Prediction And Identification Of The Antigen Epitopes Of Treponema Pallidum Memberane Protein TprF

ObjectiveTo screen and identify the B-cell, Th-cell and combined Th-B-cell antigen epitopes ofTreponema pallidum (Tp) membrane protein TprF, providing experimental foundationfor further development of syphilis multivalent epitope vaccines.Methods1. Predition of cell epitopes of TprF The TprF sequences were obtained inGenbank. The signal peptide and transmembrane domain of TprF were predictedby SignalP4.0and TMHMM2.0. MHC-II restrictive Th cell epitopes of outermembrane protein of TprF were predicted by IEDB, HLAPrep and SYFPEITHI.The physical and chemical properties of protein were analyzed by IDEB andMobyle. The B-cell epitopes candidates were predicted basing on analysis ofhydrophilicity, accessibility, flexibility and antigenicity. The epitopes in overlapareas were regarded as B-Th cell combined epitopes.2. Synthetize and identification of epitopes of TprF The predicted epitope weresynthesized by LifeTein LLC in Beijing and purified by reverse-phase highperformance liquid chromatography (RP-HPLC). MS was used to identify itsnature.3. Expression and identification of antigenicity of recombinant TprF21-284aaThe recombinant prokaryotic expression vector pET30a(+)/TprF21-284aawasconstructed and transformed into expression host strain BL21(DE3) and forinduced expression. The recombinant plasmid was identified by PCR, restrictionenzyme digestion and nucleotide sequencing. The products were analyzed andidentified by SDS-PAGE and Western blotting, purified by affinitychromatography. The BCA kit was used to measure protein concentration.4. Identification of immunogenicity of recombinant TprF21-284aaNew Zealand rabbits were immunized with recombinant protein for5times. The titersantibody against TprF21-284aawere tested.10day after last immunity, the rabbitswere killed, and the splenocytes were separated to identify Th cell epitopes.5. Identification of B-cell epitopes of TprF The96well microtiter plates werecoated with synthetic B/B-Th cell epitopes or recombinant protein TprF21-284aa,respectively. Indirect ELISA was performed to identify B/B-Th cell epitopes ofTprF.6. Identification of Th-cell epitopes of TprF: PBS(-), ConA(+), recombinantprotein TprF21-284aa(+) and synthetic T/Th-B cell epitopes were used to stimulaterabbit splenocytes, respectively. CCK-8assay was used to measure theproliferation level of splenocytes.Results1. General analysis showed that the candidate B-cell epitopes of TprF may be B1(43-62AA), B2(89-103AA), B3(231-251AA), the candidate Th-cell epitopes ofTprF may be T1(81-88AA), T2(132-146AA), T3(183-198AA) and candidatecombined Th-B cell epitopes may be L1(57-71AA), L2(125-138AA),L3(268-279AA).2. The synthesized peptide was beyond90%in purity and the measure value ofmolecular weight was consistent with the theoretical value.3. The insert in the plasmid were proven to be the target gene by PCR, restrictionenzyme digestion and nucleotide sequencing SDS-PAGE showed that the geneexpression product is in solubility with approximate36-kDa molecular weight.Western blot indicated positive specific reaction of the combined protein withanti-His antibody, syphilis sera from patients or immunized rabbits. ELISArevealed that TprF21-284aaantibody titer is above1:12000.4. ELISA showed that the predicted epitope B1, T1had a positive reaction to thesera from immunized rabbits or syphilis patients but a negative reaction to thesera from health animals (human or rabbit) or leptospirosis patients.5. No obvious proliferation of rabbit splenocytes stimulated by the predicted epitope were observed by CCK-8assay.Conclusions1. Recombinant TprF21-284aaproteins were efficiently expressed in E.coli withsoluble form, showing good immunogenicity and antigenicity.2. The predicted epitope B1, T1may be the B-cell epitope of TprF.3. The predicted epitope T1, T2, T3, L1, L2, L3may not be TprF Th-cell epitopepresentated by rabbit MHC molecules.