隨機改變重鏈 CDR3 片段的類風濕性因子 之突變與特徵分析
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。
Mutagenesis and Characterization of Rheumatoid Factor in Human Heavy Chain Complementarity-D
etermining Region 3 (CDR3)
Mutagenesis and Characterization of Rheumatoid Factor in Human Heavy Chain Complement arity-Determining Region 3 (CDR3)
Tsu-Po Yu
Rheumatoid arthritis (RA) is an extravascular immune complex disease with chronic joint infl ammation of unknown etiology. Many of patients with this autoimmune disorder have a chara cteristic marker, termed rheumatoid factor (RF), in their sera. RF is an autoantibody binds to t he 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 combinatoria l antibody library technique to randomize the heavy chain CDR3 (HCDR3) region by using ov erlap extension PCR of RF L1. A semisynthetic combinatorial antibody library was constructe d and contained about 1.16×104 mutants in size. After 4th panning against human IgG Fc port ion, 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 rev ealed they had heavy chain inserts of right size. Western blotting and ELISA analysis of 6 ran domly 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 m utant with high specificity and binding capacity against human IgG Fc portion. Besides, 3 clon es 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.
