胰岛人类白细胞抗原-A2限制性T淋巴细胞表位体外筛选方法的建立

目的 应用表位多肽与人类白细胞抗原(HLA)Ⅰ类分子结合力和解离率分析建立新型T淋巴细胞表位体外筛选方法.方法 采用基于矩阵算法的SYFPEITHI和BIMAS数据库预测6种胰岛细胞自身抗原包括谷氨酸脱羧酶65(GAD65)、胰岛素瘤相关抗原2(IA-2)、前胰岛素原(PPI)、胰岛特异性葡萄糖6-磷酸酶催化亚基相关蛋白(IGRP)、胰岛淀粉样多肽(IAPP)、神经胶质纤维酸性蛋白(GFAP)]的表位序列,根据预测的结合力指数和已有数据分析筛选并合成15个HLA-A2限制性候选表位多肽.采用HLA-A2转基因的T2细胞检测候选肽与HLA-A2的分子结合力,通过多肽/HLA复合物解离率实验分析复合物的稳定性.采用单因素方差分析进行数据统计.结果 T2细胞肽结合力分析显示,15个候选表位多肽中,IGRP152～160、IGRP215～223、IGRP228-236、PPI2～10、胰岛素B10～18、IA-2172～180、GFAP143～151与HLA-A2的分子结合力＞80%.肽/HLA复合物解离率分析显示,上述结合力较强的7个表位多肽中,胰岛素B10-18、IGRP228～236、GFAP143～151、IA-2 172～180 4 h解离率低于20%.结论 本实验建立的候选多肽结合力与解离率相结合的T淋巴细胞表位体外筛选方法有助于减少研究中目标肽数量,推进1型糖尿病T淋巴细胞诊断方法的研究.

Identification of human leukocyte antigen-A2 restricted T cell epitopes for islet cell autoantigens with combined screening method in vitro

Objective To establish a screening method in vitro based on affinity and dissociation assay for candidate peptides of epitopes to mapping T cell epitopes of beta cell autoantigens. Methods Six islet cell autoantigens, including 65-kDa isoform of glutamic acid decarboxylase ( GAD65 ), islet antigen ( IA-2 ), preproinsulin (PPI), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), islet amyloid polypeptide (IAPP) and glial fibrillary acidic protein (GFAP), were analyzed using SYFPEITHI and BIMAS algorithms. Fifteen peptides were identified as human leukocyte antigen( HLA)-A *0201 candidate epitopes for synthesis and screening. The peptide binding assay and temporal stability assay of peptide/HLA-A2 complexes were detected in HLA-A2 transgenic T2 cell One-way analysis of variances was used for data analysis. Results In peptide binding assays for T2 cell, 7 peptides which were IGRP152-160, IGRP215-223, IGRP228-236, PPI2-10, insulin B10-18, IA-2172-180 and GFAP143-151 showed ＞ 80%affinity with HLA-A2 molecule. Dissociation assay showed that 4 hour-dissociation rates of insulin B10-18,IGRP228-236, GFAP143-151 and IA-2172-180 were ＜ 20%. Conclusion This study suggests that affinity evaluation of candidate of epitope peptide with HLA molecule in vitro should consider both binding assay and dissociation assay, which would be helpful to minimize the number of target peptides in experiments and facilitate novel T cell based-diagnosis for type 1 diabetes.