TRAG-3 HLA-A2.1限制性CTL表位的预测及结合力分析

Prediction and peptide binding assay of HLA-A2.1-restricted CT L epitope from TRAG-3

目的 寻找并鉴定TRAG 3来源的HLA A2 .1限制性CTL表位 ,为临床开展基于TRAG 3表位的特异性免疫治疗奠定基础。方法 应用超基序和量化基序方案预测TRAG 3HLA A2 .1限制性CTL表位 ;采用标准Fmoc方案合成侯选表位 ,RP HPLC纯化、分析多肽 ,质谱鉴定各肽 ;最后 ,用T2细胞株测定各肽与HLA A2 .1分子的结合力。结果 超基序和量化基序方案预测出了 4个侯选表位肽 ;标准Fmoc方案合成的各肽经纯化后纯度大于 90 % ,各肽的相对分子质量与理论值一致 ;在 4个侯选表位肽中 ,ILLRDAGLV( 58- 6 6 ) 九肽与HLA A2 .1的结合力最强。结论 表位预测的结果与结合力分析实验结果一致性较好 ,两者联合应用初步认为ILLRDAGLV( 58- 6 6 ) 九肽为TRAG 3HLA A2 .1限制性CTL表位的可能性最大。

ObjectiveThis study was made to isolate HLA A2.1 res tr icted epitopes from a new cancer/testis antigen,taxol resistance associated gen e 3 (TRAG 3), which will contribute much to the application of specific immunot herapy on the basis of TRAG 3 derived epitope.MethodsSupermot if and quantitative motif methods were used in the prediction of HLA A2.1 rest ricted cytotoxic T lymphocyte epitope from TRAG 3 antigen. The peptides were sy nthesized with solid phase strategies, purified with reverse phase HPLC and ide ntified, determined with mass spectrometry. T2 cell line was used to determine t he peptide binding with HLA A2.1 molecule.Results Four candid ate nonameric epitopes were predicted by supermotif combined with quantitative m otif. Four synthetic nonameric peptides were above 90% in purity and the determi ned values of molecular weight conformed to their theoretical values. Among four predicted epitopes, ILLRDAGLV (58-66) was determined as the one with the most HLA A2.1 affinity.Conclusion Our results suggest that ep itope prediction with supermotif and quantitative motif accorded with peptide bi nding assay. In this study, both epitope prediction and peptide binding assay sh owed that ILLRDAGLV (58-66) may be the HLA A2.1 restricted epitope from TRAG 3.