Molecular Cloning and Partial Characterization of a New Autoimmune Thyroid Disease-Related Antigen*

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Abstract
To clone and characterize antigens to autoantibodies in Hashimoto''s thyroiditis we constructed a cDNA library in the expression vector .lambda.gt11 using mRNA prepared from Graves'' thyroid tissue. This library was screened using serum from a patient with Hashimoto''s thyroiditis which had an antimicrosomal antibody titer greater than 1:106. Five positive recombinants were identified and cloned. Of these, 3 reacted with 7 of 17 normal serum samples. The 2 other clones (IL-28 and IL-33) reactd with none of the 17 normal serum samples. IL-28 reacted with 4 of 15 and IL-33 with 2 of 15 Hashimoto''s thyroiditis serum samples (antimicrosomal antibody titers, > 1:6400). The specificity of the interaction between the Hashimoto''s thyroiditis samples and the fusion protein was demonstrated by Western blot analysis. In addition, neither 10-6 M human thyroglobulin nor 100 mU/ml bovine TSH inhibited binding of the serum samples to these 2 clones. Lysate from clones IL-28 and IL-33 did not reduce the antimicrosomal antibody titer in a hemagglutination assay. Absorption of Hashimoto''s thyroiditis serum with purified thyroid microsomes reduced the serum antimicrosomal antibody titer, but not binding to these 2 clones. The cDNA inserts of clones IL-28 and IL-33 were approximately 0.6 and 0.4 kilobases (kb), respectively. The 0.6-kb IL-28 insert was used to probe human thyroid and human liver poly(A)+ mRNA. A single band of 3.3 kb was evident only with the thyroid mRNA. The IL-28 insert was subcloned into M13 and sequenced in both directions by the dideoxy technique and found to be 572 basepairs in length. When tested against the GenBank and Dayhoff gene banks, no significant homology with any known sequence was determined. In summary, 1) a cDNA fragment of a previously unrecognized gene coding for an autoimmune thyroid disease-related antigen has been cloned and partly characterized; and 2) the protein produced by this clone is not thyroglobulin, the thyroid microsomal antigen, or the TSH-binding site of the TSH receptor. We have, therefore, identified a new autoimmune thyroid disease-related antigen, the pathogenetic significance of which remains to be determined.