Identification Of B-Cell Epitopes On The Structural Proteins Of Senecavirus A

Senecavirus A(SVA)belongs to the family of Picornavidae and Senecavirus genus.It causes clinical symptoms of blister-like diseases such as foot-and-mouth disease in pigs of all ages,and can cause acute death of newborn piglets,which hinders the prevention and control of foot-and-mouth and seriously endangers the development of pig industry in china.Since SVA is a newly emerged infectious disease,previous studies mainly focused on its biological characteristics,but there are few studies on SVA vaccines,invasion mechanism and immune mechanism.VP1,VP2 and VP3 are capsid proteins located on the surface of SVA,which contain both receptor-binding domains that mediate the invasion into host cells and antigenic epitopes that stimulate the immune response to produce neutralizing antibodies.Therebefore,in this study,B-cell epitopes of the three capsid proteins of SVA were screened and identified,which laid a foundation for the study of the etiology,diagnostic methods,antigen structure and novel epitope-based vaccines of SVA.In the early stage of study in our laboratory,SVA inactivated vaccine was developed by using CHFJ-2017 isolate,a field epidemic isolate of SVA,and then immune sera,including SOW6-1?PC6-2 and PC4-3,were prepared by immunizing pigs.In this study,the biological characteristics of three immune sera were analyzed and identified.The affinity between the three immune sera and viral structure proteins was determined by indirect ELISA.The immunoreactivity of the sera with SVA isolate and structural proteins was determined by the IFA and WB assays,respectively.The neutralization titers of the sera were determined by VNT.The results indicated that the sera had good immunoreactivity and high affinity with SVA and viral structure proteins,and all the viral neutralization titers were greater than 1024.Then,combining with the bioinformatic prediction method and the overlapping peptide biosynthesis method,the B-cell epitopes of three structural proteins of SVA,VP1,VP2 and VP3,were screened and identified using immune sera.The physicochemical properties and secondary structures of the three viral structural proteins,VP1,VP2 and VP3,were analyzed by Protparam and PSIPRED online softwares.The B-cell epitopes of each structural protein were predicted using 7 bioinformatic prediction softwares,including Bcepred,ABCpred,Bepipred,IEDB,SVMpred,Ellipro and SEPPA2.0.The predicted epitopes were synthesized and verified by indirect ELISA.The results showed that the immune sera could specifically recognize the predicted epitopes,and 3 potential antigenic epitopes on VP1 protein,6potential antigenic epitopes on VP2 protein,and 3 potential antigenic epitopes on VP3 protein were identified.To further improve the accuracy of identification,based on the amino acid sequences of the three structural proteins of SVA,96 overlapping 16 peptides with GST188 tags were constructed,and 8amino acid residues were overlapped between adjacent overlapping peptides.These peptides were cloned into prokaryotic expression vector pXXGST-1 and expressed by E.coli.The immunoreactivity of these overlapping peptides with immune sera was analyzed by WB.Finally,7 positive fragments on VP1 protein,13 positive fragments on VP2 protein and 5 positive fragments on VP3 protein were identified,respectively.Finally,the immunogenicity of the B-cell epitopes was analyzed.According to the screening results of above two methods,the B-cell epitopes with good consistency were selected and synthesized.These epitopes were conjugated with keyhole limpet hemocyanin(KLH)protein,and sera were prepared by immunizing guinea pigs.These immune sera had good immunoreactivity with SVA structural proteins and could specially recognize SVA in IBRS-2 cells.In summary,2 linear B-cell epitopes on the VP1 protein,VP1-1(6-21 aa)and VP1-2(221-233 aa),4 linear B-cell epitopes on the VP2 protein,VP2-1(5-16 aa),VP2-2(35-57 aa),VP2-3(175-187 aa)and VP2-4(267-282 aa),and 1 linear B-cell epitope on the VP3 protein,including VP3-1(135-153 aa),were identified.And all of these epitopes had the ability to stimulate the immune response.These data could provide the basis for the further study of diagnostic methods,epitope-based vaccines and the function of VP1,VP2 and VP3 proteins.