摘要
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。
EWS-FLI1 fusion protein expressed in Ewing＇s sarcoma（ES） is a new therapeutical target for ES treatment.The aim of this study is to predict and screen the HLA-A2.1-restricted CTL epitopes of Ewing＇s sarcoma EWS-FLI1 fusion protein,and to primarily identify the predicted HLA-A2.1-restricted CTL epitope candidates.The HLA-A2.1-restricted CTL epitopes of EWS-FLI1 fusion protein were synthetically predicted by BIMAS,SYFPEITHI,Predep and IEDB methods,and then screened by the polynomial and quantitative motif method combined with supermotif prediction method as well.Docking of the screened peptides and HLA-A2.1 was observed by molecular dynamics simulation,while all peptides were synthesized with standard Fmoc strategy.After being stimulated by peptides,the CTL granulysin release test and the test of killing effects of CTL on target cells were performed to validate the immunological effects of screened epitopes.Eight CTL epitopes were predicted,among which 4 peptides were the candidates to be synthesized.The candidates docking with HLA-A2.1 were primarily validated by 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 conformed to match their theoretical values by mass spectrometry（MS）.The granulysin release test and killing effects of CTL on target cells confirmed that all screened peptides were able to induce the cytotoxic effect of T lymphocytes.Moreover,peptide QIQLWQFLL（EWS-FLI1 304） was the most effective epitope of EWS-FLI1. All the results demonstrates that our work efficiently predict,screened,and identified the peptides of EWS-FL11.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2010年第1期10-15,共6页
Immunological Journal
基金
国家自然科学基金资助项目(30760254)
