人Ⅱ型胶原的HLA-A~＊0201限制性CTL表位的预测及初步鉴定

Primary identification of HLA-A~＊0201-restricted CTL epitopes from human collagen Ⅱ

目的预测和初步鉴定类风湿关节炎(rheumatoid arthritis,RA)主要自身抗原Ⅱ型胶原(CollagenⅡ,CⅡ)的HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)表位,为基于CⅡ抗原表位的特异性免疫治疗奠定基础。方法选取BIMAS、SYFTEPITHI、IEDB、SVMHC、AntiJen预测工具预测该抗原HLA-A*0201限制性结合肽;人工合成待测表位肽,利用T2细胞株通过直接免疫荧光法测定各肽与HLA-A*0201分子的结合力。利用酶联免疫斑点检测(enzyme-linked immunospotassay,ELISPOT)方法检测候选肽刺激关节滑液单个核细胞(synovial fluid mononuclear cell,SFMC)分泌IFN-γ的能力。结果综合BIMAS、SYFTEPITHI、IEDB、SVMHC、AntiJen预测结果筛选出来可能与HLA-A*0201结合的5条肽。MHC亲和力实验表明,在候选的5条肽中,P1261、P1365及P1399具有与HLA-A*0201分子结合的能力,平均荧光强度分别为:1.35、2.53、1.78。ELISPOT试验结果表明,P1365具有刺激SFMC分泌IFN-γ的能力。结论表位预测结果与初步鉴定结果具有一致性,两者联合应用初步认为P1365是HLA-A*0201限制性CTL表位的可能性最大,为下一步表位鉴定及基于人CⅡ抗原表位的特异性免疫治疗奠定理论基础。

This study is aimed to identify HLA-A＊0201-restricted CTL epitopes from human collagen Ⅱ,which will lay a foundation for specific immunotherapy based on epitope. We predicted HLA-A＊0201 restricted cytotoxic T lymphocyte （CTL） epitope from amino acid sequence of human collagen Ⅱ by methods of BIMAS,SYFTEPITHI,IEDB,SVMHC,and AntiJen molecular modeling. Then the predicted peptides were synthesized with solid phase strategies,purified with reverse phase HPLC,and finally identified with mass spectrometry. Furthermore,T2 cell line was used to determine the peptide binding with HLA-A＊0201 molecule. Finally five candidate peptides were predicted with HLA-A＊0201 peptide binding motif and software,and among them P1261,P1365 and P1399 could bind to HLA-A＊0201 with moderately affinity （fluorescence indexes were 1.35,2.53 and 1.78 respectively）. ELISPOT assay suggested that P1365 was capable of priming human CTL responses. Our results suggest that HLA-A＊0201 epitope predicted result of peptide binding motif and software is partly consistent with that of peptide binding assay. In this study,both epitope prediction and binding assay showed that P1365 may be the HLA-A＊0201-restricted epitope from human collagen Ⅱ.