Screening And Identification Of B Cell Epitopes Of VP1 And VP2 Proteins Of Senecavirus A

Senecavirus A Disease(SVAD)is a viral blister infection caused by Senecavirus A(Senecavirus A,SVA),which mainly infects pigs and can lead to acute death of newborn piglets.In this study,using antigen epitope prediction method combined with overlapping synthetic polypeptide method to locate and analyze the main B cell epitope on SVA epidemic strain CH-FuJ-2017 VP1 and VP2 structural protein,selecting the dominant epitope for tandem expression according to the identification results,and verifying its reactionogenicity by the method of Western blot and ELISA,which laid the foundation for the study of immune properties and functions of SVA structural proteins and the research of epitope vaccine vaccines.1.Prediction and screening of B cell antigenic epitopes: sequence analysis of structural protein VP1 and VP2 genes of SVA was performed by DNAStar and IEDB.16 potential epitopes were predicted and cloned respectively into pGEX-4T-1 plasmids for prokaryotic expression and purification.6 dominant epitopes were identified by Western blot and ELISA.2.Screening B cell epitopes by overlapping synthetic peptides: 283 amino acids of VP1 based on SVA CH-FuJ-2017 strain and 263 amino acid sequences of VP2 were designed and synthesized with 67-segment polypeptides composing of 16 amino acids and overlapping with 8 amino acids each other.These short peptides are used for ELISA analysis to perform B cell epitope mapping.Through the ELISA,the VP1 protein screened out 9 epitope peptide segments and the VP2 protein screened out 15 epitope peptide segments.3.Screening B cell epitopes with better conservatism and reactogenicity based on the results of the above two methods: Several epitopes on the VP1(7-26aa、48-72aa、92-109aa)and VP2(38-57aa、141-148aa、154-172aa、249-284aa)proteins with promiscuous T-cell epitope,and then the SVA multi-epitope gene combination was designed and constructed,cloned into the pET-30 a vector for prokaryotic expression and purification.Through the verification of Western blot and ELISA the recombinant multiepitope proteins in this study have good reactionogenicity with polyclonal antibodies SVA VP1 and SVA VP2,which provide technical reserves for further preparation of SVA epitopal vaccines and clinical applications.