Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92209
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dc.contributorJung-Yie Kaoen_US
dc.contributor高振益zh_TW
dc.contributor.author蘇彥兆zh_TW
dc.contributor.authorYan-Zhao Suen_US
dc.contributor.other生物化學研究所zh_TW
dc.date2015zh_TW
dc.date.accessioned2015-12-15T05:30:04Z-
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dc.identifier.urihttp://hdl.handle.net/11455/92209-
dc.description.abstract吲哚胺2,3雙加氧酶(IDO)是色胺酸代謝途徑中重要的胞內酵素,癌細胞若有高的IDO表現量會將癌細胞周圍微環境的色胺酸代謝成犬胺酸,在色胺酸(必需胺基酸)減少與致細胞凋亡的犬胺酸存在下會抑制周圍T細胞的增生,因此讓癌細胞擺脫T細胞的追踪與獵殺並獲得免疫逃脫能力。 利用IDO抗體來抑制其活性使癌細胞再度被識別是一種新穎的抗癌策略,然而抗體受限於分子量太大無法進入細胞中,因此將專一性抗體修改成分子量較小的單鏈抗體將可成為此方略下的抗癌手段。 本研究成功地自表現IDO抗體之融合瘤細胞中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,在篩選最佳之大腸桿菌表現宿主後,以BL21(DE3)pLysS大量表現並使用Ni-resin親和性管柱純化獲得IDO的單鏈抗體。藉由抗體競爭性試驗同時證實了IDO單鏈抗體對IDO的親和力並證實了單鏈抗體與其單株抗體競爭相同區域。未來更可分析IDO單鏈抗體在細胞內抑制IDO活性的能力,並進一步地利用易錯聚合酶鏈反應(error prone PCR)來增加親和力或融合蛋白質轉導區(PTD domain)使單鏈抗體更準確投遞到癌細胞以提升抑制癌細胞內IDO活性的能力。zh_TW
dc.description.abstractIndoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme which plays an important role in tryptophan catabolism. In case of highly IDO-expressed tumor cells, the micro-environmental tryptophan was metabolized into proapoptotic kynurenines. The decline of tryprophan level and accumulation of kynurenines results in suppression of the surrounding T cell's proliferation. Hence, this phenomenon has made the cancer cell flee from the tracking and killing of the T cells thus contributed to the gain-of-function immune-escape of the tumor cells. It is an innovative anti-cancer strategy to reboot T cell's tumor cells recognition by inactivating IDO using a specific monoclonal antibody. Due to the fact that the antibody is too large to enter the cytosol, employing recombinant DNA technology to generate a small size single-chain variable fragment (scFv) other than large antibody might help breakthrough this bottleneck. In this project, we have successfully cloned IDO-scFv gene from hybridoma mRNA and fused the gene with a His-tag gene onto an expression vector. The optimized expression of this protein in E. coli BL21(DE3) pLysS was utilized to produce the recombinant IDO-scFv protein. Additionally, the IDO-scFv protein was purified with Ni-IMAC. Using the competition assay with IDO monoclonal antibody and its derived scFv has demonstrated that IDO-scFv could contend with its source monoclonal antibody in the same antigen site. In the future, the inhibition of IDO activity can be monitored by metabolic methods. Furthermore, to enhance scFv's binding affinity by error-prone PCR approach or to improve scFv's cancer-cell-targeting specificity by fusing the tumor specific protein transduction domain (PTD) flank to the scFv could promisingly inactivate IDO in the cancer cell.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 Indoleamine 2,3-dioxygenase (IDO) 之生物功能簡介 2 2.1.1 IDO的免疫調節功能與機制 3 2.1.2 腫瘤的免疫逃脫與免疫治療 4 2.2 抗體之簡介 5 2.2.1單株抗體在癌症治療上之應用 7 2.2.2單鏈抗體的介紹 7 2.2.3單鏈抗體在醫療上之應用 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 11 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) 24 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IDO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 33 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 35 第五章 參考文獻 37 圖 45 表 60 附錄 66zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2015-02-02起公開。zh_TW
dc.subject吲哚胺2,3雙加氧酶zh_TW
dc.subject單鏈抗體zh_TW
dc.subjectIDO,single-chain variable fragmenten_US
dc.subjectscFven_US
dc.title重組抗-吲哚胺2,3雙加氧酶單鏈抗體之製備與分析zh_TW
dc.titlePreparation and characterization of recombinant anti- Indoleamine 2,3-dioxygenase single-chain variable fragment (scFv)en_US
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2018-02-02zh_TW
dc.date.openaccess2015-02-02-
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item.languageiso639-1zh_TW-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
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