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研究生： 黃馨儀
Huang, Xin-Yi
論文名稱： 選殖與鑑定體外轉錄訊息RNA用酵素
Cloning and characterization of mRNA in vitro transcription enzymes
指導教授： 張晃猷
Chang, Hwan-You
口試委員: 張壯榮
Chang, Chuang-Rung
張晉源
Chang, Chin-Yuan
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2023
畢業學年度： 111
語文別： 中文
論文頁數： 76
中文關鍵詞： 重組 T7 RNA 聚合酶 、E. coli poly(A) 聚合酶 、非洲豬瘟病毒mRNA-加帽酶 (NPR868R) 、非放射性T7 RNA 聚合酶活性檢測方法 、檢測鳥苷酸轉移酶活性的可視方法
外文關鍵詞： recombinant T7 RNA polymerase, E. coli poly(A) polymerase, African swine fever virus NP868R capping enzyme, Radioisotope-free T7 RNA polymerase assay, visual detection method for guanylyltransferase assay
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    • 近年來，RNA 藥物與RNA疫苗的應用逐漸成為生醫領域的另一種選擇。一開始領軍的是由RNAi製成的抗癌藥物，而後奮發突起的則是藉由2019年末爆發的嚴重特殊傳染性肺炎Covid-19而廣為使用的Covid-19 mRNA疫苗。鑒於mRNA的應用與研究將越來越廣泛，因此本論文旨在嘗試建立實驗室用mRNA製作平台。首先，將製作mRNA步驟所需的酵素以重組基因的方式在大腸桿菌中表達: T7 RNA polymerase使用trc/lac系統，大腸桿菌poly(A) polymerase (pcnB)使用T7/lac 系統，非洲豬瘟病毒mRNA-capping enzyme (NPR868R)則兩種表現系統皆進行測試。重組蛋白使用親和性管柱純化後，以肝素管柱減少核酸酶汙染，並採用非放射性方法檢測酵素活性。本研究成功表達並純化具活性之T7 RNA polymerase與大腸桿菌poly(A) polymerase。非洲豬瘟病毒的mRNA-capping enzyme作用方式屬於經典加帽反應，酵素可單體完成cap0。此蛋白在一般溫度或低溫表達皆不可溶，與GST、thioredoxin (Trx)融合仍難以增加其可溶且可純化性。使用尿素與鹽酸胍(guanidine HCL)變性Trx融合蛋白也無法得到可純化之蛋白。本研究亦修改產生短片段RNA的方法，並用來檢測鳥苷酸轉移酶活性的可視方法。

    In these days, RNA drugs and vaccines progressively became an alternative of the biomedical field. The anti-cancer drugs composed of RNAi first caught the eyebrows, and then the widely used mRNA vaccine against Covid-19 rose to fame. The application and analysis of mRNA is becoming more and more extensive. Thus, the aim of this study is to build a mRNA synthesis platform. First, recombinant enzymes for the synthesis of mRNA were generated in E. coli: T7 RNA polymerase was expressed by the trc/lac system; E. coli poly(A) polymerase (pcnB) was expressed by the T7/lac system; African swine fever virus NP868R capping enzyme was expressed using both of the above systems. Our enzyme purification strategy was first using nickel-charged resin chromatography, then with heparin affinity chromatography to eliminate ribonuclease contamination. Radioisotope-free assays for these enzymes were also established. This study successfully expressed and purified recombinant T7 RNA polymerase and poly(A) polymerase. African swine fever virus is a dsDNA virus. Its mRNA-capping enzyme reaction follows the conventional capping pathway, and this single-subunit enzyme can synthesize cap0 by itself. Regardless the expression temperature (37°C or 16°C) and fusion partner (glutathione transferase or thioredoxin), soluble protein could not be obtained. Using urea and guanidine-HCl to dissolve the inclusion body still could not obtain soluble recombinant protein for purification. Finally, this study also modified a visual detection method for guanylyltransferase assay using short RNA as the substrate.

