近年來信使核糖核酸疫苗已成為對付新冠肺炎疫情的利器，也促使此領域的蓬勃發展。要生產用於疫苗的高效率信使核糖核酸需要透過體外轉錄、5′ 端加帽、3′端多腺苷酸化等步驟。其中5′帽結構與3′端多腺苷酸結構對於轉錄後修飾、轉譯起始效率和信使核糖核酸的穩定性有很大的相關性。由於信使核糖核酸5′ 端加帽反應的量化並不容易測定，本論文嘗試使用能辨認5′端帽結構的真核起始因子4E(eIF4E)與酸性磷酸酶AphA 融合報導蛋白，以量化5′端帽結構合成的效率。最初我們在大腸桿菌中產生並過表達重組 AphA-eIF4E 融合蛋白。通過組氨酸標籤親和性鎳離子管柱，使用不同濃度的咪唑沖洗，分離出帶有組氨酸標籤的融合蛋白AphA-eIF4E。eIF4E 部分能夠特異性地結合加帽的 信使核糖核酸，而 AphA 可以作為報告酶以光學估計加帽步驟的效率。我們使用鏈親和素塗層於96 孔盤做為固定相，並加入生物素化的多聚胸腺嘧啶，與核糖核酸樣品結合，篩選出帶有3′端多腺苷酸的核糖核酸樣品，然後我們使用融合蛋白 AphA-eIF4E 來識別 5′加帽結構。 最後，我們使用 AphA 催化的螢光產物來估計 核糖核酸修飾的比例。在反應干擾物的測定上顯示三磷酸鳥苷與S-腺苷甲硫氨酸會干擾本偵測反應，並須先去除。本偵測系統可以有效地分辨出信使核糖核酸與非信使核糖核酸的測試樣品，偵測靈敏度最低偵測限制為200 奈克的信使核糖核酸，而偵測線性範圍約落在200 至1600 奈克的信使核糖核酸之間。

Messenger RNA vaccines have been demonstrated to be an effective tool against Covid-19 infections and many other infections. The synthesis of mRNA typically involves steps including in vitro transcription, 5′ capping, and 3′ A-tailing. The 5′ capping is essential for translation initiation and mRNA stability. The 3′ poly A tail is necessary for nuclear export, translation and stability of mRNA. Because mRNA capping is difficult to measure, this study planned to develop a 5′ capping detection system. Initially, a recombinant AphA (acid phosphatase)-eIF4E (eukaryotic initiation factor 4E) fusion protein was generated and overexpressed in E. coli. The eIF4E portion is capable of binding capped mRNA specifically, whereas AphA could serve as a reporter enzyme to optically estimate the efficiency of the capping step. We used a streptavidin-coated 96-well plate to immobilize biotin-labeled oligo d(T), which in turn can capture mRNA with 3′poly(A) tail. Then, AphA-eIF4E was added to quantify the 5′ cap structure that can be quantitated by adding AphA substrate 4-MUP. The results show that the detection system can effectively distinguish mRNA and non-mRNA samples. Nucleotides GTP and SAMe could interfere the assay and therefore need to be removed before conducting the assay. The limit of detection of the detection system was approximately 200 ng mRNA. The linear range of detection was approximately between 200 to 1600 ng mRNA.

摘要: I
Abstract: II
致謝 III
縮寫表 IV
目錄 VII
表目錄 X
圖目錄 X
壹、 前言: 1
1.1真核生物中的mRNA修飾 1
1.2參與mRNA修飾之蛋白 1
1.3 mRNA疫苗的重要性與製造 2
1.4現有5'端帽結構偵測技術 2
1.5真核起始轉譯因子4E (eIF4E) 3
1.6 B類酸性磷酸酶(AphA) 4
1.7蛋白質連接頭 4
1.8實驗目的 5
貳、材料與方法 6
2.1 菌株培養 6
2.1.1 細菌培養液製備 6
2.1.2 勝任細胞 (Competent Cell) 製備 6
2.2 質體建構 7
2.2.1 引子 (Primer) 設計與合成 7
2.2.2 載體 (Vector) 7
2.2.3 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 7
2.2.4 限制酵素處理 8
2.2.5 接合作用 8
2.2.6 熱休克轉型作用 8
2.3 蛋白質誘導表現與分析 8
2.3.1 IPTG (Isopropyl β-D-thiogalactopyranoside) 誘導基因表現 8
2.3.2 GST-tag 親和性管柱純化蛋白質 9
2.3.3 組氨酸標籤親和性管柱純化蛋白質 9
2.3.4肝素瓊脂糖管柱純化蛋白 10
2.3.4 SDS 聚丙烯醯胺膠體電泳 10
2.3.5考馬斯亮藍染色 (Coomassie Brilliant Blue) 11
2.3.6 蛋白質的透析 11
2.3.7 蛋白質定量分析 11
2.4 蛋白活性分析 12
2.4.1重組蛋白eIF4E與AphA-eIF4E, eIF4E活性測試 (RNA斑點雜交) 12
2.4.2重組融合蛋白AphA-eIF4E， AphA端活性測試 (pNPP asaay) 12
2.4.3重組蛋白AphA-eIF4E活性測試電泳遷移率實驗(EMSA) 13
2.5 mRNA樣品製備 13
2.5.1 RNA合成 13
2.5.2 RNA 3'端多腺苷酸化 13
2.5.3 RNA 5'端加帽修飾 13
2.5.4 3T3細胞總RNA提取 14
2.6 5'帽結構檢測系統建立 14
2.6.1鏈親和素塗層96孔盤置備 14
2.6.2生物素化的多聚胸腺嘧啶 15
2.6.3微盤分析儀檢測 15
2.6.4 5'端帽檢測系統流程 15
2.7 5'帽結構檢測系統問題檢測 16
2.7.1鏈親和素塗層確認 16
2.7.2鏈親和素與生物素結合確認 (斑點雜交) 16
2.7.3 RNA測試樣品與生物素化多聚胸腺嘧啶結合確認電泳遷移率實驗(EMSA) 16
2.8 5'帽檢測系統反應物置換4-MUP assay 17
2.9 RNA測試樣品純化 17
參、結果 18
3.1 RNA測試樣品的處理與驗證 18
3.1.1 FGF2 RNA合成與轉錄後修飾 (5'加帽， 3'端多腺苷酸化) 18
3.1.2短序列RNA合成 18
3.1.3 3T3細胞 total RNA 18
3.2起始轉譯因子eIF4E 19
3.2.1構築和純化真核起始轉譯因子eIF4E 19
3.2.2真核起始轉譯因子eIF4E的活性評估 19
3.3重組蛋白AphA-eIF4E 20
3.3.1構築重組蛋白AphA-eIF4E 20
3.3.2重組蛋白AphA-eIF4E，AphA活性評估(pNPP assay) 20
3.3.3重組蛋白AphA-eIF4E，AphA活性極限測試 21
3.3.4 RNA對於重組蛋白AphA-eIF4E之AphA活性之影響 21
3.4 5'帽結構檢測系統 21
3.4.1 5'帽結構檢測系統建立 21
3.4.2 5'帽結構檢測系統測試 22
3.4.3檢測系統問題排除 22
3.4.4 5'帽結構檢測系統反應物置換4-MUP assay 24
肆、討論 25
伍、參考資料 29

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