Cloning of cDNA encoding the DNA-terminal binding protein (Ku)

PSS-PM重叠综合征患者的抗ku自身抗体识别70kD/80kD蛋白异源二聚体，该异源二聚体选择性结合到dsDNA的末端区域。我已经分离并鉴定了编码Ku自身抗原的cDNA克隆。在本项目中，我利用cDNA克隆定位自身表位，检测基因多态性，并开发灵敏的ELISA检测抗ku自身抗体。从抗ku患者血清用限制性内切酶酶切的Ku80-6（编码80kD-Ku亚基的全长cDNA）和K68（编码70kD-Ku亚基的部分cDNA）表位文库中分离到编码ku -表位的cDNA片段，并测定其氨基酸序列。在70kD-Ku亚基的c端定位了一个表位，在80kD-Ku亚基的c端定位了两个表位。含有抗ku抗体的患者血清与这三个表位表现出不同的反应性，并且它们的反应性似乎与临床表现有关。从患者和正常人的外周血白细胞中提取基因组DNA，用各种限制性内切酶消化，琼脂糖凝胶分离，转移到尼龙膜上，与编码Ku自身抗原的放射性标记cDNA杂交。Hind - 1酶切后，与Ku80-6 cDNA杂交，鉴定出2.8kb的多态性条带。这种多态性似乎编码在80kD-Ku基因组的一个内含子中。值得注意的是，2.8kb多态性条带在所有具有抗u1rnp抗体的SLE患者中都被识别（11/11），但在抗u1rnp阴性的SLE患者（4/9）和正常健康对照（7/16）中较少出现。利用编码70kD-(KI4)和80kD-(K71)Ku亚基的部分cdna表达纯化的β -半乳糖苷酶融合蛋白，建立了灵敏的ELISA检测抗Ku自身抗体。ELISA法筛查各种结缔组织疾病患者血清中，抗ku抗体在重叠综合征中检出最多，而在其他疾病中未检出，与常规免疫扩散试验结果一致。编码自身抗原的cdna将成为研究自身免疫性疾病病因和发病机制的有用探针。少

Anti-Ku autoantibodies in patients with PSS-PM overlap syndrome recognize a 70kD/80kD Protein heterodimer which selectively binds to terminal region of dsDNA. I have isolated and characterized cDNA clones that encode the Ku autoantigens. In this project, I utilized the cDNA clones for mapping of auto-epitopes, detection of gene polymorphism, and development of sensitive ELISA to detect anti-Ku autoantibocdies.cDNA fragments encoding Ku-epitopes were isolated from epitope library which were made by restriction enzyme-digested Ku80-6(full-length cDNA encoding the 80kD-Ku subunit)and K68(partial cDNA encoding the 70kD-Ku subunit)using anti-Ku patient sera, and their amino acid sequences were determined. One epitope was mapped on the C-terminus of the 70kD-Ku and two epitopes were mapped on the C-terminal region of the 80kD-Ku subunit. Patient sera containing anti-Ku antibodies revealed various reactivities with these three epitopes, and their reactivities appeared to beassociated with cli … More nical manifestations.Genomic DNA extracted from periferal leukocytes of patients and normal subjects were digested with various restriction enzymes, fractionated with agarose gel, transfered to a nylon membrane and hybridized with radiolabeled cDNA encoding the Ku autoantigens. When DNA was digested with Hind Ill and hybridized with Ku80-6 cDNA, a 2.8kb-polymorphic band was recognized. This Polymorphism appeared to be encoded in an intron of the 80kD-Ku genome. It was noted that the 2.8kb-polymorphic band was recognized in all patients with SLE who had anti-U1RNP antibodies(11/11), but less frequent in antiU1RNP-negative SLE patients(4/9)and normal healthy controls(7/16).Using purified fusion proteins with beta-galactosidase expressed from partial cDNAs encoding the 70kD-(KI4)and 80kD-(K71)Ku subunit, sensitive ELISA was developed to detect anti-Ku autoantibodies. In screening of sera from patients with various connective tissue diseases by ELISA, anti-Ku antibodies were detected in overlap syndrome most frequently, but not in other diseases, consisted with the result of a conventional immunodiffusion assay.The cDNAs encoding autoantigens will be useful probes to study etiologic and pathogenetic mechanisms of autoimmune diseases. Less