中文摘要：

目的 观察人表皮生长因子受体通路底物8（Eps8）抗原改造表位是否有人白细胞抗原A（HLA-A）＊0201限制性抗肿瘤能力。方法 替换Eps8抗原锚定位点氨基酸获得改造肽,用BIMAS、SYFPEITHI在线数据库及Aotodock 4.2软件预测改造表位与HLA-A＊0201分子的结合力,筛选出稳定结合的改造表位E1-9V（ILDDIEFFV）、E1-1Y9V（YLDDIEFFV）。用经典肽结合力实验检测各改造表位与HLA-A＊0201分子的亲和力。制备天然表位（E1）、E1-9V、E1-1Y9V特异性杀伤性T淋巴细胞（CTLs）。用乳酸脱氢酶（LDH）释放试验检测并比较E1-9V、E1-1Y9V特异性CTLs对MCF-7细胞、沉默Eps8的MCF-7细胞（MCF-7/shRNA）和抗HLA-A2抗体预孵育的MCF-7细胞（MCF-7/A2Ab）的杀伤率,E1-9V、E1-1Y9V特异性CTLs对SW480、U251、K562、IM-9细胞的杀伤率,E1、E1-9V、E1-1Y9V特异性CTLs对T2细胞的杀伤率。结果 E1-9V、E1-1Y9V均可与HLA-A＊0201分子B、F对接口袋的氨基酸形成稳定氢键。肽结合力实验结果显示E1-9V、E1-1Y9V与HLA-A＊0201均具有高亲和力,且高于天然表位E1,P均〈0.05。E1-9V、E1-1Y9V特异性CTLs对MCF-7/shRNA、MCF-7/A2Ab细胞杀伤率明显低于MCF-7细胞,P均〈0.05。E1-9V、E1-1Y9V特异性CTLs对SW480、U251细胞杀伤率明显高于K562、IM-9细胞（P均〈0.05）。E1-1Y9V特异性CTLs对T2细胞的杀伤率显著高于E1、E1-9V特异性CTLs,P均〈0.05。结论 Eps8抗原改造表位E1-9V、E1-1Y9V与天然表位E1相比具有更高的HLA-A＊0201分子亲和力,保留了原有的免疫原性,并且E1-1Y9V抗肿瘤免疫效应强于天然表位E1。

英文摘要：

Objective To observe whether modified epitopes from the human epidermal growth factor receptor pathway substrate 8 （Eps8）antigen have leucocyte antigen （HLA -A）＊0201 restrictive anti-tumor ability.Methods We obtained the modified peptides from Eps8 containing HLA-A＊0201-binding anchor motifs by replacing anchor residues,then we se-lected stable binding affinity peptides E1-9V （ILDDIEFFV）and E1-1Y9V（YLDDIEFFV）by using different computer al-gorithms such as BIMAS,SYFPEITHI database and Aotodock 4.2 software to predict the modified peptides and their bind-ing affinity to HLA-A＊0201,respectively.T2 binding assay were performed to verify modified epitopes＇ binding affinity to HLA-A＊0201.Cytotoxic T lymphocytes （CTLs）were induced from peripheral blood mononuclear cells （PBMCs）of HLA-A＊0201 positive healthy donors stimulated by modified epitopes E1-9V and E1-1Y9V.The kill rates on SW480, U251,K562,IM-9 and T2 cells were compared between E1-9V and E1-1Y9V peptide-specific CTLs,and meanwhile,the kill rates on T2 cells were compared among E1,E1-9V and E1-1Y9VE1-9V peptide-specific CTLs.Results Both E1-9V and E1-1Y9V formed stable hydrogen bonds with amino acids of HLA-A＊0201 molecular B,F docking pockets.T2 binding assay showed that E1-9V and E1-1Y9V showed significantly higher affinity for HLA-A＊0201 than the native peptide E1 （all P 〈0.05）.Modified peptide-specific CTLs of E1-9V and E1-1Y9V had lower kill rates on MCF-7 /shRNA and MCF-7 /A2Ab cells than that of MCF-7 cells （all P 〈0.05）.In addition ,the CTLs expressed higher cytotoxicity against tumor cells SW480 and U251 than that of K562 and IM-9 （all P 〈0.05）.E1-1Y9V peptide-specific CTLs showed higher cytotox-icity against tumor cells T2 than E1 and E1-9V peptide-specific CTLs （all P 〈0.05）.Conclusion Compared with native peptide E1,modified epitopes E1-9V and E1-1Y9V have higher binding affinity with HLA-A＊0201 and retain immunoge-necity.In addition,the anti-tumor immunity effect of modified epitope E