利用水稻細胞之懸浮培養建立蛋白質高效率分泌系統

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姓名

劉馨如(Hsin-Ju Liu) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

利用水稻細胞之懸浮培養建立蛋白質高效率分泌系統
(Development of a high efficient protein secretory system in rice cell suspension cultures)

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摘要(中)

近數十年來，有許多研究致力於利用不同的表達系統來生產具有醫療用價值的基因重組蛋白質。而一個理想的表達系統必須能以最低的成本來生產安全、具有生物活性的蛋白質。近年來植物的表達系統已然成為具有許多優勢的選擇，其中水稻系統的成本低，且不具毒性，目前已經應用於許多蛋白質的表現；除了選擇一個理想的表達系統之外，我們必須集結系統中的其他優勢使其更有效率。無論是真核或原核生物，蛋白質的信號肽已被許多研究利用來有效生產蛋白質藥物，使蛋白質的N端附加一段分泌信號肽，讓蛋白質得以分泌至細胞外，而簡化下游純化的工程並降低生產成本。動植物中的蛋白質信號肽有某些保守的特徵，但其氨基酸組成的變異大，因此不同蛋白質的信號肽被認為有不同的辨認機制，而使分泌效率有所不同。為了增加蛋白質在水稻細胞培養系統中的分泌效率，我們篩選水稻中的內生性的蛋白質信號肽，分別是αAmylase3、CIN1(cell wall invertase1)以及33kD，C端各別接上GFP (green fluorescent protein)作為報導基因，於水稻細胞中大量表現。利用西方點墨法偵測蛋白質並比較三種信號肽的分泌效率，分析的結果發現αAmylase3之信號肽有最佳的分泌效率。為了確定αAmylase3信號肽的分泌效率是否專一於GFP，抑或可以適用在其他蛋白質，因此我們將會分析這三組信號肽對於mGM-CSF (mouse granulocyte-macrophage stimulation factor)蛋白質的分泌效率。除了初步篩選水稻中的內生性信號肽，未來我們會從其他動植物，甚至酵母菌中進行篩選，希望集結這些資訊而使發展一套高分泌效率的水稻細胞表達系統。

摘要(英)

Research in the past few decades has worked on the use of therapeutically valuable proteins from different protein expression systems. The ideal foreign protein expression system would be the one that produces the most safe, biologically active material at the lowest cost. Plant-based expression systems have emerged as a serious competitive force in the large-scale production of recombinant proteins. Most recombinant genes can be expressed in cultured rice cells; therefore, it is essential to determine which expression cassettes offer the most advantages for the production of the recombinant protein. The knowledge of protein signal sequences has become a crucial tool for pharmaceutical scientists who genetically modify bacteria, plants, and animals to produce effective drugs. By adding a specific tag to the desired proteins, one can, for instance, tag them for excretion, making them much easier to harvest. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition. To increase the secretion of recombinant protein in rice suspension cells, we generated overexpression vectors using different endogenous N-terminal signal peptides (αAmylase3,CIN1,33kD) fused with green florescence protein (GFP) and mouse granulocyte-macrophage stimulation factor (mGM-CSF), respectively. We detected the secreted GFP by Western blotting. Comparison of different signal peptides for secretion of GFP in rice suspension culture, we found that αAmylase3 is the better signal peptide for secreted the GFP out. To examine whether the supremacy of the αAmylase3 signal is specific for GFP, we’ll subsequently analyze the secretion efficiency of mGM-CSF and intend to develop a high efficiency secretion system in rice suspension cells.

關鍵字(中)

★ 蛋白質高效率分泌系統

關鍵字(英)

