抗雙股去氧核醣核酸抗體的重組 Fab 片斷之表現及其特性分析
全身性紅斑性狼瘡 (Systemic Lupus Erythematosus ， SLE) 是一種具有各式 各樣臨床症狀的自體免疫疾病，患者最特別的地方在於血清中有各種自體抗 體的產生，其中最常見到的自體抗體是抗雙股去氧核醣核酸抗體 (anti-dsDN A antibodies) ，它也被認為是導致 SLE 最主要的自體抗體，尤其是和狼瘡性 腎炎 (lupus nephritis) 有關。在本實驗中，我們利用聚合酶鏈鎖反應 (Polym erase Chain Reaction ， PCR) 來放大老鼠融合瘤細胞 (hybridoma cell) 所分泌 的腎原性 (nephritogenic) 抗雙股去氧核醣核酸抗體的重鏈 (Heavy chain) 和 輕鏈 (Light chain) 基因片段，我們將反應產物選殖入 pComb3H 載體中，然 後利用限制酵素作用於重組 DNA ，以分析在隨意挑選的菌株中，確認已含有 重鏈及輕鏈基因片段，接著再將它們以大腸桿菌來表現 Fab 片段並分析其免 疫特性。利用西方墨點法 (Western blot) 、抗老鼠 IgG 抗體分析後，結果證 實 8 個能夠表現 Fab 片段的菌株 (T1, T3, T4, T5, T8, T9, T10, 及 T11) 都有 5 0 kDa 的 Fab 蛋白存在。酵素連結免疫吸附分析法 (enzyme-linked immunosorb ent assay, ELISA) 則顯示這 8 個能夠表現 Fab 的菌株與 dsDNA 呈現較低的結 合能力。由於 DNA 序列分析的結果顯示這些菌株的重鏈和輕鏈來自於相同 g ermline 的免疫球蛋白基因，因此我們的實驗結果指出抗雙股去氧核醣核酸抗 體的 Fc 片段對於其抗原結合力具有關鍵性的影響。
Expression and characterization of recombinant Fab fragments against double-stranded DNA
Systemic Lupus Erythematosus (SLE) is a clinically diverse autoimmune disease. S LE is characterized serologically by the presence of a wide variety of autoantibodie s. Among them, anti-double-stranded DNA antibodies (anti-dsDNA antibodies) are the most common and well defined as the pathogenic autoantibodies of SLE, especi ally for lupus nephritis. In this study, we amplified the heavy and light chain antibo dy genes of a mouse hybridoma-secreting IgG nephritogenic antibodies to dsDNA by polymerase chain reaction (PCR), and cloned them into the phagemid vector pC omb3H. DNA restriction analysis revealed that randomly selected clones contained both heavy and light chain inserts. Moreover, the Fab fragments were expressed in Escherichia coli and immunologically characterized. A 50 kDa protein band was id entified in 8 bacterial clones (T1, T3, T4, T5, T8, T9, T10, and T11) by Western bl ot using anti-mouse IgG antibody. Enzyme-linked immunosorbent assay (ELISA) f ound that all the eight Fab expressing clones showed lower dsDNA-binding activiti es. Sequence determination revealed that the heavy and light chain genes of all the analyzed clones are derived from identical germline Ig genes. Taken together, our r esults indicated the Fc portion of anti-dsDNA antibody may be crucial in the antige n-binding activity.
