Design And Evaluation Of The Multi-epitope Antigens Against Peste Des Petits Ruminants

Peste des Petits Ruminants Virus(PPRV)belongs to Morbillivirus,which was the cause of acute infectious diseases of small ruminants such as goats and sheep.The high morbidity and high mortality are the characteristics of the disease.It was listed as a notifiable animal infectious disease by the world organization for animal health(OIE),and it also was classified as A list of animal diseases in China.Initially,PPRV infected goats and sheep only.In recent years,there had been reports of interspecies transmission.Although only one serotype,PPRV can be divided into four genetically distinct lineages(?,?,?and?)with the characteristics of regional distribution.At present,PPRV mainly distributed in Africa and Asia,and Europe was facing a huge threat.HN and F proteins are embedded in the viral envelope,which constitute the fibers of the virion surface.HN protein can interact with SLAM receptors on the lymphocyte to mediate viral invasion,which may be the main cause of immunosuppression.In the process of viral invasion,F protein can play a role in the fusion of cell membrane and viral envelope.In addition,PPRV can cause strong cellular and humoral immune responses,while HN and F were the major protective antigens.In this study,we hoped to identify the B cell epitopes of HN and F proteins,and then to design the candidate vaccine antigens against PPR.pET21a-rPPRV-HN-F plasmid constructed by our laboratory was transformed into E.coli.The rPPRV-HN-F protein was induced to express and purified.The result of the detection showed the rPPRV-HN-F protein had obvious reactionogenicity with PPRV positive serum in sheep.Monoclonal antibodies and polyclonal antibodies in mouse with good reactionogenicity were prepared using rPPRV-HN-F protein.In addition,we extracted the glycoprotein from the VeroE6 cell culture medium by inoculating PPRV,and the polyclonal antibody in mouse was prepared,which had good reactionogenicity and specificity.The structure of PPRV HN-sheep SLAM complexes were simulated using Discovery Studio V4.5,and the key amino acids of interaction was determined.Combined with the simulated structure,the B cell epitopes of HN and F protein were predicted by using the immune information tool.The reactionogenicity of peptides with PPRV specific antibody was detected by indirect ELISA method with an amino ELISA plate,and we discovered 11 peptides with the reactionogenicity.The antigens of the multiple epitope-based vaccine were obtained by biosynthesis.The level of antibody and the proliferation of T lymphocytes were detected in the immunized mouse.The results showed that the mouse could produce specific antibodies stimulated by the multi-epitope antigens.Flow cytometry analysis showed that CD3~+CD4~+T lymphocytes and CD3~+CD8~+T lymphocytes increased in different degrees,especially CD3~+CD8~+T lymphocytes.Therefore,this study was of great significance to the prevention and control of PPR,and even to the implementation of the global eradication program.