CT抗原KM-HN-1 HLA-A * 0201限制性表位预测及其与HLA-A * 0201结合强度分析

目的通过对CT抗原（cancer-testis antigen）KM-HN-1进行HLA-A＊0201限制性表位预测，并对候选表位肽与HLA-A＊0201分子结合亲和力及复合物稳定性进行分析，为探索基于KM-HN-1的免疫治疗奠定基础。方法利用基于蛋白酶体剪切位点特异性的算法PAProc及基于肽MHC-I结合的算法BIMAS和SYFPEITHI对KM-HN-1进行HLA-A＊0201限制性表位预测．合成KM-HN-1相关候选表位肽KM-HN-I321-329（KLLPFRETV），KM-HN-I303-211，（FLPTAPPNV），KM-HN-I629-637。（TLLQIIETV），KM-HN-I87-95（ILNKSIIEV），KM-HN-I538-596。（QMMEALDQL）及阳性对照肽HBVcAg18-27（FLPSDFFPSV）；对这些合成肽与HIA-A＊0201分子结合亲和力及其复合物稳定性根据文献报道的方法进行分析。结果KM-HN-I321-329（KLLPERETV）结合亲和力最低，KM-HN—I203-211（FLPTAPPNV）结合亲和力最高，其余3条肽结合亲和力介于2者之间；稳定性实验（DC50）结果显示：KM-HN-I538—546（QMMEALDQL）DC50小于2h，KM—HN-I321-329（KLLPERETV）的DC50介于2～4h之间，KM-HN-I87-95。（ILNKSIIEV）的DC50介于6～8h之间，KM-HN-I233-211（HLPTAPPNV）及KM-HN-I629—633（TLLQIIETV）的DC50均大于8h。结论基于蛋白酶体剪切位点特异性的算法及基于肽MHC-I结合的算法对KM-HN-1进行HLA-A＊0201限制性表位预测，结合候选表位肽与HLA-A＊0201分子结合的亲和力与复合物稳定性实验分析，为该抗原HLA-A＊0201限制性表位的鉴定奠定了基础。

Prediction of HLA-A * 0201 restricted epitopes from a cancer-testis antigen KM-HN-1 and peptide HLA-A* 0201 binding & complex stability assay of the candidate peptides

Objective To predict HLA-A*0201 restricted epitopes from cancer-testis (CT) antigen KM-HN-1 and assay the HLA-A*0201 peptide binding & complex stability of the predicted epitopes for developing cancer therapeutic vaccine based on KM-HN-1. Me- thods Proteasomal cleavage specificity based and peptide-MHC-I binding based algorithms were combined to predict HLA-A*0201 restricted epitopes from antigen KM-HN-1. Peptide-MHC-I binding was predicted using 2 algorithms: BIMAS and SYFPEITHI. Proteasomal cleavage specificity was predicted using algorithm PAProc. The peptides were ranked for each algorithm and sorted by a cumulative score. Five candidate epitope peptides KM-HN-1_ 321-329 (KLLPFRETV), KM-HN-1_ 203-211 (FLPTAPPNV), KM-HN-1_ 629-637 (TLLQIIETV), KM-HN-1_ 87-95 (ILNKSIIEV), and KM-HN-1_ 538-546 (QMMEALDQL)] and positive control peptide were synthesized. Peptide HLA-A*0201 binding and complex stability was assayed following previously described methods. Results KM-HN-1_ 321-329 (KLLPFRETV) showed the lowest HLA-A*0201 binding affinity among the 5 candidates, while KM-HN-1_ 203-211 (FLPTAPPNV) was of the highest HLA-A*0201 binding affinity and the rest of the peptides showed intermediate binding affinity. The complex stability assay indicated that the dissociation complex 50 (DC_ 50 ) for KM-HN-1_ 538-546 (QMMEALDQL) was below 2 h, the DC_ 50 for KM-HN-13_ 21-329 (KLLPFRETV) was 2-6 h, the DC_ 50 for KM-HN-1_ 87-95 (ILNKSIIEV) was 6-8 h, and the DC_ 50 for KM-HN-1_ 203-211 (FLPTAPPNV) and KM-HN-1_ 629-637 (TLLQIIETV) were both above 8 h. Conclusion The prediction of HLA-A*0201 restricted epitopes from CT antigen KM-HN-1 and the peptide HLA-A*0201 binding affinity & complex stability assay of the 5 candidate peptides provide experimental data for identification of HLA-A*0201 restricted epitopes from KM-HN-1.