Clinical analysis of islet antigen eluted from type 1 diabetes-susceptible MHC molecule

1型糖尿病易感MHC分子洗脱胰岛抗原的临床分析

• 批准号: 12671130
• 负责人: NAKANISHI Koji
• 金额: $ 1.86万
• 依托单位: Okinaka Memorial Institute for Medical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671130/
• 关键词: Type 1 diabetes; MHC molecule; antigenic peptide

The aim of this study is to identify the peptide which derived from pancreatic islet and presented by type 1 diabetes-susceptible MHC class II molecule. B lymphobiastoid cell line was generated from peripheral blood of type 1 diabetic patient and cultured up to 10^<10> cells. After pulsing the debris of fetal islet cell line, HLA-DR and DQ molecule was recovered by affinity chromatography, treated by acid and subsequently applied to reverse phase HPLC. From the HPLC peaks specific for pulsing the debris of fetal islet cell line, I identified a peptide which consists of 14 amino acids. Homology search of this peptide identified Heparan sulfate/Heparin Interacting Protein as the native protein. The cDNA of this protein was cloned by RT-PCR from fetal islet cell line and the protein was expressed in E coil. Western blotting was performed using this bacterially expressed protein. However, the autoantigen against this protein was not detected by this westemblottig. The possible reasons of this are 1) T cell recognition of this peptide and production of autoantibody is far aparted events, 2) This peptide is T cell epitope exclusively for cell-mediated autoimmunity 3) Conformational epitope, not linear epitope, is recognized by autoantibody. Thus, the T cell response to this peptide or protein and immunoprecipitation for autoantibody detection will be needed in the future study.

本研究的目的是鉴定1型糖尿病易感的MHC Ⅱ类分子所提呈的胰岛源肽。从1型糖尿病患者的外周血产生B淋巴母细胞样细胞系，并培养至10 μ <10>g细胞。将人胎胰岛细胞破碎物脉冲处理后，亲和层析回收HLA-DR和DQ分子，酸处理后用于反相HPLC。从对胎儿胰岛细胞系的碎片进行脉冲的特异性HPLC峰，我鉴定了由14个氨基酸组成的肽。该肽的同源性搜索鉴定出硫酸乙酰肝素/肝素相互作用蛋白为天然蛋白。用RT-PCR方法从人胎胰岛细胞系中克隆了该蛋白的cDNA，并在大肠杆菌中表达。使用该细菌表达的蛋白进行Western印迹。然而，针对该蛋白的自身抗原未被该westemblottig检测到。其原因可能是：1）T细胞对该肽的识别与自身抗体的产生是两个相隔甚远的事件; 2）该肽是细胞介导的自身免疫的T细胞表位; 3）自身抗体识别的是构象表位，而不是线性表位。因此，T细胞对该肽或蛋白的反应和免疫沉淀检测自身抗体将在未来的研究中需要。