烟曲霉抗原Asp f16HLA-A*0201限制性的CD8+CTL抗原表位生物信息学预测与实验室鉴定

目的 预测与鉴定烟曲霉抗原Asp f16的HLA-A *0201限制性CD8+细胞毒性T细胞(CTL)抗原表位.方法 以国人常见的HLA-A*0201位点为靶点,依据生物信息学软件扫描烟曲霉特异性抗原Asp f16的全部427个氨基酸序列.使用HLA-A *0201转基因小鼠制备骨髓来源的树突状细胞(DC)和CTL.流式细胞仪技术检测DC表面MHC Ⅱ类抗原,CD80,CD86和CD11c的表达来验证其是否成熟.ELISPOT试验检测烟曲霉抗原多肽特异性CTL产生的细胞因子IFN-γ.四聚体(Tetramer)试验证实烟曲霉特异性CTL与抗原肽,HLA-A*0201分子复合体的亲和性.结果 根据与MHC I类分子结合的半衰期评分,选择了3个HLA-A*0201限制性抗原表位.流式细胞仪分析示成熟DC高表达HLA Ⅱ类抗原,CD80,CD86和CD11c.Tetramer试验证实烟曲霉特异性T细胞受体与抗原肽,HLA-A*0201分子复合体的高亲和性.ELISPOT实验结果 表明烟曲霉抗原肽体外可以活化CD8+CTL,被负载了抗原肽的DC刺激活化后可以产生IFN-γ.结论 本研究成功鉴定烟曲霉抗原Asp f16的HLA-A*0201限制性CD8+CTL表位,可作为疫苗设计的候选表位,为进一步研发新型抗烟曲霉疫苗提供参考.

Prediction and identification of new HLA-A * 0201-restricted CD8+ CTL epitopes from Aspergillus fumigatus antigen Asp f16

Objective To predict and identify potential HLA-A＊0201-restricted CD8＋ cytotoxic T lymphocyte（CTL） epitopes in Aspergillus fumigatus antigen Asp f16.Methods A total of 427aa Asp f16 amino acid sequences were scanned using HLA peptide binding prediction software on the target of HLA-A＊0201.Bone-marrow-derived dendritic cells（DC） and peptide specific CTLs were prepared in HLA A＊0201 transgenic mice.Expressions of MHC class II antigen,CD80,CD86,and CD11c were analyzed by flow cytometry.IFN-γ produced by Asp f16 peptides specific CTLs was detected by ELISPOT assay.Tetramer assays were performed to investigate the affinity of the CD8＋ CTLs to the Asp f16 peptides complexed HLA-A＊0201.Results Three potential HLA-A＊0201 binding 9-mer peptides were selected based on their estimated dissociation half-life from MHC class I molecular.DCs highly expressed MHC class II antigen,CD80,CD86 and CD11c.Strong affinity of the TCR to the peptide/MHC complex was demonstrated by tetramer staining.Asp f16 peptides specific CD8＋ CTL actively produced IFN-γ after exposure to peptide-loaded DCs.Conclusions Three HLA-A＊0201-restricted CD8＋ CTL epitopes of Asp f16 successfully identified might be candidates for anti-Apergillus fumigatus vaccine designing.