隨機改變重鏈

隨機改變重鏈 CDR3 片段的類風濕性因子之突變與特徵分析

Mutagenesis and Characterization of Rheumatoid Factor in Human

Heavy Chain Complementarity-Determining Region 3 (CDR3)

隨機改變重鏈 CDR3 片段的類風濕性因子之突變與特徵分析 于思泊

類風濕性關節炎 (rheumatoid arthritis, RA) 為一種血管外的免疫複合性疾病，引 發慢性關節發炎，其致病機轉尚不甚清楚。大部分罹患此種自體免疫疾病的病 人，在他們的血清中發現一種特有的標記，稱之為類風濕性因子 (Rheumatoid factors; RFs)。類風濕性因子為一種自體抗體，能與 IgG 的 Fc 區域結合，造成 類風濕性患者關節滑膜的發炎及組織損傷。為了進一步研究類風濕性因子 CDR3 於抗體結合親和力上所扮演的角色，我們使用半合成聯合抗體基因組庫技術，以 overlap extension PCR 來隨機改變類風濕性因子 L1 的重鏈 CDR3 區域。本實 驗中，我們建構了一個含有 1.16×104 個突變體的半合成聯合抗體組庫，經過四 次針對於人類 IgG Fc 部分的篩選過程後，結果顯示了隨機所挑選的 15 個菌 株，其大小正確的比例由 80% 增加到 100%。除此之外，6 個來自最後一次篩 選完畢的 15 個菌株經限制酵素分析後，發現確有大小正確之重鏈片段存在。西 方點墨法及 ELISA 分析經 IPTG 誘導蛋白產生後的 6 個隨機選取菌株，顯示 它們的確為抗體 Fabs，而且其中有 2 個， Y#1 與 Y#9，對 Fc 有較佳的結合 能力。更進一步經胺基酸序列分析後發現，此 2 個具專一性的菌株中，Y#1 同 於類風濕性因子 L1，而 Y#11 則與 MT#9 完全相同。總觀以上可知，菌株 Y#9 為一經篩選出對於人類 IgG Fc 部分有高專一性與結合能力的突變體。此外，這 些突變體中的 3 個菌株；Y#9，Y#1 及 MT#9；其 H-CDR3 之起始第二個胺基 酸皆為 arginine，顯示其可能有某種重要性存在。 英文摘要

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.