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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。
Objective To predict and preliminarily identify HLA-A * 0201-restricted cytotoxic T lymphocytes (CTL) epitopes that are the major autoantigen of rheumatoid arthritis (RA) Based on C Ⅱ antigen epitope-based immunotherapy lay the foundation. Methods BIMAS, SYFTEPITHI, IEDB, SVMHC and AntiJen prediction tools were used to predict the HLA-A * 0201-restricted binding peptide of this antigen. The epitope peptide was synthesized and tested by direct immunofluorescence with T2 cell line. * 0201 molecular binding. Candidate peptides were tested for their ability to stimulate the secretion of IFN-γ by synovial fluid mononuclear cells (SFMCs) using enzyme-linked immunospotassay (ELISPOT). Results Based on the predictions of BIMAS, SYFTEPITHI, IEDB, SVMHC and AntiJen, five peptides that might bind to HLA-A * 0201 were screened out. MHC affinity experiments showed that among the five candidate peptides, P1261, P1365 and P1399 had the ability of binding to HLA-A * 0201 molecules with average fluorescence intensities of 1.35, 2.53 and 1.78, respectively. ELISPOT test results showed that P1365 has the ability to stimulate SFMC to secrete IFN-γ. Conclusions The results of epitope prediction are consistent with the results of preliminary identification. The combination of the two suggests that P1365 is the most probable HLA-A * 0201-restricted CTL epitope and is the most promising candidate for epitope identification and epitope-based human CII epitope Specific immunotherapy laid the theoretical foundation.