Screening And Identification Of Senecavirus A Structural Proteins T Cell Epitopes

Senecavirus A(SVA)is the only member of the genus Senecavirus within the family Picornaviridae.SVA infection causes fever,salivation and blister-like sores on the tongue and lips,in the mouth,on the teats and between the hooves in pigs.Ruptured blisters could result in extreme lameness and reluctance to move or eat.The clinical manifestation of the disease caused by SVA is indistinguishable with Foot-and-Mouth disease,Swine Vesicular Visease and Vesicular Stomatitis Diseases.SVA was discovered in the United States in 2002.Beginning in late 2014,SVA was reported in many swine herds in Canada,Brazil,China,Thailand and Colombia,which caused significant economic consequence.In China,the first case of SVA infection was observed in Guangdong Province in 2015.Subsequently,SVA infections were reported on other farms in Hubei,Heilongjiang,Henan,and Fujian Provinces,which seriously affected pig industry and caused huge economic losses to the livestock industry.At present,there are few researches about SVA cell-mediated immunity,T cell response is a crucial part of the adaptive immune system in the fight against infections.T-cell epitope prediction aimed to identify the shortest peptides within an antigen that is able to stimulate T-cells.They are particularly important for anigen epitopes to screen and identify.The rapid development of bioinformatics make it possible for people to use immune epitope databases to screen epitopes.Fortunately,researchers have developed in silico prediction methods that dramatically reduce the burden associated with epitope mapping by decreasing the list of potential epitope candidates for experimental testing.There are great interest in identifying epitopes in antigens for a number of practical reasons,including understanding disease etiology,immune monitoring,developing diagnosis assays,and designing epitope-based vaccines.Studies have shown that the virus particle immunity of Senecavirus A could stimulate the proliferation of T lymphocytes,and IFN-?-specific T cells can be detected after stimulation.Under this premise,by predicting and identifying the T cell epitope of Senecavirus A,this project hope to pave the way for the development of Senecavirus A T cell vaccines and studyed the role of T cell immune response in Senecavirus A infection.First,we used the bioinformatics website to predict the epitopes of the Senecavirus A structural protein,selected the peptides synthesis with excellent scores and high repetition rate,and then constructed the eukaryotic expression vector plasmid of the Senecavirus A structural protein(pc DNA3.1-P1),verifyed its expression in vitro by Western blotting,indirect immunofluorescence and indirect ELISA experiments,and then it was used as a DNA vaccine to immunize mice,and DNA vaccine stimulated the ability of mice to produce antibodies is detected by virus neutralization experiment and enzyme-linked immunosorbent assay.Afterwards,flow cytometry was used to detect the ability of plasmid-immunized mice to promote lymphocyte proliferation,and enzyme-linked immunospot technology was used to detect the number of polypeptides that stimulated the production of IFN-? in splenocytes.Finally,a repetitive and effective peptide was selected to test the ability of the peptide to stimulate spleen cells to produce IFN-?.This peptide may be a T cell epitope of Senecavirus A.Experiments had shown that although the ability of DNA vaccines to stimulate mice to produce antibodies was weak,there were still antibodies produced in mice,and then synthetic peptides were used for in vitro stimulation.The IFN-? ELISPOT kit was used to screen the above peptides.Four peptides with better effects had been developed,incuding peptides 1(SFDTASGTF),9(MPFQSLGTY),11(ITLTFCGPM),and 21(TSVDISVPYI).These data will pave ways for SVA peptide vaccines development.