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

编码 DNA 末端结合蛋白 (Ku) 的 cDNA 的克隆

基本信息

批准号：
01570369
负责人：
MIMORI Tsuneyo
金额：
$ 1.41万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570369/
关键词：
cDNA cloning DNA-terminal binding protein anti-Ku antibody Autoantibody Autoantigen Antigenic epitope Gene polymorphism Overlap syndrome cDNA 融合蛋白 Ku抗原 ELISA RFLP 自己抗体自己免疫疾患

项目摘要

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

PSS-PM重叠综合征患者中的抗Ku自身抗体识别选择性结合dsDNA末端区域的70 kD/80 kD蛋白异二聚体。我已经分离和表征编码Ku自身抗原的cDNA克隆。本研究利用已克隆的Ku抗原表位cDNA进行自身抗原表位的定位、基因多态性的检测以及建立检测抗Ku自身抗体的ELISA方法用抗Ku患者血清克隆了K68（编码80 kD-Ku亚基的全长cDNA）和K68（编码70 kD-Ku亚基的部分cDNA），并测定了它们的氨基酸序列。一个表位定位在70 kD-Ku亚基的C-末端，两个表位定位在80 kD-Ku亚基的C-末端区域。含有抗Ku抗体的患者血清显示与这三个表位的各种反应性，并且它们的反应性似乎与抗Ku抗体相关。 ...更多信息临床表现：从患者和正常人外周血白细胞中提取的基因组DNA用各种限制性内切酶消化，用琼脂糖凝胶分离，转移到尼龙膜上，与放射性标记的编码Ku自身抗原的cDNA杂交。DNA经Hind III酶切后与Ku 80 -6 cDNA杂交，识别出一条2.8kb的多态性条带。这种多态性似乎编码在80 kD-Ku基因组的内含子中。结果表明，抗U_1RNP抗体阳性的SLE患者均出现2.8kb的多态性条带（11/11），而抗U_1RNP抗体阴性的SLE患者（4/9）和正常对照组（7/16）的出现率较低。建立了灵敏的ELISA检测抗Ku自身抗体。用ELISA法检测各种结缔组织病患者血清，发现抗Ku抗体在重叠综合征中检出率最高，而在其他疾病中未检出，与常规免疫扩散法的结果一致，编码自身抗原的cDNA将为研究自身免疫性疾病的病因和发病机制提供有用的探针。少