ANTIGENIC DETERMINANTS OF THE KU-ANTIGEN

本提案的目标是建立氨基酸 Ku抗原抗原决定簇的序列分析 与自身免疫性疾病有关。Ku抗原已经被 其特征是一种DNA结合蛋白复合体 多肽，86/70kD。它被患者的血清所识别 患有硬皮病和系统性红斑狼疮。这个 提议的实验的先决条件是：1)单抗 已研制出针对该抗原的抗体；2)该蛋白质 已纯化并鉴定了复合体，3)克隆了 该抗原的表达表位已被克隆并部分 已排序。识别的表位的数目 将确定单抗并将其映射到 蛋白质复合体。这将为分析 结合人类自身抗体的特异性。患者血清 将对患有自身免疫性疾病的人进行抗体筛查 用纯化的抗原在酶联免疫吸附试验、免疫印迹和 放射免疫分析。自身免疫识别的表位 血清将在竞争实验中与 单克隆抗体。这些表位将被映射到 利用重组DNA技术和单克隆化抗原 抗体。表位的氨基酸序列如下 来源于其片段的cDNA克隆和亚库 在表达载体中构建。或者，抗原性 通过蛋白质降解抗原产生的多肽将 进行纯化和测序。这些实验将使我们能够 调查表位之间可能的相关性 波谱和临床参数在发展过程中 自身免疫性疾病。已建立的氨基酸序列 可用于开发潜在的合成肽 诊断探头。

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.