顆粒巨噬細胞集落刺激因子 (Granulocyte-macrophage colony-stimulating factor, GM-CSF) 是細胞激素的一種，目前主要使用基因重組方式生產老鼠的GM-CSF (mGMCSF) 做基礎醫學研究。mGMCSF可利用哺乳類動物細胞生產，但有成本高、產量與生物安全性低等缺點，因此我們期望利用水稻細胞生產mGMCSF。此重組蛋白可以在水稻細胞中表現，但是在蛋白質的醣基化後修飾中會加上植物特有的木醣 (xylose) 與岩藻醣 (fucose)，可能引發過敏反應，因此我們利用基因工程的方法，在水稻細胞內以RNA干擾 (RNA interference, RNAi) 的模式降低木醣轉移酶 (XT) 與岩藻糖轉移酶 (FT) 的表現量，並導入人類醣基化模式以生產mGMCSF。由於減少這兩個植物特有的醣基轉移酶，可能會影響植物細胞的生長與生理，因此我們將持續表現的啟動子置換為受糖所調控的α- AMY3啟動子，在細胞培養液中利用糖的有無來調控RNAi的表現，目前我們得到16株水稻轉植細胞株。
我對懸浮細胞做連續多天的缺糖處理，分析水稻轉植細胞株內XT與FT基因的表現量與醣蛋白質的修飾情況。利用Q-RT-PCR分析，缺糖6小時後，水稻轉植細胞株之XT與FT mRNA已經下降，而用西方墨點 (Western blot) 分析醣蛋白的醣化情況，發現缺糖處理12小時後，木醣與岩藻醣添加在醣蛋白的程度，已明顯下降。為了得知分泌至培養基中的蛋白質是否會加上木醣與岩藻醣，分別取細胞缺糖不同時間的培養基來分析醣蛋白質的醣化程度，缺糖24小時後，分泌至細胞外的醣蛋白質已難偵測到木醣與岩藻醣的存在。本研究的目標為利用可誘導性動物醣基化系統的水稻懸浮細胞來生產mGMCSF。

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein and a cytokine that stimulates stem cells to produce granulocytes and monocytes. Recombinant GM-CSF is widely applied in basic medical research. Recombinant mouse GM-CSG (mGMCSF) can be produced by mammalian cells, but the drawbacks of this expression system are high cost and biosafety risk. In our research we choose rice suspension cell system to produce mGMCSF. We have successfully expressed mGMCSF in rice cell. However, plant specific β-1,2-xylosyltransferase (XT) and α-1,3-fucosyltransferse (FT) add xylose and fucose to glycoproteins, respectively, and these two sugar residues may induce human allergic reactions. Based on our experiences with rice expression system, RNA interference (RNAi) was used to reduce XT and two FTs to humanize mGMCSF production in rice cell.
In our original design, the triple RNAi knockdown system was controlled by a strong and constitutive promoter to generate hairpin loops of double strand RNA molecules that are able to continuously induce gene silence for three plant specific glycosyltransferases. However, these enzymes maybe essential for plant cells, and their absences may lead to abnormal physiological responses. To construct an inducible system, α-AMY3 promoter was chosen because of its tight regulation by sugar. Sofar, we have obtained 16 transgenic rice cell lines. At RNA level, FT and XT transcripts were reduced after 6 hours of sugar starvation. At protein level, the secreted glycoproteins contained low β-1,2-xylose and α-1,3-fucose after 12 hours of sugar starvation. After refreshing the sugar free medium and further starving the cells for another 12 hours, we were not able to detect any xylose and fucose in secreted glycoproteins with our inducible RNAi rice cell lines, suggesting that sugar free medium can turn on RNAi to block plant specific N-glycosylation pathways.

