| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1988: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1987: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Anti-ku autoantibodies in patients with PSS-PM overlap syndrome recognize a 70kD/80kD protein heterodimer which binds to ends of dsDNA. In this project, we isolated and characterized cDNA clones that encode the Ku autoantigen, and intended their clinical application.
In the first year, a human hepatoma cell cDNA library inserted into the EcoRl site of lambda gtll phages were screened with an anti-Ku serum to identify plaques expressing Ku epitopes. Three positive clones (K14, K68 and K71) were isolated and deomnstrated to express fusion proteins recognized by anti-Ku antibodies. In the second year, Okayama-berg cDNA library was screened by using a previously isolated cDNA encoding the partial 80kD sequence (K71) as a probe for colony hybridization. The clone Ku80-6 that contained the longest cDNA insert (3.4kb, ldentical with the larger mRNA from two mRNAs hybridized by K71) was isolated, and its nucleotide sequence was determined by Sanger's dideoxy method. The Ku80-6 cDNA contained a
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single long open reading frame encoding 732 amino acids (Mr=82,713) followed by 1082 bases of 3'-non-coding region and a poly(A) tail. The sequence of the Ku80-6 was compared with previously described sequences using the computer search, but no significant homology was found either in DNA (GenBank) or protein (NBRF) data bank. In Southern blot, the Ku80-6 hybridized with 4-5 DNA bands from human leukocyte DNA digested with various restriction enzymes. It was noted that RFLP was seen in the 3.4kb-HindIII fragment and likely to associate with SLE patients who had anti-ULRNP antibodies. Using purified fusion proteins expressing from K68(70kD) and K71(80kD) clones, we developed ELISA to detect anti-Ku antibodies. When sera from various collagen diseases were screened by this assay, anti-Ku antibodies were mostly detected in patients with overlap syndrome, consisted with the result of a conventional immunodiffusion assay.
Our cDNA encoding the Ku antigenic proteins will be a powerful tool to study not only the structure and function of the Ku antoantigen but also pathogenetic mechanisms of autoimmune diseases. Less
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