论文部分内容阅读
目的预测、筛选及合成尤文肉瘤EWS-FLI1蛋白HLA-A2.1限制性细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte,CTL)表位,初步鉴定EWS-FLI1表位肽。方法综合运用BIMAS、SYFPEITHI、Predep和IEDB方案对EWS-FLI1蛋白进行HLA-A~*0201限制性CTL表位的预测,应用多项式方案、量化基序方案对预测的CTL表位进行筛选,并将筛选的CTL表位与HLA-A~*0201分子进行动力学模拟,应用标准Fmoc方案合成CTL表位肽;通过表位多肽刺激CTL释放颗粒溶素的检测,以及靶细胞杀伤实验验证表位多肽的免疫效应。结果综合预测得出了8个可能的表位肽,进一步筛选确定其中4个肽为候选合成表位,应用分子动力模拟初步验证了4个候选表位肽与HLA-A2.1分子的结合力,合成的各条肽经色谱分析纯度均在98%以上,经质谱分析各肽的分子量测定值与理论值相符,颗粒溶素释放实验及靶细胞杀伤实验证实了筛选的表位均能产生刺激效应,其中,表位肽QIQLWQFLL(EWS-FLI1 304)的刺激效应最为显著。结论综合运用多个方案可提高预测效率,分子动力学模拟可初步验证表位肽与HLA-A~*0201的结合力,所合成的多肽为高纯度肽,通过免疫学实验初步鉴定了EWS-FLI1的表位。
Objective To detect, screen and synthesize EWS-FLI1 epitope of HLA-A2.1 cytotoxic T Lymphocyte (CTL) in Ewing’s sarcoma, and to identify EWS-FLI1 epitope peptide. Methods The HLA-A * 0201 restricted CTL epitopes of EWS-FLI1 protein were predicted by BIMAS, SYFPEITHI, Predep and IEDB protocols. The predicted CTL epitopes were screened by polynomial and quantified motifs. Screening CTL epitopes and HLA-A * * 0201 molecular dynamics simulation, the application of standard Fmoc program CTL epitope peptide synthesis; CTL release granule lysin by epitope peptide detection and target cell killing experiments to verify the epitope peptide Immune effect. RESULTS: Eight potential epitope peptides were obtained by comprehensive prediction. Four of the peptides were further screened to be candidate synthetic epitopes. The binding kinetics of the four candidate epitope peptides and HLA-A2.1 molecules were preliminarily verified by molecular dynamics simulation , And the purity of each peptide synthesized was over 98% by chromatographic analysis. The molecular weight of each peptide was confirmed by mass spectrometry. The results of granulysin release and target cell killing confirmed that the selected epitopes could stimulate Effect, where the stimulation effect of the epitope peptide QIQLWQFLL (EWS-FLI1 304) is most pronounced. Conclusions The multiple prediction methods can be used to improve the prediction efficiency. Molecular dynamics simulation can initially verify the binding ability between epitope peptide and HLA-A * 0201. The synthesized peptide is a high-purity peptide. Through immunological experiments, the EWS- The epitope of FLI1.