★ protein secretory system

論文目次

縮寫與全名對照表 I
中文摘要 II
Abstract III
本文目錄 IV
表目錄 VII
圖目錄 VIII
壹、研究動機 1
貳、前言 1
1. 基因重組蛋白質表達系統 2
1.1 原核生物表達系統 3
1.2 酵母菌表達系統 3
1.3 昆蟲及哺乳動物細胞表達系統 3
1.4 植物蛋白質表達系統 4
1.5 植物細胞蛋白質表達系統 6
2. 水稻懸浮細胞表達系統 10
3. 信號肽 (Signal peptides) 11
3.1 水稻基因的信號肽 (Signal peptides of Oryza sativa) 13
4. 綠色螢光蛋白 (Green Fluorescent Protein) 14
5. 顆粒細胞一巨噬細胞集落刺激因子 (granulocyte-macrophage colony stimulating factor，GM-CSF) 14
6. 實驗架構 17
參、研究方法與材料 18
第一部份建構表達質體 18
1. 選擇蛋白質在細胞外大量存在的基因 18
2. 利用SignalP server 預測蛋白質信號肽的位置 18
3. 反轉錄PCR分別合成三組signal peptide 18
3.1 RNA的抽取 18
3.2 RNA的純化(去除DNA的污染) 19
3.3 反轉譯PCR ( Reverse-transcription PCR) 19
3.4 Primers的設計 19
3.5 以PCR合成signal peptides 19
3.6 PCR產物的純化及限制酶處理 20
3.7 瓊脂膠體回收限制酵素切過之DNA 21
4. 接合反應 23
4.1 限制酵素作用 23
4.2 線性載體DNA的去磷作用 23
4.3 黏頭端載體DNA與插入DNA片段的接合 23
5. 以Gateway系統快速建構mGM-CSF之表達載體 24
5.1 以Gateway system製備含有mouse GM-CSF的topo entry vector 24
5.2 利用LR recombination的方式製備mGM-CSF的expression clone 25
6. 細菌的轉殖作用 26
6.1 製備E.coli competent cell勝任細胞 ( JM109 strain) 26
6.2 細菌的轉殖 (transformation) 27
6.3 小量純化細菌質體DNA 27
6.4 大量純化細菌質體DNA法 27
7. DNA序列分析 29
第二部份農桿菌之水稻基因轉殖 29
8. 利用農桿菌 (Agrobacterium) 進行水稻細胞的轉殖 29
8.1 從水稻未成熟的胚誘導出癒傷組織 (callus) 29
8.2 製備Agrobacterium competent cells ( EHA 105 strain ) 30
8.3 農桿菌的轉殖方法 30
8.4 檢測農桿菌 (Ketolactose test) 31
8.5 水稻的轉殖作用 31
8.6 水稻轉植株的再生 32
第三部份、轉殖株的分子生物學分析 32
9. GUS染色篩選 32
10. 以PCR放大genomic DNA中GFP的基因 32
10.1 基因組DNA的抽取與純化 33
10.2 基因組DNA之PCR篩選 33
11. 南方墨點法分析( Southern blot) 33
11.1 DNA墨點轉印 33
11.2 探針(probe)的製備 (Roche DIG-labeled DNA probe) 34
11.3 純化探針 35
11.4 探針雜交 ( probe hybridization) 35
第四部份、轉殖株的蛋白質表現分析 36
12. 建立水稻懸浮細胞培養 36
13. 細胞內外表現蛋白質的分析 36
13.1 收集細胞內外分泌蛋白質及純化 37
13.2 蛋白質的定量 37
13.3 培養液之蛋白質濃縮 37
13.4 西方墨點法分析( Western blot) 38
肆、結果 40
1. 大量表現信號肽融合GFP之表達載體的構築 40
1.1 預測蛋白質的信號肽部分 40
1.2 蛋白質的信號肽的選殖 40
1.3 構築信號肽融合GFP之表達載體 40
2. 建立信號肽融合GFP之轉殖水稻細胞株 41
2.1 利用農桿菌轉殖系統建立信號肽融合GFP之轉殖水稻細胞株 41
2.2 轉殖細胞的基因組DNA鑑定 42
3. 水稻中信號肽融合GFP蛋白質的分泌效率分析 43
3.1 建立水稻懸浮細胞培養 43
3.2水稻懸浮細胞株其GFP之RNA表現量 43
3.3 水稻懸浮細胞培養下GFP蛋白質的分析 43
4. 大量表現信號肽融合mGM-CSF之表達載體的構築 44
5. 利用Gateway系統快速建構由αAmy3啟動子調控mGM-CSF的表達載體 45
伍、討論 46
陸、參考文獻 49
附錄一質體建構圖(1) 72
附錄二質體建構圖(2) 73
附錄三質體建構圖(3) 74
附錄四本研究所使用之引子序列 75
附錄五信號序列及其氨基酸組成 76
附錄六研究材料配製 77

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指導教授

陸重安(Chung-An Lu)

審核日期

2006-9-20

推文