| Summary: | 碩士 === 台北醫學院 === 細胞及分子生物研究所 === 88 === Abstract
Mutagenesis and Characterization of Rheumatoid Factor in Human Heavy Chain Complementarity-Determining Region 3 (CDR3)
Tsu-Po Yu
Rheumatoid arthritis (RA) is an extravascular immune complex disease with chronic joint inflammation of unknown etiology. Many of patients with this autoimmune disorder have a characteristic marker, termed rheumatoid factor (RF), in their sera. RF is an autoantibody binds to the Fc region of IgG may cause inflammation and tissue damage in the rheumatoid synovium. To further study the role of RF CDR3 on binding affinity, we used semisynthetic combinatorial antibody library technique to randomize the heavy chain CDR3 (HCDR3) region by using overlap extension PCR of RF L1. A semisynthetic combinatorial antibody library was constructed and contained about 1.16×104 mutants in size. After 4th panning against human IgG Fc portion, the results on 15 randomly chosen clones showed that the clones of correct size increased from 80% to 100%. Otherwise, restriction enzyme analysis of 6 of 15 final panning clones revealed they had heavy chain inserts of right size. Western blotting and ELISA analysis of 6 randomly selected clones after IPTG induction suggested they are Fabs and 2 of them, Y#1 and Y#9 have better binding capacity to Fc. Moreover, Y#1 was RF L1 and clone Y#11 is equal to MT#9 after analysis of amino acid sequences. Viewed as a whole, clone Y#9 is one selected mutant with high specificity and binding capacity against human IgG Fc portion. Besides, 3 clones of the mutants, Y#9, Y#11 and MT#9 have the same amino acid, arginine, at the initial 2nd residue of the H-CDR3, revealed that it probably plays an important role.
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