Research Project
3488332
We propose to develop a methodology by which the genes coding for autoimmune disease-specific protein antigens can be rapidly cloned using recombinant DNA techniques. The system which we are using lends itself to high level expression of these antigens as hybrid proteins in E. coli. This facilitates production of these antigens for generating antibodies or developing immunodiagnostic tests. In Phase I a cDNA library will be constructed from human thyroid tissue. Cloning will be into the lambda gt11 system. Clones carrying Graves' specific sequences will be identified by differential immuno-screening with sera from Graves' patients and sera from normal individuals. Graves' positive clones can be induced with the lambda gt11 system, leading to expression of substantial amounts of the Graves' antigen fused to beta-galactosidase (hybrid protein). Because of their large size, they can subsequently be purified easily. In Phase II the cloned Graves' antigens will be characterized. They will be tested extensively for the ability to bind specifically with autoantibodies present in the sera from Graves' patients. Cloned antigens determined to be Graves' specific will be used to develop diagnostic tests directed for use in clinical diagnostic laboratories.
