臺灣博碩士論文加值系統

二苯胺(Diphenylamine；DPA)是一種抗氧化劑，它的化學結構屬於非固醇類抗炎藥（Nonsteroidal Anti-inflammatory Drµgs; NSAIDs）的骨架結構，亦是一種致癌物（mutagen）。在農業上常做為蘋果收成時浸泡的溶液，以防止蘋果表皮燙斑現象（superficial scald）。
由於二苯胺是一種殺菌劑（fungicides），在農業上被高濃度的使用於殺菌劑中，亦是BTX（Benzene，Toluene，xylene）的重要衍生物之一，可做為橡膠助劑、染料中間體，而應用於染髮劑上。因此二苯胺可能因為農藥殘留問題而污染自然環境，或因人體接觸造成毒性傷害。
因為二苯胺分子量很小（MW=169.2），屬於半抗原（hapten），在免疫反應中無法引發抗體產生，故本實驗使用胎牛血清蛋白（BSA），作為攜帶蛋白，並利用螯合劑Disuccinimidyl Suberate（DSS）將兩者接合，形成一個完全抗原，再利用分生技術來製備DPA單鏈抗體。
將經免疫後的小鼠脾臟取出，與已知的抗體基因序列的引子（primer）以 RT-PCR 得到 cDNA。再將 cDNA 分別以抗體可變區重鏈（VH）及輕鏈（VL）之混合引子，經 PCR 放大後再將 VH 和 VL 進行組裝成單鏈抗體片段（scFv）基因，將 scFv 轉殖（clone）至 pHEN2 載體中，再感染 E. coli。利用噬菌體展示系統（phage display system）來構築單鏈抗體基因庫，以期得到抗二苯胺的單鏈抗體。經 BstNI 限制酶圖譜分析（BstNI Restriction fingerprint）確認基因庫具有多樣性。

Diphenylamine is an antioxidant, having the skeleton structure of non-steroidal anti-inflammatory drugs. In the use of agriculture, it is as a dipping solution to prevent superficial scald in the surface of apple.
Diphenylamine also is a fungicide and was used in the pesticides with high concentration. It is an important derivative of BXT, and often used in hair color dye. It is toxic, possible mutagen and teratogen, and harmful in contact with skin.
Because diphenylamine is a hapten, in this experiment, we use conjugate reagent to synthesize the bovine serum albumin with diphenylamine together as an immunogen. After immunization, and confirmed with ELISA, we take out the spleen of mouse, and use a series of degenerate primers covering recently known murine immunoglobulin gene family and performing RT-PCR to amplify variable region of heavy chain (VH) and light chain (VL) gene.
The amplified VH and VL DNA fragments were assembled to perform single chain fragment of variable region (scFv). The scFv DNA were cloned to the pHEN2 vector, and then transformed to E. coli. We use the technique of phage display system to construct the library of scFv for further selection. The constructed antibody phage display library was confirmed the diversity by BstNI restriction fingerprinting analysis.

目 錄
中文摘要 1
英文摘要 3
研究背景
二苯胺的物質安全資料表 4
二苯胺在日常生活中的應用 5
抗原抗體免疫反應 6
單鏈抗體 9
融合瘤細胞技術 13
噬菌體展示系統 14
實驗流程 18
實驗材料與方法
1. Primer 20
2. Vector 25
3. Bacterial strain 25
4. Baceriophage strain 25
5. Media 26
6. Synthesis of immunogen 27
7. Immunization 28
8. Tail bleeding of mice 29
9. Indirect ELISA 29
10. Total RNA extraction from spleen cell. 31
11. Reverse transcription… 33
12. Agarose DNA electrophoresis 34
13. PCR amplification of VH / VL 35
14. Assembly of scFv. 37
15. Restriction enzyme digestion of DNA 38
16. Extraction of pHEN2 plasmid 40
17. Ligation of scFv DNA 41
18. Preparation of electrocompetent cell 42
19. Electroporation and construction scFv library 43
20. BstNI fingerprinting analysis of scFv library 45
結果與討論
1. 抗原合成 47
2. 免疫小鼠 47
3. 抗體可變區域基因庫構築 48
4. 單鏈抗體的組裝 50
5. 單鏈抗體基因庫的分析 52
6. 討論 54
縮寫字對照表 58
參考文獻 60

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