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研究生:夏可強
研究生(外文):Ko-Chiang Hsia
論文名稱:由噬菌體呈現抗體庫中篩選並探討具有lipase活性的催化性抗體
論文名稱(外文):Isolation and characterization of lipase abzyme from phage displayed antibody library
指導教授:宣大衛宣大衛引用關係
指導教授(外文):David Shiuan
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:67
中文關鍵詞:lipase催化性抗體噬菌體呈現抗體庫
外文關鍵詞:lipasecatalytic antibodyphage display antibody library
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抗原與抗體之間的專一性辨認和結合,與酵素和受質之間的結合類似,因此某些抗體也有類似於酵素的催化活性,可催化特定的反應,稱之為催化性抗體。在本研究中,我們利用lipase (脂肪分解酵素) 受質的TSA (transition stats analog,過渡狀態類似物) 為餌,從phage display antibody library (噬菌體呈現抗體庫) 中篩選具有催化脂類水解活性的催化性抗體。TSA的主要結構有兩個部份,一個是生物素 (biotin) 端,可與白蛋白 (avidin) 結合固定在immunotube上作篩選;另一個則是p-nitrophenyl phosphonate抑制端,此端類似過渡狀態受質的架構,可與lipase活性區結合並抑制其活性。因此端為TSA結構所以有潛力去篩選出我們所要的催化性抗體。經過數次的篩選,其得到了六株phage clone可以與TSA專一結合。但以此六株噬菌體顆粒去做酵素活性分析,並沒有觀察到明顯的酵素活性。接著將此六株噬菌體感染大腸桿菌HB2151 (K12 ara Δ(lac-proAB) thi/F' proA+B lacIq lacZΔM15),表達出抗體片段蛋白。經過his-tag的純化之後,將這六種抗體蛋白做進一步的測試及分析,發現其與TSA的結合能力,以及對lipase受質p-nitrophenol butyrate之微弱水解能力。經過DNA定序,進一步以生物資訊軟體探討獲得之catalytic antibody,比較其與已知3D結構的lipase催化活性區之特性。
Interactions of antigen and antibody resemble to some extents the recognition and specific binding between enzyme and substrate. For this reason, antibodies may have certain enzymatic activity and were named abzymes or catalytic antibodies. In the present work we aimed to get lipolytic abzymes through the selection from phage displayed antibody library. A transition state analog for selection of lipolytic abzymes was synthesized. The structure composed of 4-nitrophenyl activated phosphonate, the transition state analog, (TSA) of lipases/esterases, connected to a biotin moiety through a 15 carbon chain spacer. After several rounds of selection, we obtained six monoclonal phage particles capable of binding significantly to the TSA. However, no apparent enzyme activity was observed using the solution of these phage particles for lipase enzymatic assay. Then, we used strain HB2151 (K12 ara Δ(lac-proAB) thi/F' proA+B lacIq lacZΔM15) to express the soluble antibody fragment. Through the His-tag purification, the purified antibody protein was examined further for its binding capability to TSA and the possible enzymatic activity. The present method presents a more efficient and convenient means to find new abzyme. Catalytic antibodies may have the potential to replace current enzymes and may have very diverse application.
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