| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術研究所 === 94 === Autoantibodies against double-stranded DNA (anti-dsDNA) have been detected in patients of systemic lupus erythematosus (SLE). These abnormal antibodies may play an important role in lupus nephritis. The monoclonal anti-dsDNA 9D7 has been demonstrated to penetrate into cells, induce the production of inflammatory cytokines (IL-1β, IL-6, IL-8, IL-10, and TNF-α),inhibit IL-2 expression and cross-react with hnRNP A2 (human heterogeneous nuclear ribonucleoproteins A2) and PGK (phosphoglycerate kinase). In addition, recombinant single-chain Fv 9D7 (scFv 9D7) antibody was also established and found to be have specific binding capability to dsDNA. According to previous study, the binding capability increase with the ratio of argine (R) in CDRs. Therefore, we analyzed the structure of the scFv 9D7 by computer and then changed various Rs of VH CDR2 and CDR3 into alanine (A) by site-directed mutagenesis in this study. Seven mutations of CDR2 and CDR3 were set up: R52A, R61A, R103A, R52A-R61A, R61A-R103A, R52A-R103A and R52A-R61A-R103A. By ELISA and competitive ELISA, scFv 9D7 was not only found to bind to dsDNA but also to other antigens including ssDNA, oligonucleotide, and PGK. Among these antigens, oligo-dG12 had the strongest binding affinity to scFv 9D7. The mutations of CDRs in R52A and R61A-R103A significantly reduced antigen binding affinity. Based on these findings, the recombinant scFv Abs expression system may be used as a tool for investigation of antibody-antigen binding sites and the consensus peptide of CDR for SLE treatment.
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