GENETIC ORIGIN AND STRUCTURE OF INSULIN ANTIBODIES

胰岛素抗体的遗传起源和结构

• 批准号: 3245439
• 负责人: James W Thomas
• 金额: $ 14.52万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 1997-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3245439
• 关键词: antibody formation; antibody specificity; autoantibody; cross immunity; gene expression; hybridomas; immunoglobulin genes; insulin; insulin dependent diabetes mellitus; insulin receptor; laboratory mouse; molecular genetics; monoclonal antibody; polymerase chain reaction; protein structure function; site directed mutagenesis; transfection /expression vector

Anti-insulin antibodies arise after administration of exogenous hormone and spontaneously in the autoimmune prodrome of insulin dependent diabetes (IDDM). These autoantibodies are one of the best indicators of beta cell destruction in both human and murine (NOD) diabetes. In this project, we propose to characterize the structure and molecular composition (germline genes, somatic mutation, etc.) of insulin autoantibodies. In our previous studies and preliminary data, we produced anti-insulin mAb using protocols that induce T cell dependent (TD, CFA insulin) and T cell independent (TI, Brucella-insulin) responses in BALB/c mice. Analysis of the IgG1 mAb from TD responses revels some unusual features that include utilization of underrepresented V genes and the presence of tandem prolines in CDR3 of VKs. IgG2 and IgM anti-insulins from TI immunization have characteristics of the preimmune repertoire that include germline encoded V genes, some of which are used by other autoantibodies. Many anti-insulins from both TI and TD repertoires use VK5 related L chains, and these L chains share amino acid sequence motifs with the hormone receptor for insulin. We propose to extend these observations by producing mAb from NOD mice and using reverse transcriptase-polymerase chain reaction (RT/PCR) to study the pathological anti-insulin repertoire. These data will characterize the relationship between the germline repertoire and the repertoires selected by insulin immunization and autoimmune beta cell destruction. Experiments using eukaryotic expression vectors and chain recombination will assess the effect of germline structures and specific motifs (e.g., Pro-Pro) on autoreactivity, epitope specificity, and polyreactivity. These studies will identify structural features of insulin antibodies that may be used to modify adverse immunological reactions and provide new information on genetics and structure of pathological autoantibodies for diagnosis and intervention in autoimmune diabetes.

注射外源性激素后会产生抗胰岛素抗体 胰岛素依赖型糖尿病自身免疫前驱症状中的自发性 (IDDM)。这些自身抗体是β细胞的最佳指标之一。 对人类和小鼠(NOD)糖尿病都有破坏性。在这个项目中，我们 建议表征结构和分子组成(生殖系 基因、体细胞突变等)胰岛素自身抗体。在我们之前的 研究和初步数据，我们使用方案生产了抗胰岛素单抗 诱导T细胞依赖(TD，CFA胰岛素)和T细胞非依赖(TI， 布鲁氏菌-胰岛素)对BALB/c小鼠的反应。猪瘟病毒IgG1mAb的分析 TD响应提供了一些不同寻常的功能，包括利用 猪瘟病毒CDR3中V基因表达不足和串联蛋白的存在 VKS。TI免疫产生的IgG2和IgM抗胰岛素具有一定的特点 在包括生殖系编码的V基因的免疫前谱系中，一些 被其他自身抗体所使用。来自两个TI的许多抗胰岛素药物 TD曲目使用VK5相关的L链，这些L链共享氨基酸 与胰岛素的激素受体具有酸序列基序。我们建议 通过从NOD小鼠生产mAb并使用反向 转录酶-聚合酶链式反应(RT/PCR)用于病理研究 抗胰岛素曲目。这些数据将描述这种关系的特征 在生殖系谱系和胰岛素选择的谱系之间 免疫和自身免疫的β细胞破坏。使用以下工具进行实验 真核表达载体和链重组将评估 生殖系结构和特定基序(如Pro-Pro)对 自身反应性、表位特异性和多反应性。这些研究 将确定胰岛素抗体的结构特征，这些抗体可用于 修改不良免疫反应并提供新的信息 诊断和诊断病理性自身抗体的遗传学和结构 对自身免疫性糖尿病的干预。