    摘要 i Abstract ii 致謝 iii 目錄 iv 表目錄 viii 圖目錄 ix 壹、前言 (Introduction) 1 1.1 RNA therapy [13-15] 1 1.2 In vitro transcription 2 1.2.1 In vitro transcribed (IVT) mRNA 2 1.3 T7 RNA polymerase 3 1.4 Poly(A) polymerase (PAP I, pcnB) 4 1.5 Guanylyltransferase (NP868R) 5 貳、結果(Results) 6 2.1 Recombinant plasmid with T7RP and purification 6 2.1.1 質體pGEX5X1-T7RP的建構 6 2.1.2 測試GST-T7RP 重組蛋白在不同菌株的表達差異 6 2.1.3 GST-T7RP蛋白的表達與純化 7 2.1.4 蛋白活性單位之測試 8 2.2 Recombinant plasmid with pcnB and protein purification 11 2.2.1 含pcnB之重組質體建構 11 2.2.1.1 pET30-pcnB 11 2.2.1.2 pET28-pcnB 12 2.2.2 測試E. coli PAP I 在不同菌株與溫度的表現 12 2.2.3 蛋白活性的初測試 12 2.2.4 不含 6×His-tag的PAP I的表達與純化 13 2.2.5 蛋白活性的初測試 13 2.2.6 誘導蛋白6×His-PAP I的表達與純化 14 2.3 Recombinant plasmid with NP868R and purification 15 2.3.1 含NP868R之重組質體建構 15 2.3.1.1 pET28-NP868R 15 2.3.2 改善蛋白的溶解度與表達 16 2.3.2.1 降低誘導蛋白產量 16 2.3.2.2 Fusion protein 17 2.3.2.2.1 GST-GTase 17 2.3.2.2.1.1 誘導與純化測試 17 2.3.2.2.1.2 改變液態培養基pH值與滲透壓 18 2.3.2.2.1.3 測試破菌buffer 18 2.3.2.2.2 Trx-GTase 18 2.3.2.2.2.1 以變性方法純化 19 2.3.2.3 片斷化ASFV GTase 20 2.3.2.3.1 蛋白表達測試 20 2.3.2.3.2 純化測試 20 2.3.2.4 不同E. coli 菌株的表達 21 2.3.2.4.1 Origami2(DE3) 21 2.3.2.4.2 Rosetta-gami2(DE3) 21 2.3.2.4.3 BL21(DE3)pLysS 22 2.3.2.4.3.1 低溫蛋白表達與純化測試 22 2.3.2.4.3.2 添加少量酒精以增加重組蛋白之表達 22 2.3.2.4.3.3 以西方墨點法檢測目標蛋白ASFV GTase在BL21(DE3) pLysS的表達 23 2.3.2.5 在細胞裂解過程中添加穩定劑以增進重組蛋白的溶解度 23 2.4 Ribonuclease Assay Using Methylene Blue 25 2.4.1 在有/無RNA存在下，光譜偏移量 25 2.4.2 比較 RNA 被不同濃度的RNaseA降解後的光譜變化 25 2.4.3 不同濃度RNase A隨著時間在688 nm的吸光值變化 26 2.4.4 利用methylene blue RNase assay 偵測純化後的T7 RNA polymerase中核糖核酸酶汙染的程度 28 2.4.5 利用methylene blue RNase assay 偵測純化後的E. coli poly(A) polymerase I中核糖核酸酶汙染的程度 29 2.5 Short RNA synthesis and GTase assay establishment 29 2.5.1 DNA模板設計 29 2.5.2 plasmid construction 30 2.5.3 IVT的結果 31 2.5.3.1 pUC19ᵠ2.5-42G16U IVT 31 2.5.3.2 pUC19ᵠ2.5-42G15U IVT 32 2.5.4 小片段 IVT的條件測試 33 2.5.4.1 尋找PAGE圖中高背景的來源 33 2.5.4.2 不同MgCl2濃度與反應時間對small RNA IVT 的影響 34 2.5.4.3 不同限制酵素切割位與NTPs的比率對IVT的影響 