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

Expression and characterization of recombinant Fab fragments against double-stranded DNA

鍾幸芳(Shing-Fang Chung)

指導教授 ： 楊沂淵

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2004年)

摘要

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-dsDNA 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 (Polymerase 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50 kDa的Fab蛋白存在。酵素連結免疫吸附分析法(enzyme-linked immunosorbent 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germline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。

關鍵字

抗雙股去氧核醣核酸抗體 ； 重組Fab片段

並列摘要

Systemic Lupus Erythematosus (SLE) is a clinically diverse autoimmune disease. SLE is characterized serologically by the presence of a wide variety of autoantibodies. Among them, anti-double-stranded DNA antibodies (anti-dsDNA antibodies) are the most common and well defined as the pathogenic autoantibodies of SLE, especially for lupus nephritis. In this study, we amplified the heavy and light chain antibody genes of a mouse hybridoma-secreting IgG nephritogenic antibodies to dsDNA by polymerase chain reaction (PCR), and cloned them into the phagemid vector pComb3H. 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 identified in 8 bacterial clones (T1, T3, T4, T5, T8, T9, T10, and T11) by Western blot using anti-mouse IgG antibody. Enzyme-linked immunosorbent assay (ELISA) found that all the eight Fab expressing clones showed lower dsDNA-binding activities. 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 results indicated the Fc portion of anti-dsDNA antibody may be crucial in the antigen-binding activity.

並列關鍵字

anti-dsDNA antibody ； recombinant Fab fragment

參考文獻

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