ANTIGENIC DETERMINANTS OF THE KU-ANTIGEN

本提案的目标是建立氨基酸 Ku抗原的抗原决定簇序列 与自身免疫性疾病有关 Ku抗原已经 其特征为两个DNA结合蛋白复合物， 多肽，86/70 kD。 它被患者的血清所识别 硬皮病和系统性红斑狼疮 的 本实验的前提条件是：1）单克隆 已经开发了针对抗原的抗体; 2）蛋白质 复合物已纯化和表征，和3）cDNA克隆 已经克隆了表达抗原的表位， 测序 由抗原识别的表位的数量 单克隆抗体将被确定和映射在 蛋白质复合物 这将为分析 人自身抗体的结合特异性。 患者血清 自身免疫性疾病的患者将进行筛查， 在ELISA中使用纯化的抗原，免疫印迹和 放射免疫测定 自身免疫系统识别的表位 血清将在竞争实验中测定， 克隆抗体 这些表位将被定位在 利用重组DNA技术， 抗体的 表位的氨基酸序列将是 来源于cDNA克隆及其片段的亚文库 在表达载体中构建。 或者，抗原性 由抗原的蛋白水解降解产生的肽将 进行纯化和测序。 这些实验将使我们能够 研究表位之间可能的相关性， 光谱和临床参数在发展过程中， 自身免疫性疾病 已确定的氨基酸序列 可用于开发潜在的合成肽 诊断探针

The goal of the present proposal is to establish the amino acid sequences of the antigenic determinants of the Ku-antigen involved in autoimmune diseases. The Ku-antigen has been characterized as a DNA binding protein complex of two polypeptides, 86/70 kD. It is recognized by sera from patients with scleroderma and systemic lupus erythematosus. The prerequisites for the proposed experiment are: 1) monoclonal antibodies to the antigen have been developed; 2) the protein complex has been purified and characterized, and 3) cDNA clones expressing epitopes of the antigen have been cloned and partially sequenced. The number of the epitopes recognized by the monoclonal antibodies will be determined and mapped on the protein complex. This will provide the basis for analysis of the binding specificities of human autoantibodies. Sera from patients with autoimmune diseases will be screened for antibodies to the antigen using purified antigen in ELISA, immunoblot and radioimmunoassays. The epitopes recognized by the autoimmune sera will be determined in competition experiments with the monoclonal antibodies. These epitopes will be mapped on the antigen by using recombinant DNA technology and the monoclonal antibodies. The amino acid sequences of the epitopes will be derived from the cDNA clones and sublibraries of their fragments constructed in expression vectors. Alternatively, antigenic peptides produced by proteolytic degradation of the antigen will be purified and sequenced. These experiments will allow us to investigate the possible correlations between the epitope spectrum and the clinical parameters during the development of the autoimmune diseases. The established amino acid sequences can be used for developing synthetic peptides as potential diagnostic probes.