Primary biliary cirrhosis (PBC) is a chronic idiopathic autoimmune liver disease characterized by progressive inflammatory obliteration of intrahepatic bile ducts and is now the commonest disease for which liver transplantation is carried out. Although the etiology remains unknown, essentially all patients with this disease have anti-mitochondrial autoantibodies (AMA). We developed a novel expression vector, lambda-foo, which is based on bacteriophage-lambda. This vector is able to express foreign proteins on the surface of phage particles and is suitable for the construction of cDNA libraries. The cDNA library can be screened efficiently by antibodies, DNAs, or enzymes immobilized on solid supports such as a microtiter plate or paramagnetic beads. The system may be powerful for the efficient identification of autoantigens in autoimmune diseases. PBC sera have AMA as major autoantibodies and would be ideal to establish experimental conditions of the lambda-foo system for the isolation of autoantigens.
Our aims i n this proposed project are to isolate cDNAs coding for AMA epitopes and for minor autoantigens that have not been isolated. (1) We will construct cDNA libraries from mRNA of a humanHEp-2 cell line with the lambda-foo vector. (2) The libraries will then be searched for specific antigens to PBC patients' sera. (3) Clones that have not been isolated or identified before will be analyzed by DNA sequencing, Northern and Western blots, and antibody staining of HEp-2 cells. (4) We will also develop an efficient method for the epitope mapping of antigens. Our long-term objective is to isolate autoantigens on the biliary epithelial cell surface in PBC, using the lambda-foo system. The proposed project may provide us with clones which have not been previously isolated or identified. These clones are useful in formulating rapid, sensitive and specific diagnostic tests for the presence of autoantibodies in PBC. The amino-acid sequence of the antigens will allow the mapping of the epitopes recognized by both T and B cells of PBC patients. Furthermore, the clones may provide a clue to an understanding of etiology and/or pathogenesis of PBC. Once the system is adapted to the screening of autoantigens of PBC in this proposed project, the technology will be a sensitive, efficient, and economical alternative to conventional methods for the identification of autoantigens, particularly low abundant molecules, in other autoimmune diseases.
| Niwa, M; Maruyama, H; Fujimoto, T et al. (2000) Affinity selection of cDNA libraries by lambda phage surface display. Gene 256:229-36 |
| Kuwabara, I; Maruyama, H; Mikawa, Y G et al. (1997) Efficient epitope mapping by bacteriophage lambda surface display. Nat Biotechnol 15:74-8 |
