The Study Of F Protein Immunogenicity And Interaction Of F And H Protein Of PPRV

Peste des petits Ruminants is deadly infectious disease,which is ruled class A bythe world organization for animal health (OIE). There is no good way to treat the PPRat present. Currently inactivated vaccine is mainly vaccine,but the safety is not verygood. H and F protein are two kinds of surface glycoproteins of Peste des petitsRuminants virus, H protein is the main protective immunogen and can induce thebody to produce neutralizing. F protein can make virus to pass cell membrane andenter into cell, at the same time can assist H protein to play role and is also a majorprotective immunogen. Therefore, F protein is a good candidate for the the study ofnucleic acid vaccine of proveting PPR.In this study，according to the F gene sequence of Nigera75/1to synthetizeprimers and to amplify F gene that don’t contain signal peptide and transmembraneregion.Then cloned F gene to pET-30a that is a prokaryotic expression vector andconstructed prokaryotic expression plasmid pET-30a/F. The F protein was expressedand purificated,then used the purified protein to immune chinchilla rabbit inorde toacquire polyclonal antibodies.At last,analyse the specificity of the antibodies byELISA and Western-blot,results showed that the polyclonal antibody had goodimmune reactivity.Similarly,H gene and F gene sequence were amplified and the HA tag and FLAGtag were respectively added the N-terminal of the two genes.Then the H gene and Fgene were respectively cloned to vector pCAGGS that is a eukaryotic expressionvector and constructed recombinational plasmids pCAGGS-H、 pCAGGS-F、pCAGGS-H/HA、pCAGGS-F/FLAG.Then the H gene and F gene were expressedrespectively in cells and were detected by Western-blot and indirectimmunofluorescence,at the same time,the interaction of H protein and F protein weredected by immune co-precipitation and fusion experiment.The results showed that Hprotein and F protein can be expressed normally and can interact effectively.Furthermore, the immunogenicity was evaluated in BALB/c mice with pCAGGS-Fvia intracutaneous injection in the foot and the antibody was detected through ELISA.In conclusion, the pCAGGS-F was able to induced specific immune responses inmice.So the study laid a solid foundation for PPR vaccine development of new type and efficient.