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dc.contributorJung-Yie Kaoen_US
dc.contributor.authorYu-Han Huangen_US
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dc.description.abstract在過去,氯黴素(CAP)是廣泛應用在獸醫與人醫的抗生素,然而氯黴素殘留會造成人體骨髓抑制與再生不良性貧血等副作用,因此現在已被禁止用於產肉動物的治療,台灣也對農畜產品制定了零容忍政策。 在食品安全議題受到重視的現今,利用有效且不昂貴的免疫方法快速檢測抗生素殘留以取代昂貴費時的LC-MS分析應更具有發展價值。 因此開發一個高感度的抗體並研製成檢測套組成為確保食品安全的重要方針。 本研究自表現CAP抗體之融合瘤細胞中成功地取得其可變區基因並進一步建構成融合His-tag之單鏈抗體之表現質體,在最適化之大腸桿菌表現宿主BL21(DE3)pLysS表現後可利用Ni-resin親和性管柱純化並獲得CAP之單鏈抗體。利用ELISA方法分析可證實CAP單鏈抗體可以專一識別CAP,在抗體競爭性試驗同時也證實了CAP單鏈抗體與其單株抗體均會競爭相同結構。在未來更可利用易錯聚合酶鏈反應(error prone PCR)來增加靈敏度並輔以融合不同螢光蛋白或結合其他偵測技術以增加免疫檢測的偵測極限與多元應用性。zh_TW
dc.description.abstractChloramphenicol (CAP) is a potent and efficient antibiotic widely used in pharmacological treatments in veterinary and human. Despite being highly effective, it shows severe toxicity as the residual CAP results in Aplastic anemia (AA) and bone marrow suppression. Due to the potential risk in public health of CAP utilization, the use in food-producing animal's therapy has been banned. Taiwan government has established a zero tolerance policy to CAP in livestock products. Recently, the food crises have grabbed huge public attention in Taiwan. Replacing the expensive LC-MS analysis by cheap and efficient rapid immunoassay to detect the residual illegal compound can better fit to public interest. Therefore, to generate high sensitivity antibodies for immunoassay kits play a key role in improving the food safety. This study has successfully cloned CAP-scFv gene from hybridoma and constructed CAP-scFv gene onto an expression vector fused with a His-tag gene. The expression of this protein in E. coli BL21(DE3) pLysS was optimized and the expressed recombinant CAP-scFv protein was purified with Ni-IMAC. We confirmed the specificity between CAP-scFv and chloramphenicol via ELISA assay. Furthermore we also demonstrated that CAP-scFv recognizes the same molecular structure as CAP monoclonal antibody does by performing the competition assay with CAP monoclonal antibody. In the future, using the error-prone PCR approach further improve the CAP-scFv sensitivity and fusion of fluorescent to the protein flank on scFv or combination with various detection methods will be the next target to enhance the detection limit of immunoassay and create novel applications to numerous aspects.en_US
dc.description.tableofcontents圖目錄 vii 表目錄 viii 附錄目錄 ix 第一章 緒論 1 第二章 文獻回顧 2 2.1氯黴素(chloramphenicol; CAP)簡介 2 2.1.1氯黴素的特性與用途 2 2.1.2氯黴素的獸藥殘留與對人體的危害 3 2.1.3 台灣對氯黴素之法律規範 4 2.2 抗體之簡介 5 2.2.1單株抗體在檢測上之應用 7 2.2.2單鏈抗體的介紹 7 2.2.3單鏈抗體在檢測上之應用 8 2.2.4抗體的其他應用 9 2.3 研究動機與目的 9 第三章 材料與方法 11 3.1 材料 11 3.1.1酵素類 11 3.1.2試劑類 12 3.1.2套組與其他類 13 3.2 方法 14 3.2.1 單株抗體之取得 (Obtain the monoclonal antibody) 14 3.2.2 Total RNA之萃取 (Total RNA isolation) 14 3.2.3 反轉錄反應取得cDNA (Reverse transcription) 15 3.2.4 VH與VL的取得 (Amplify the variable region of the monoclonal antibody) 16 3.2.5 連接子組裝 (Linker assembly) 17 3.2.6 最終組裝 (Final assembly) 18 3.2.7 單鏈抗體片段再放大 (Re-amplification of scFv fragment) 19 3.2.8 T/A 選殖 (T/A cloning) 20 3.2.9 接合(Ligation) 21 3.2.10 Colony PCR check 22 3.2.11 PCR產物的純化 (Purification of PCR products) 23 3.2.12 質體製備 (Mini-prepare of plasmid) 23 3.2.13 勝任細胞的製備 (prepare of competent cell) 24 3.2.14 轉形作用(Transformation) 25 3.2.15 限制酶處理( Restriction enzyme digestion ) 25 3.2.16 DNA瓊脂凝膠膠體電泳 (Agarose gel electrophoresis) 26 3.2.17 蛋白表現 (ScFv protein expression) 26 3.2.18 可溶性蛋白質製備 (Prepare of the soluble ScFv protein) 27 3.2.19 金屬親和性層析純化 (Immobilize metal ion chromatography purification) 27 3.2.20 蛋白A親和性層析純化 (Protein A resin purification) 28 3.2.21蛋白L親和性層析純化 (Protein L resin purification) 28 3.2.22 蛋白質SDS-PAGE膠體電泳(SDS-PAGE electrophoresis) 29 3.2.23 西方轉漬法(Western blotting) 30 3.2.24酵素連接免疫吸附法(ELISA assay) 30 第四章 結果與討論 32 4.1 單株抗體純化與分型鑑定 32 4.2建構可表現CAP之單鏈抗體基因 32 4.3 取得並定序單鏈抗體基因 33 4.4 構築單鏈抗體表現質體 34 4.5 單鏈抗體的表現與純化 34 4.6 單鏈抗體之親和性分析 35 4.7 討論與未來展望 36 第五章 參考文獻 37 圖 42 表 59 附錄 66zh_TW
dc.subjectsingle-chain variable fragmenten_US
dc.titlePreparation and characterization of recombinant single-chain variable fragment (scFv) against chloramphenicolen_US
dc.typeThesis and Dissertationen_US
item.openairetypeThesis and Dissertation-
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