GENETIC ORIGIN AND STRUCTURE OF INSULIN ANTIBODIES

胰岛素抗体的遗传起源和结构

• 批准号: 3245438
• 负责人: James W Thomas
• 金额: $ 14.95万
• 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/3245438
• 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.

抗胰岛素抗体在给予外源性激素后产生， 在胰岛素依赖型糖尿病的自身免疫前驱症状中自发地 （胰岛素依赖型糖尿病）。 这些自身抗体是β细胞的最佳指标之一， 在人类和小鼠（NOD）糖尿病中的破坏。 本课题 建议表征结构和分子组成（种系 基因、体细胞突变等）胰岛素自身抗体 在我们以前 研究和初步的数据，我们生产的抗胰岛素单克隆抗体使用协议 其诱导T细胞依赖性（TD，CFA胰岛素）和T细胞非依赖性（TI， 布鲁氏菌-胰岛素）应答。 来自的IgG 1 mAb的分析 TD的回应揭示了一些不寻常的功能，包括利用 代表性不足的V基因和在CDR 3中串联脯氨酸的存在， VKs TI免疫产生的IgG 2和IgM抗胰岛素具有特征性 在包括种系编码的V基因的免疫前库中， 其他自身抗体也会使用这些抗体。 许多抗胰岛素来自TI和 和TD库使用VK 5相关的L链，并且这些L链共享氨基 酸序列基序与胰岛素的激素受体。 我们建议 通过从NOD小鼠中产生mAb并使用逆转录病毒来扩展这些观察结果。 采用逆转录-聚合酶链反应（RT/PCR）技术， 抗胰岛素药物 这些数据将描述 在生殖细胞谱系和胰岛素选择的谱系之间 免疫和自身免疫性β细胞破坏。 实验中使用 真核表达载体和链重组将评估 生殖系结构和特定基序的影响（例如，Pro-Pro）上 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， 改变不良免疫反应，并提供新的信息， 病理性自身抗体的遗传学和结构， 自身免疫性糖尿病的干预。