CAJ | 학술논문

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.
목적 응용표위다태여인류백세포항원(HLA)Ⅰ류분자결합력화해리솔분석건립신형T림파세포표위체외사선방법.방법 채용기우구진산법적SYFPEITHI화BIMAS수거고예측6충이도세포자신항원[포괄곡안산탈최매65(GAD65)、이도소류상관항원2(IA-2)、전이도소원(PPI)、이도특이성포도당6-린산매최화아기상관단백(IGRP)、이도정분양다태(IAPP)、신경효질섬유산성단백(GFAP)]적표위서렬,근거예측적결합력지수화이유수거분석사선병합성15개HLA-A2한제성후선표위다태.채용HLA-A2전기인적T2세포검측후선태여HLA-A2적분자결합력,통과다태/HLA복합물해리솔실험분석복합물적은정성.채용단인소방차분석진행수거통계.결과 T2세포태결합력분석현시,15개후선표위다태중,IGRP152～160、IGRP215～223、IGRP228-236、PPI2～10、이도소B10～18、IA-2172～180、GFAP143～151여HLA-A2적분자결합력＞80%.태/HLA복합물해리솔분석현시,상술결합력교강적7개표위다태중,이도소B10-18、IGRP228～236、GFAP143～151、IA-2 172～180 4 h해리솔저우20%.결론 본실험건립적후선다태결합력여해리솔상결합적T림파세포표위체외사선방법유조우감소연구중목표태수량,추진1형당뇨병T림파세포진단방법적연구.
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.