA wassuccessfully constructed, which lays foundation for effectively rescuing PPRV.6. Twenty-five male and female6-month-old goats were grouped randomly into five experimentalgroups of five animals each, and immunized with the conventional PPR vaccine, DNA vaccine(pcDNA3.1/N, pcDNA3.1/P, pcDNA3.1/L and pOK12-PPRV), three plasmids (pcDNA3.1/N,pcDNA3.1/P and pcDNA3.1/L), pOK12/full-length cDNA and PBS. The results of antibody total levels,cytokine levels, levels of T cell proliferation, CD4+/CD8+T cell ratio and neutralizing antibody levelsshowed that at21d after the first immunization and14d after the boost, DNA vaccines inducedsignificant changes in the above indexes, and that neutralizing antibodies could reach1:32after theboost. These showed that like traditional vaccines, DNA vaccines could induce specific humoral andcellular immune responses, which provides new ideas and directions for the prevention and treatment ofPPR.
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