Experimental Study Of EWS-FLI1 Fusion Region And The Limited Epitope Of CTL About HLA-A*0201

Objective:Prediciting, screening, and synthesizing the HLA-A2.1-restricted CTL epitopes derived from Ewing'sarcoma EWS-FLI1 fusion protein, then identifying them by immunological method..Methods:1 Epitopes prediction ,molecular dynamics simulation and synthesis:The HLA-A2.1 restricted CTL epitope of the EWS-FLI1 fusion protein were synthetically predicated by BIMAS, SYFPEITHI, Predep and IEDB methods, and then screened by the polynomial and quantitative motif method. Docking the screened CTL epitopes with HLA-A2.1 were simulated by molecular dynamics,All peptides were synthesized and purified according to standartd Fmoc strategy,then confirmed by RP-HPLC and MS.2 The test of CTL granulysin release stimulated by epitopes:Separated HLA-A2.1 positive PBMCs derived from healthy person peripheral blood. One part PBMCs induced into DCs which followed to be pulsed with Ewings sarcoma cell lysates(TP-DCs). The other part PBMCs were induced into CTL, under the condition of co-incubating with TP-DCs and epitopes successively. The ability of CTL producing granulysin was detected by the ELISA test.3 The test of effect cells killing target cells :Under different effector/target ratio, the effect cells were co-incubated with target cells-- A673. The killing effect was detected by LDH release ELISA test.Results:1. Eight HLA-A2.1 restricted CTL epitopes derived from EWS-FLI1 fusion protein were predicted according to the methods of BIMAS,SYFPEITHI,Predep and IEDB. Four epitoes were further screened under the polynomial and quantitative motif methods.2. The screened epitopes were primarily confirmed to be high affinity to HLA-A2.1 by the molecular dynamics simulation. All synthesized peptides were detected beyond 98 % in purity by reverse phase high performance liquid chromatography (RP-HPLC) and the values of molecular weight of them were measured to conform to their theoretical values by mass spectrometry(MS).3. The granulysin release test confirmed that all screened peptides were able to induce the cytotoxic effect of T lymphocytes. Especially, the peptide of QIQLWQFLL(EWS-FLI1 304) was the most effective.4. The test of LDH release confirmed all screened peptides were able to produce killing effect. The peptide of QIQLWQFLL(EWS-FLI1 304) had stronger effect than others, and the killing rate was increasingly related with the effector / target ratio.Conclusion1. Synthetical methods can elevate the efficiency in predicting the HLA-A2.1 restricted CTL epitopes of EWS-FLI1 fusion protein. Molecular dynamics simulation can primarily confirm the epitopes affinities to HLA-A2.1.2. The tests of CTL granulysin release and killing effect primarily confirm the peptide of QIQLWQFLL(EWS-FLI1 304) is the HLA-A2.1-restricted CTL epitopes of EWS-FLI1.