Bioinformatic Prediction And Identification Of Dominant Epitopes Of The Antigenic 14-3-3 Protein Of Echinococcus Multilocularis

Objective:To evaluate the immunogenicity of the recombinant antigen protein14-3-3 and its preventive effect/therapeutic effect in secondary infection of Echinococcus multilocularis protoscolex in BALB/c mice,and to identify the dominant T and B cell epitopes of Echinococcus multilocularis antigen protein14-3-3.Methods:1.The prokaryotic expression system of Em-14-3-3 was established.IPTG induction and Ni²⁺-affinity chromatography were used to obtain high-purity14-3-3 protein which was used as a vaccine to immunize BALB/c mice.The level of antibody and cytokine in serum of mice were detected by ELISA.The preventive and therapeutic effects in mice secondary infected with Echinococcus multilocularis protoscolex were assessed according to the number and weight of the cysts.2.The amino acid sequence information of Echinococcus multilocularis antigen protein14-3-3 was analyzed by SOPMA and the homology MODEL was established by Swiss-Model;IEDB and SYFPEITHI were used to predict 14-3-3 potential T cell epitopes.According to the Bcepred and ABCpred,the 14-3-3 B cell epitopes were comprehensively predicted and analyzed.The predicted epitopes were compared with the amino acid sequence of human,dog and mouse sources to exclude common epitopes.The dominant epitopes predicted by bioinformatics were identified by splenic lymphocyte proliferation,ELISA,ELISpot,flow cytometry.Results:The recombinant 14-3-3 antigen protein of Echinococcus multilocularis was successfully expressed and purified.Immunogenicity studies showed the 14-3-3protein has good immunogenicity and induce a Th1/Th2 mixed type immunological response.In preventive evaluation model,the number of cysts has decreased by 59.84±15.09%(P=0.049)and the weight of cysts has decreased by 83.28±18.01%(P=0.000)when treated with 14-3-3.In the therapeutic evaluation model,the weight of cysts has decreased by 72.28±11.47%(P=0.006).Eight potential epitopes of14-3-3 were screened according to the results of prediction software and excluding the common epitopes of human,mouse and dog:14-3-3_1-15?14-3-3_6-21?14-3-3_71-86?14-3-3144-157?14-3-3145-166?14-3-3146-160?14-3-3153-161?14-3-3164-177.The 3D structure model of the 14-3-3 antigen protein was constructed and the potential epitopes location distribution were located.The epitopes of dominant antigen of B cells were identified as 14-3-3_145-166 and 14-3-3_164-177;Th1 dominant antigen epitopes were14-3-3_6-21,14-3-3_145-166;Th2 dominant antigen epitope was 14-3-3_145-166.Conclusion:The recombinant 14-3-3 antigen protein of Echinococcus multilocularis has preventive and therapeutic effects against the secondary Alveolar Echinococcosis of mice,2 dominant antigen epitopes of B cells,2 Th1 dominant antigen epitopes and 1 Th2 dominant antigen epitope were identified.This study provides theoretical foundation and experimental basis for the development of effective and safe Echinococcus multilocularis epitope vaccine.