製作 anti-Zelda antibody 與分析 Zelda 表現量

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姓名

黃柏翰(Po-Han Huang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

製作 anti-Zelda antibody 與分析 Zelda 表現量
(anti-Zelda antibody production and Zelda expression analysis)

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至系統瀏覽論文 (2028-6-1以後開放)

摘要(中)

在所有動物中，初期胚胎發育於缺乏轉錄的情況下進行，且由母源物質（maternal components）控制與調節，在一段時間的發育之後，合子基因組將被啟動（zygotic genome activation, ZGA），並調控之後的胚胎發育，這個轉換過程被稱之為（maternal to zygotic transition, MZT）。以黑腹果蠅（Drosophila melanogaster）為例，ZGA 起始於受精後一小時（於25 ℃）並伴隨著兩波的合子基因表現。於2008年，Liang等人發現轉錄因子 Zelda 在 MZT 時期作為 ZGA 的關鍵激活因子，活化了75%以上的早期基因。隨後，多項證據顯示 Zelda 亦可促進其他轉錄因子與其下游基因的 enhancers 結合，進而調控果蠅早期胚胎發育的 gene network，但目前對 Zelda 蛋白結構與分子功能尚不明瞭。
本實驗室曾經使用桿狀病毒表現系統，在昆蟲細胞異源表達 6xHis-tagged Zelda蛋白，然而 anti-His antibody 卻無法穩定檢測 Zelda 蛋白表達，為了能有較的偵測 Zelda 蛋白，在我的研究中測試了對昆蟲細胞或組織更具靈敏度的anti-His antibody，同時也製作並測試新的 anti-Zelda antibody，希望能在不同的樣本中更精確檢測 Zelda 蛋白。
在實驗中，我們使用大腸桿菌表達系統生產並純化 truncated c-terminal Zelda protein （從amino acid 1240到1596包涵了 C-terminal 的4個 zinc fingers, Zelda1240-1596），並委託生技公司製作 anti-Zelda antibody，之後利用 Immunostaining 與 Western blot 檢測這個新的 anti-Zelda antibody 之特異性與敏感度。

摘要(英)

In all animals, early embryogenesis take place in the absence of transcription, and the development is controlled by maternal components. Later, the zygotic genome will be activated and take over the control in a process called maternal to zygotic transition (MZT). In Drosophila, zygotic genome activation (ZGA) initiated at one hour after fertilization (at 25 ℃) with 2 waves of gene expression. In 2008, Liang, et al. discovered a transcription factor, Zelda, which acts as a key activator of ZGA during MZT. Subsequently, multiple evidences suggested that Zelda also promotes chromatin accessibility and facilitates the binding of the other transcription factors. However, the structure and molecular function of Zelda is still not clear.
Our lab previously established a platform ectopically expressing 6xHis-tagged Zelda in insect cells using Bac-to-Bac system. However, most anti-His antibody was unable to detect Zelda protein stably. In order to have a better detection of Zelda protein, I tested anti-His antibody which has better performance for insect cells or tissues (Dr. Z. Y. Wu, personal communication, 2022), and also detected the expression of Zelda by the new anti-Zelda antibody.
In my research, I also generated and purified truncated c-terminal Zelda protein (from amino acid 1240 to 1596 and includs C-terminal 4 zinc fingers, Zelda1240-1596) using E. coli protein expression system and sent for anti-Zelda antibody production. I also conduct anti-body staining and western blot for checking anti-Zelda antibody function.

關鍵字(中)

★ Zelda 蛋白
★ Zelda 抗體
★ Zelda 表現量

關鍵字(英)

★ Zelda protein
★ anti-Zelda antibody
★ Zelda expression

論文目次

中文摘要 i
Abstract ii
誌謝 iii
圖目錄 vi
表目錄 viii
中英文對照表 ix
壹、前言 1
1.1 黑腹果蠅（Drosophila melanogaster）早期胚胎發育 1
1.2 Maternal-to-zygotic transition（MZT）1
1.3 果蠅轉錄因子 Zelda 2
1.3.1 Zelda 為 MZT 時期所需的重要轉錄因子 2
1.3.2 Zelda調控發育相關基因 3
1.3.3 Zelda促進其他轉錄因子與基因組結合 4
1.3.4 Zelda 調節染色質可及性（Chromatin accessibility） 5
1.4 Zelda 蛋白檢測 6
1.5 大腸桿菌重組蛋白表達系統 6
1.6 研究動機與目的 6
貳、實驗方法與材料 9
2.1. 實驗材料 9
2.1.1 菌種與質體 9
2.1.2 細胞株 9
2.1.3 果蠅品系 9
2.2. 實驗方法 9
2.2.1 構築 Zedla c-terminal 之表現載體 9
2.2.2 限制酶切割 10
2.2.3 DNA 膠體電泳（DNA gel electrophoresis） 10
2.2.4 細菌轉型作用（Transformation） 10
2.2.5 Zelda C-terminal蛋白之過度表達 10
2.2.6 破菌處理大腸桿菌表達系統（E. coli expression system）之沉澱物 11
2.2.7 純化 Zelda1240-1596蛋白 11
2.2.8 測定 Zelda1240-1596蛋白濃度 12
2.2.9 蛋白濃縮 13
2.2.10 估計濃縮之 Zelda1240-1596蛋白濃度 13
2.2.11 異位表達 6xHis-tagged full-length Zelda 蛋白於不同的昆蟲細胞株 13
2.2.12 SDS-PAGE 之蛋白質樣品製備 14
2.2.13 蛋白質膠體電泳 15
2.2.14 西方墨點法（Western blot） 16
2.2.15 免疫組織化學染色（Immunohistochemical staining） 16
參、實驗結果 24
3.1 設計與確認質體 pGEX_zldCzfAgeHis 24
3.2 以大腸桿菌蛋白表達系統表達 Zelda1240-1596蛋白 27
3.3 以 Ni-NTA agarose beads 純化 Zelda1240-1596蛋白 30
3.4 利用 electro-elution 純化 Zelda1240-1596蛋白 33
3.5 濃縮 Zelda1240-1596蛋白並委託生產多株客製抗體 34
3.6 測試anti-His antibody靈敏度與專一性 35
3.7 Anti-Zelda antibody 檢測不同來源之 Zelda 蛋白 37
3.7.1 檢測於 E. coli 表現之 Zelda1240-1596蛋白 37
3.7.2 檢測於果蠅胚胎、細胞與草地貪夜蛾細胞異位表達的6xHis-tagged Zelda 38
肆、實驗討論 45
4.1 於異源蛋白表達系統大量表達 Zelda 45
4.2 純化與濃縮 Zelda1240-1596蛋白 45
4.3 Zelda 蛋白檢測 46
4.4 未來應用 47
伍、結語 49
參考資料 50

參考文獻

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指導教授

粘仲毅(Chung-Yi Nien)

審核日期

2023-7-27

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