Identification Of Dominant Epitope And Evaluation Of Prevention And Treatment Effect Of Leucine Aminopeptidase In Echinococcus Multilocularis

Objective The prokaryotic expression system of recombinant Leucine Aminopeptidase（LAP）was constructed and its enzyme activity was analyzed.Immunogenicity and efficacy of LAP were demonstrated in a BALB/c mice model.The present study also aims to identify dominant T cell and B cell epitopes in Leucine Aminopeptidase of Echinococcus multilocularis.Methods The gene sequence of LAP was obtained from NCBI-Gen Bank.The expression vector p Czn1 was connected with the r Em-LAP and transformed into the BL-21 strains.High purity recombinant target protein LAP was obtained by Ni-IDA-Sepharose CL-6B column affinity chromatography after IPTG was added in the fermentation process to induce the expression of target protein.Em-LAP activity was determined using L-leucine-p-nitroaniline（Leu-p NA）as a substrate and measuring the release of p NA at 405 nm.Efficacy（number and weight of cysts,serum antibody and cytokine levels）of r Em-LAP combined with Freund’s adjuvant in the prevention and treatment of Echinococcus multilocularis infection was evaluated.Then,Lymphocyte-proliferation assay,flow cytometry,ELISApot,and ELISA were used to identify the dominant antigen epitopes.Finally,the 3D structure was modeled with SWISS-MODEL software,and Py MOL software was used to accurately locate the LAP dominant epitopes.Results The p Czn1-LAP plasmid was successfully constructed by gene sequencing and enzyme digestion,and the r Em-LAP was successfully transformed into Escherichia coli.LAP protein（57.4KD）was obtained by Ni²⁺column affinity purification.The optimal activity of Em-LAP is p H 9 and 60℃.The order of Em-LAP activity in different metal ion chloride solutions was Co²⁺>Zn²⁺>Ca²⁺>Mn²⁺>Mg²⁺>Ba²⁺>K⁺.Serum indirect ELISA demonstrated that Em-LAP could induce a Th1 and Th2 mixed-type immunological response and produce high levels of Ig G,Ig G1,Ig G2a,Ig M,and Ig A.Furthermore,serum IFN-γand IL-4 secretion were increased compared with the control groups.Vaccination with r Em-LAP significantly decreased both the number and size of the cysts in Echinococcus multilocularis metacestode infected mice model.The dominant antigen epitopes of Th1 were identified by flow cytometry as LAP_79-63,LAP_396-410,and LAP_106-120.The epitopes of Th2 were LAP_13-28,LAP_495-510,and LAP_396-410.The dominant antigen epitopes identified by ELISApot as LAP_396-410,LAP_504-518,and LAP_106-120.The epitopes of Th2 were LAP_504-518,LAP_313-328,and LAP_396-410.The identification of predominant B cell epitopes were LAP_464-479,LAP_495-510,and LAP_313-328.The bioinformatics software showed that the antigenic peptides were mostly located on the surface of the protein.Conclusion The current study provides evidence that Em-LAP could be a potential vaccine antigen of Echinococcus multilocularis.The dominant T cell and B cell epitopes of LAP were successfully identified which maybe contribute to develop a multivalent epitope vaccine against Echinococcus multilocularis infection.