STRUCTURAL BASIS FOR AUTOIMMUNE ANTI-DNA SPECIFICITY

自身免疫性抗 DNA 特异性的结构基础

基本信息

批准号：
6032834
负责人：
Marko Z Radic
金额：
--
依托单位：
MCP HAHNEMANN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 1999-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6032834
关键词：
DNA DNA footprinting affinity chromatography antibody specificity antinuclear autoantibody gene expression gene mutation gene rearrangement histones molecular cloning molecular pathology nucleosomes protein structure function site directed mutagenesis small nuclear ribonucleoproteins systemic lupus erythematosus transfection

项目摘要

Antigen selection and affinity maturation of autoantibodies leave a genetic imprint indicative of the type of antigen that is bound in vivo. Therefore, a detailed investigation of genetic mechanisms that generate the immunological diversity of autoantibodies should provide crucial information about the induction of autoimmunity. The principle of antigen selection has been tested with murine anti-DNA antibodies and recent studies suggest that it also applies to a wide range of autoantibodies that arise in human autoimmune disease. This application proposes to analyze by in vitro mutagenesis three autospecificities that are typical of Systemic Lupus Erythematosus: anti- double stranded DNA, anti-histone, and anti-snRNP. In particular, the effects of V gene use, rearrangement, and somatic mutation will be evaluated in three antibodies that have dual specificity and bind either histones or snRNP in addition to DNA. The focus of the studies will be to learn: a) the relative contributions of the different diversity-generating mechanisms, and b) the order in which the three specificities arise. It is hoped that conclusions from these studies will be relevant for the etiology and possible treatment of Systemic Lupus Erythematosus. The experiments described have the following aims: Specific Aim 1: Clone and express anti-DNA V genes. The heavy (H) and light (L) chain V genes used by three hybridomas 3H9, LG8-1, and 1-42 will be cloned and manipulated in vitro, in order to determine the structural basis for dsDNA, nucleosome, and SmD binding. These experiments will involve chain recombination, VHCDR3 grafting, and site-directed mutagenesis. Specific Aim 2: Test antibody specificity for dsDNA conformation. Purified mutant anti-dsDNA antibodies will be used to establish whether DNA curvature improves DNA binding or not. If conformational preference is found, then: a) The size and location of bound DNA sequences will be established using Exonuclease III and ethylation interference footprinting. b) Single chain antibody fragments (scFv) will be constructed to distinguish whether preference is intrinsic to each combining site or is a consequence of IgG structure. Specific Aim 3: Explore antibody specificity for dsDNA sequence. In order to evaluate the potential for DNA sequence specificity, oligonucleotides of initially random composition will be processed through several cycles of affinity enrichment and PCR purification. Affinity chromatography will be on anti-dsDNA antibodies or anti-dsDNA scFv linked to functional domains from the transcriptional factor GCN4 and from protein A. Substrates containing a palindromic core sequence will be used to reduce the lateral displacement of bound antibodies.

自身抗体的抗原选择和亲和力成熟离开遗传烙印，指示体内结合的抗原类型。因此，对产生的遗传机制进行了详细研究自身抗体的免疫学多样性应提供至关重要的有关自身免疫性诱导的信息。抗原原理选择已用鼠抗DNA抗体测试和最近研究表明，它也适用于多种自身抗体这在人类自身免疫性疾病中产生。该应用建议通过体外诱变进行分析三全身性红斑狼疮典型的自动特异性：抗双链DNA，抗抗酮和抗SNRNP。特别是 V基因使用，重排和躯体突变的影响将是以三种具有双重特异性并结合的抗体进行评估除DNA外，组蛋白或SNRNP。研究的重点将是学习：a）不同多样性产生的相对贡献机制，b）三个特异性的顺序。这是希望这些研究的结论将与病因和可能治疗全身性红斑狼疮。这描述的实验具有以下目的：特定目标1：克隆和表达抗DNA V基因。重（h）和光（L）链V基因由三个杂交瘤3H9，LG8-1和1-42使用在体外被克隆和操纵，以确定结构 DSDNA，核小体和SMD结合的基础。这些实验会涉及链条重组，VHCDR3接枝和定向诱变。特定目标2：测试DSDNA构象的抗体特异性。纯化的突变抗DSDNA抗体将用于确定是否是否 DNA曲率是否改善了DNA结合。如果构象偏好是然后发现：a）绑定的DNA序列的大小和位置将是使用核酸外切酶III和乙基化干扰建立足迹。 b）单链抗体片段（SCFV）将是构建是为了区分偏好是否对每个偏好是固有的组合位点或IgG结构的结果。特定目标3：探索dsDNA序列的抗体特异性。为了为了评估DNA序列特异性的潜力，寡核苷酸最初随机组成将通过几个周期处理亲和力富集和PCR纯化。亲和力色谱法会使用与功能相关的抗DSDNA抗体或抗DSDNA SCFV 转录因子GCN4和蛋白质A的域A。含有腔粒核序列的底物将用于减少结合抗体的横向位移。