根據世界衛生組織統計，癌症是全球第二大死因。在癌症藥物的選擇中，主流的
化學抗癌藥物大多缺乏對癌細胞的專一性，在非腫瘤部位造成不必要的副作用。也因
此，隨著精準醫療概念的興起，標靶治療成為抗癌藥物的重點研究領域之一。標靶藥
物主要由兩項要素組成，分別是對癌細胞具有辨認能力的分子以及辨認後能產生細胞
毒性的毒素，使標靶藥物在毒殺癌細胞的同時，能將對周圍正常細胞的傷害降至最低。
本研究使用細胞毒素PE38 以進行癌細胞毒殺，其源自綠膿桿菌外毒素A，藉由刪除
外毒素A 基因序列中受體辨識片段，使其缺乏主動進入細胞的能力，搭配擁有腫瘤
標靶能力的分子便可成為具癌症專一性的毒素，擁有應用於癌症標靶治療的潛力。本
研究首先建立大腸桿菌表現PE38 的系統，以得到重組PE38。隨後將PE38 蛋白及帶
PE38 基因的質體轉染至癌化的人類胚胎腎上皮細胞株HEK293T 中，其中質體形式
之PE38 帶有已知具癌症標靶能力的3 端非轉譯區，能夠在分裂快速的細胞中穩定目
標基因mRNA，使轉譯效率提升。透過轉染後細胞活性分析，發現質體形式之PE38
在轉染後能夠明顯抑制HEK293T 細胞活性，而在西方墨點法分析轉染後存活細胞之
裂解液蛋白中卻無法得到PE38 蛋白的訊號。我們亦構築T7-RNA 聚合酶的表現載
體，經生產及純化後應用於體外轉錄反應，以嘗試合成RNA 形式之PE38。在未來，
本研究結合RNA 形式之PE38 毒殺研究後，便可提供PE38 藥物形式選擇的相關資
訊。綜合以上論述，PE38 擁有應用於標靶藥物的潛力。

Accroding to the World Health Organization, cancer is the number two cause of
deaths all over the world. However, among all the cancer drugs, the most popular one,
chemotherapy drugs, lack specificity for cancer cells and thereby induce side effect in noncancerous
regions. With the rise of the concept of precision medicine, targeted therapy has
become the key research field in anticancer drug. Tumor specificity of targeting molecules
and killing ability of toxins are two key attribute of targeted drugs, by which cancer cells
can be eliminated and surrounding normal cells remain unaffected. In this study, we used
biotoxin PE38 as cytotoxic agent for tumor-killing. PE38 is a truncated toxin derived from
Pseudomonas exotoxin A. By deleting the sequence coding for the receptor binding
domain of exotoxin A, PE38 loses its ability to invade cells. Combination of PE38 and
specific tumor-recognized molecules leads to a potential application in cancer targeted
therapy. Here, we transfected PE38 protein and PE38-coding plasmid to cancerated human
embryonic kidney cell line HEK293T. Analyzing the cell viability after transfection, we
found that PE38-coding plasmid leads to decline of cell activity. But further Western blot
results showed no PE38 signal in lysate of living cell after plasmid transfection. We also
produced and purified T7 RNA polymerase from a construct expressing plasmid, and used
it for in vitro transcription for PE38 RNA synthesis. In the future, by combining with the
research on the cytotoxicity from mRNA of PE38, this study can provide valuable
information for choosing the type of PE38 as a cancer drug. Finally, this study suggests
that PE38 can be a potential anti-cancer drug in targeted therapy.

中文摘要.......................................II
Abstract......................................III
致謝...........................................IV
縮寫表.........................................V
目錄...........................................VII
表目錄.........................................IX
圖目錄.........................................IX
壹、 前言.......................................1
1.1 癌症.......................................1
1.2 蛋白質藥物..................................2
1.3 DNA藥物....................................2
1.4 RNA藥物....................................3
1.5 mRNA抗癌藥物................................4
1.6 體外轉錄反應（In Vitro Transcription）......5
1.7 PE38.......................................6
1.8 研究目的...................................6
貳、 材料與方法.................................8
2.1 菌株培養...................................8
2.2 細胞培養...................................9
2.3 質體建構...................................10
2.4 蛋白誘導表現與純化..........................13
2.5 mRNA合成...................................14
2.6 細胞轉染（Transfection）....................15
2.7 PE38轉染效果檢..............................15
參、 結果......................................18
3.1 PE38重組蛋白於HEK293T細胞毒殺能力試驗........18
3.2 PE38-UTR之DNA質體於HEK293T毒殺能力試驗......19
3.3 T7-RNA聚合?蛋白之生產純化..................21
3.4 以T7-RNA聚合?合成PE38-UTR之RNA.............22
肆、 討論......................................24
伍、 參考文獻..................................28

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