34 2.5.4.4 控制NTPs製造固定長度的小片段RNA 35 2.5.5 GTase assay 36 參、討論 (Discussion) 37 3.1 T7RP 37 3.2 PAP I 39 3.3 NP868R 40 3.4 Ribonuclease Assay Using Methylene Blue 40 3.4.1 比較 RNA 被不同濃度的RNaseA降解後的光譜變化 40 3.4.2 利用methylene blue RNase assay 偵測純化後的T7 RNA polymerase中核糖核酸酶汙染的程度 40 3.5 Short RNA synthesis and GTase assay establishment 41 肆、材料與方法(Materials and methods) 42 4.1 材料 42 4.1.1 化學藥劑 42 4.1.2 常用商用試劑套件(commercial kits)與酵素 42 4.1.3 儀器 42 4.1.4 菌株 43 4.1.5 質體(plasmid) 43 4.1.6 引子(primers)與寡核苷酸(oligonucleotide) 45 4.1.7 Stock buffer composition for preparation of SDS-PAGE 46 4.2 方法 47 4.2.1 克隆(clone) 47 4.2.1.1 設計primer 47 4.2.1.2 Fragment PCR 47 4.2.1.3 Agarose gel electrophoresis 48 4.2.1.4 PCR clean-up 48 4.2.1.5 判定DNA的濃度 48 4.2.1.6 restriction enzyme digestion (酵切與接合) 49 4.2.1.7 ligation of vector and insert (質體接合) 49 4.2.1.8 Transformation (轉型細菌) 49 4.2.1.8.1 Chemical transformation: 49 4.2.1.8.2 Electroporation transformation: 50 4.2.1.9 Competent cell preparation(勝任細胞的製備) 50 4.2.1.9.1 chemically competent cell (化學勝任細胞) 50 4.2.1.9.2 electrocompetent cells(電勝任細胞) 50 4.2.1.10 抽取細菌質體 50 4.2.1.11 Colony PCR (檢查用) 51 4.2.2 蛋白誘導、純化與不可溶蛋白樣品製備方法 52 4.2.2.1 蛋白誘導表達測試 52 4.2.2.2 SDS-PAGE 52 4.2.2.2.1 拉姆利系統(Laemmli system, Glycine-SDS-PAGE) 52 4.2.2.2.2 Tris-Acetate系統 53 4.2.2.3 不可溶蛋白的變性可溶樣品之製備 54 4.2.2.4 蛋白純化 54 4.2.2.4.1 收集菌體與裂解菌體 54 4.2.2.4.2 Nickle column純化方法 54 4.2.2.4.3 Glutathione column純化方法 55 4.2.2.4.4 Heparin column純化方法 55 4.2.2.5 蛋白定量 55 4.2.2.6 裂解添加劑測試 55 4.2.2.6.1 以自誘導培養液製造大量培養物 55 4.2.2.6.2 第一輪添加劑篩選 56 4.2.2.6.3 第二輪添加劑篩選 56 4.2.3 RNA的製備、純化與定量 56 4.2.3.1 大於300 nt RNA的製備 56 4.2.3.2 大於300 nt RNA純化 57 4.2.3.3 Small RNA的製備 57 4.2.3.4 Small RNA的純化 57 4.2.3.5 RNA的定量 58 4.2.3.6 A-tailing 58 4.2.3.7 Capping 58 4.2.4 Ribonuclease Assay Using Methylene Blue 58 4.2.4.1 在有/無RNA存在下，光譜偏移量 59 4.2.4.2 比較RNA被不同濃度的RNaseA降解後的光譜變化 59 伍、參考資料 (Reference) 60 陸、補充(supplementary) 69

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