Screening And Identification Of Treponema Pallidum Tp92Protein Antigen Epitopes

Objective: To screen and identify the B, T and combined T-B cell antigen epitopesTreponema pallidum (Tp) Tp92protein, and to lay the foundation for furtherinvestigation of syphilis epitope vaccine of these epitopes.Methods:(1) Amino acid sequences of Tp92were obtained from GenBank. The signal peptidesequence was predicted. Using hydrophilic, flexibility, antigen index and surfacepossibility schemes, B cell epitopes of Tp92were predicted by IEDB and Bcepredsoftwares; The HLA-DRBl restricted Th cell epitopes of Tp92were predicted byRANKPEP and SYFPEITHI softwares. Combined B-Th cell epitopes were predictedaccording to above prediction results.(2) Predicted epitope peptides were artificially synthesized and purified by RP-HPLC,and then analysed and identified with mass spectrum.(3) With sera from syphilis patients, and healthy human sera as negative control,predicted B cell or joint B-Th cell epitopes of Tp92were identified with indirectELISAs.(4) Peripheral blood mononuclear cells (PBMC) from syphilis patients or healthypersons were separated and cultivated. After stimulated with synthetic Th or joint Th-B cell epitopes (ConA and RPMI-1640were used as positive and negative control,respectively), lymphocyte proliferation of cultures were detected with CCK-8lymphocyte proliferation kit for identification of Tp92T cell epitopes. At the sametime with ELISA kit were performed to determine the levels of INF-gamma and IL-4of cultures for appraisal of the type of Th cell epitope.Results：(1) Comprehensive analysis of computer prediction scheme showed that P1(Tp92 24-39AA), P2(Tp92332-347AA), P3(Tp92520-536AA), P4(Tp92575-588AA), P5(Tp92103-118AA) and P6(Tp92694-712AA) were regards as the mostpromising candidate B cell epitopes. P5(Tp92103-118AA), P6(Tp92694-712AA), P7(Tp92668-680AA), P8(Tp92300-313AA)and P9(Tp92396-410AA)were predicted as a Th cell epitope and P5、P6was a joint Th-B cell epitope.(2) The purity of all synthetic peptides is more than90%by the RP-HPLCpurification, mass spectrometry analysis revealed that masured value of the syntheticpeptide molecular weight is in accordance with theoretical value.(3)ELISA analysis showed that the peptides P1, P2, P3, P4, P5, P6were active withsera from syphilis patients sera but not with that of healthy persons.(4) T cell proliferation tests indicated that the P6, P7, P9can induce syphilis patientsPBMC to proliferate and produce IFN-gamma. P1, P2, P3, P4, P5and P6failed toinduce syphilis patients PBMC to produce IL-4.Conclusions：(1) The predicted P1, P2, P3, P4, P5, P6(especcialy P3and P6) may be the B cellepitopes of Tp92protein.(2) The predicted P6, P7and P9may be the Th1cell epitopes of Tp92protein.(3) The predicted P6may be a joint B-Th1cell epitopeof Tp92protein.