| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
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
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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
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