抗體是一種被廣泛應用於基礎研究、疾病診斷及治療的生物分子。然而，大多數的應用需要抗體專一性地修飾上功能分子；先前的研究，大多是透過抗體上的一級胺或樞紐點的雙硫鍵進行修飾，這樣的修飾方式有許多缺點，例如會導致異質產物生成且會影響到抗體的結構。
為了解決這些問題，利用天然抗體位於Asn-297上的Fc寡醣，進行醣類酵素專一性修飾，成為本研究的首要目標；同時，可利用醣類相關酵素將具有疊氮基團的醣體成功地引入抗體中，而疊氮基團是一種不存在於生物系統中的生物正交分子，可與炔烴進行環狀加成反應，如此一來便可將功能分子以此方法進行連接。
在本論文研究中，我們提供了一種簡單的製備方法進行抗體修飾。首先，我們利用化學酵素法，製備帶有疊氮基修飾之胞苷單磷酸唾液酸醣予體；而後，使用一系列醣基轉移酶有效地將天然醣體及疊氮基修飾的醣體引入抗體上；最後，經過修飾的同質抗體可利用鍵擊反應將特定分子專一性地連接在抗體上。此外，為了確認修飾效率，我們透過鍵擊反應連接上螢光分子觀察訊號，同時也利用N-糖苷酶F水解Fc-上寡糖，再利用MALDI-TOF MS進行醣體的質譜分析。

Antibodies are biomolecules that are widely used as affinity reagents for many applications in research, disease diagnostics, and therapies. Most of these applications require the modification of antibody with a specific functional moiety. However, the traditional conjugation methods by targeting primary amines or hinge disulfides have a number of drawbacks including heterogeneous product profiles and structure alteration.
To overcome these drawbacks, the natural occurring Fc-glycan on the antibody is the interesting target for site-specific modification due to its presence at Asn-297 and easily modulated by sugar possessing enzymes to incorporate azido functional group. As a result, the bioorthogonal cycloaddition between azide and alkynes can be achieved to successfully introduced desired molecule on modified antibody.
In this research, we developed a facile strategy for antibody modification. First, we used chemoenzymatic approach to synthesize sialic acid donor, CMP-Neu5Az. Then, a series of glycosyltransferases were applied to orderly and high efficiently install natural glycans and azide-functionalized glycans into antibodies. Finally, the homogenous modified antibody was conjugated with desired molecule by click chemistry to achieve the site-specific conjugation. In addition, to ensure the modification efficiency, we not only made the fluorescent molecules clicked with antibody to check the signal on SDS-PAGE, but also utilized PNGase F to digest the Fc-glycan followed by MALDI-TOF-MS analysis.

摘要 I
Abstract II
縮寫對照表 (abbreviation) IV
酵素中英文名稱對照表 VII
單醣中英文名稱及代表符號 VIII
目錄 IX
圖目錄 XI
流程圖目錄 XIV
表目錄 XV
壹. 緒論 1
1.1 抗體的結構及功能 1
1.1.1 抗體的結構 1
1.1.2 抗體的功能 3
1.2 抗體的修飾方法及應用 6
1.2.1 目前已開發之抗體藥物 6
1.2.2 基因工程 7
1.2.3 抗體工程技術 8
1.2.4 醣體修飾 10
1.3 生物正交性化學 17
1.3.1 鍵擊化學 18
1.3.2 鍵擊化學的應用 19
1.4 酵素 20
1.4.1 醣激酶及磷酸醣核苷酸轉移酶 20
1.4.2唾液酸醛醇縮酶 26
1.4.3醣基轉移酶 27
1.4.4 唾液酸水解酶 39
1.4.5 N-醣苷酶F 40
1.5回顧分析醣蛋白N-寡醣之策略 40
1.6 研究目標與動機 46
貳、實驗結果與討論 47
2.1 以大腸桿菌誘導表現目標蛋白 47
2.1.1 勝任細胞 47
2.1.2 目標酵素純化與表現分析 48
2.1.3 酵素表達分析 52
2.1.4 酵素活性測試 56
2.2以化學及酵素法合成疊氮基唾液酸相關醣體 59
2.3 抗體修飾 61
2.3.1 抗體修飾策略 62
2.3.2抗體修飾結果與討論 68
2.4. 抗體藥物 77
參、結論與未來展望 81
肆. 實驗部分 82
參考文獻 98
附錄 i

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