Prediction And Identification Of The Antigen Epitopes Of Treponema Pallidum Adhesin Tp0751

Objective：To screen and identify the B-cell, Th-cell and combined Th-B-cellantigen epitopes of Treponema pallidum (Tp) adhesin Tp0751，providing theexperimental basis and laying the foundation for further development of syphilismultivalent epitope vaccines.Methods:1. Predition of cell epitopes of Tp0751Obtained the amino acid sequences ofTp0751from Genbank, the HLA-II restricted Th-cell epitopes of Tp0751werepredicted with SYFPEITH, HLAre and IEDB softwares. Using hydrophilic,flexibility, Beta-Turn, antigen index and surface possibility schemes, B-cellepitopes of Tp0751were predicted with IEDB and EMBOSS softwares.Combined B-Th-cell epitopes of Tp0751were predicted according to aboveprediction results.2. Synthetize and identification of epitopes of Tp0751RP-HPLC was used tosynthesize and purify the predicted epitopes which then were analysed by massspectrum.3. Expression and identification of antigenicity of recombinant Tp0751Theprokaryotic expression vector pET28a(+)/Tp0751without sequence of signalpeptide was constructed and transformed into E.coli BL21for Tp0751proteinexpression after identified by double digestion, PCR and gene sequencing.Antigenicity and expression form of recombinant Tp0751(rTp0751) purifiedwith Ni-NTA affinity chromatography were determined by Western blot andSDS-PAGE, respectively. Protein concentration was tested by BCA kit.4. Preparation of sera and splenocytes of immunized rabbit and evaluation ofTp0751immunogenicity. The New Zealand rabbits were injectedsubcutaneously with rTp0751for5times. Sera were collected for determination of titers of anti-rTp0751and identification of B epitopes of Tp0751.Splenocytes were harvested at10days after the last immunization foridentification of Th-cell epitopes of Tp0751.5. Identification of B-cell epitopes of Tp0751Synthesized peptides andrecombinant proteins were coated on96well microtiter plates, respectively.Using sera from syphilis patients or rTp0751-immunized rabbit sera as primaryantibodies (with nomal human serum and rabbit serum as negative control), theindirect ELISAs were performed to analyse B-cell epitopes of rTp0751protein.6. Identification of Th-cell epitopes of Tp0751Cultured splenocytes fromimmunized rabbits were stimulated with synthetic Th or Th-B cell epitopes(with PBS as negative control and ConA or rTp0751as positive control)，CCK-8lymphocyte proliferation kit was used to detect the lymphocyteproliferation of cultures and realtime-PCR was performed to evaluate themRNA levels of IL-4and INF-γ.Results1. Predition of cell epitopes of Tp0751Comprehensive analysis of computerprediction scheme indicated that P1(65-83AA), P2(90-101AA),P3(127-137AA), P4(221-237AA), P5(175-185AA) were regards as candidateB-cell epitopes. P5(175-185AA), P6(103-113AA), P7(162-171AA) werepredicted as a Th-cell epitope and P5was a combined Th-B cell epitope.2. Synthetize and identification of epitopes of Tp0751Mass spectrometryanalysis showed that masured value of the synthetic peptide molecular weightwas in accordance with that of theoretical value. RP-HPLC indicated that thepurity of synthetic peptides is more than90%, reaching the requires forfollowing experiments.3. Expression and identification of recombinant Tp0751Enzyme digestion andsequencing confirmed that sequence of gene inserted into recombinant plasmidpET28a(+)/Tp0751was completely consistent.with that logined on GenBank Soluble26kDa-size recombinant protein was expressed with above95%ofpurity. The result of Western-blot indicated that only syphilis sera were activewith rTp0751. The titers of anti-rTp0751were more than12,000after the4thimmunization.4. Identification of B-cell epitopes of Tp0751ELISA results showed that thepredicted P4, P5were strongly active with sera from patients with syphilis andimmunized rabbits, but not with sera from healthy human and unimmunizedrabbits.5. Identification of Th-cell epitopes of Tp0751Lymphocyte proliferation assayshowed that no significant lymphocyte proliferation was observed.realtime-PCR revealved that there were no significant increase of levels of IL-4and INF-γ mRNA in peptide-stimulated splenocytes from immunized rabbits.Conclusions1. Recombinant Tp0751proteins were efficiently expressed in E.coli with solubleform, showing good immunogenicity and antigenicity.2. The predicted epitope P4and P5may be the potential B-cell epitopes of Tp0751antigen.3. The predicted epitope P4and P5may be the B-cell epitopes of Tp0751antigen.4. The predicted epitope P5, P6and P7may not be the B-cell epitopes of Tp0751antigen presented by rabbit MHC.