GENETIC ORIGIN AND STRUCTURE OF INSULIN ANTIBODIES

胰岛素抗体的遗传起源和结构

• 批准号: 2143407
• 负责人: James W Thomas
• 金额: $ 15.35万
• 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/2143407
• 关键词: NOD mouse; 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.

服用外源激素后会产生抗胰岛素抗体， 自发地出现胰岛素依赖型糖尿病的自身免疫前驱症状 （IDDM）。 这些自身抗体是β细胞的最佳指标之一 人类和小鼠（NOD）糖尿病中的破坏。 在这个项目中，我们 提议表征结构和分子组成（种系 胰岛素自身抗体的基因、体细胞突变等）。 在我们之前的 研究和初步数据，我们使用协议生产抗胰岛素单克隆抗体 诱导 T 细胞依赖性（TD、CFA 胰岛素）和 T 细胞非依赖性（TI、 BALB/c 小鼠中的布鲁氏菌-胰岛素反应。 IgG1 mAb 的分析 TD 响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 CDR3 中串联脯氨酸的存在 VK。 TI免疫的IgG2和IgM抗胰岛素有特点 免疫前库包括种系编码的 V 基因，其中一些 被其他自身抗体使用。 TI 的许多抗胰岛素药物 和TD库使用VK5相关的L链，并且这些L链共享氨基 具有胰岛素激素受体的酸序列基序。 我们建议 通过从 NOD 小鼠中产生 mAb 并使用反向 转录酶聚合酶链式反应（RT/PCR）用于研究病理学 抗胰岛素曲目。 这些数据将描述关系的特征 种系库和胰岛素选择库之间 免疫和自身免疫β细胞破坏。 实验使用 真核表达载体和链重组将评估 种系结构和特定基序（例如 Pro-Pro）对 自身反应性、表位特异性和多反应性。 这些研究 将鉴定胰岛素抗体的结构特征，可用于 修改不良免疫反应并提供新信息 用于诊断和治疗的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。