摘要 i
Abstract ii
誌謝 iii
表目錄 ix
圖目錄 x
附表目錄 xii
附圖目錄 xiii
一.前言 1
1-1 實驗動機 1
1-2 實驗目的 1
1-3 實驗架構 2
二.文獻回顧 3
2-1 生物醫療蛋白 (biopharmaceutical) 3
2-2 重組蛋白表達系統 3
2-3 植物分子農場 5
2-4 顆粒細胞集落刺激因子 (GM-CSF) 5
2-5 真核細胞的蛋白質醣基化後修飾 6
2-6 醣基對於醣蛋白的重要性 8
2-7 人類化醣蛋白生產系統 8
2-8 植物特有醣基xylose與fucose可能誘發過敏反應 10
2-9 植物醣蛋白的“人類化” : Humanization 11
2-9-1 去除植物特有beta-1,2xylosyltransferase 和 alpha-1,3 fucosyltransferase的策略 12
2-9-1-1 將重組蛋白滯留在ER中 12
2-9-1-2 去除植物特有的醣基化酵素 12
2-9-2 在植物細胞表現哺乳動物特有醣基化酵素 13
2-10 誘導系統 14
2-10-1 持續表現的啟動子 (constitutive promoter) 14
2-10-2 可誘導而表現的啟動子 (inducible promoter) 14
2-11 水稻 15
2-12 水稻懸浮細胞表達系統 15
三.材料方法 17
3-1 實驗設備 17
3-2 實驗藥品 18
3-3 實驗材料 19
3-3-1 實驗水稻 19
3-3-2 水稻培養條件與培養基 19
3-3-3 本研究所用之引子 19
3-3-4 本研究所用之抗生素 20
3-3-5 本研究所用之抗體 20
3-4 實驗方法 20
3-4-1 菌體前培養 20
3-4-2 Plasmid DNA 純化 20
3-4-3 限制酶之切割 20
3-4-4 DNA電泳分析 20
3-4-5 洋菜膠體萃取DNA 20
3-4-6 LR 重組：LR recombination 21
3-4-7 勝任細胞製作 21
3-4-8 細菌轉殖：熱休克 (Heat shock) 21
3-4-9 菌種保存 21
3-4-10 水稻轉殖 22
3-4-10-1 水稻癒傷組織誘導 22
3-4-10-2 農桿菌轉型：電穿孔 (Electroporation) 22
3-4-10-3 農桿菌生化檢測 22
3-4-10-4 水稻感染 22
3-4-10-5 轉殖水稻細胞株篩選 23
3-4-10-6 水稻繼代培養 23
3-4-10-7 水稻再生 23
3-4-11 GUS染色分析 23
3-4-12 Genomic DNA的抽取 23
3-4-13 聚合酶連鎖反應 24
3-4-14 RNA的萃取 24
3-4-15 RNA DNase 處理 24
3-4-16 cDNA的製備 25
3-4-17 逆轉錄聚合酶連鎖反應：RT-PCR 25
3-4-18 即時定量聚合酶連鎖反應：Real-time qRT-PCR 25
3-4-19 蛋白質分析 25
3-4-19-1 蛋白質萃取 25
3-4-19-2 蛋白質濃度測定 25
3-4-19-3 SDS-PAGE 26
3-4-19-4 Comassion blue呈色 26
3-4-19-5 銀染 26
3-4-19-6 西方墨點法 26
3-4-20 水稻原生質體 (protoplast) 的分離 27
3-4-21 利用基因槍於水稻細胞短暫表現重組蛋白基因 27
3-4-21-1 水稻細胞與DNA的準備 27
3-4-21-2 金粒子與DNA附著製備 27
3-4-21-3 基因槍設定 27
3-4-21-4 轉殖植物樣品處理 28
3-4-22 水稻懸浮細胞 28
3-4-22-1 建立水稻懸浮細胞 28
3-4-22-2 繼代水稻懸浮細胞 28
3-4-22-3 測試水稻懸浮細胞生長速率 28
3-4-22-4 減糖處理懸浮細胞 (細胞內蛋白質) 28
3-4-22-5 懸浮細胞減糖處理 (胞外蛋白質) 28
3-4-22-6 懸浮培養液中的澱粉降解 28
3-4-23 碘染 29
四.結果 30
4-1 水稻細胞生產重組蛋白 30
4-1-1 確認重組蛋白p33KD-mGMCSF質體 30
4-1-2 GUS染色分析初步篩選之轉殖水稻細胞 30
4-1-3確認轉殖水稻癒傷組織細胞基因組片段 30
4-1-4 轉殖水稻癒傷組織細胞基因表現情形 30
4-1-5 轉殖水稻癒傷組織細胞重組蛋白表現情形 31
4-1-6 重組蛋白mGMCSF-GFP6在水稻細胞中的表現位置 31
4-2 利用”人類化”植物醣蛋白水稻細胞生產重組蛋白 31
4-2-1 利用已減少植物特有Xylose醣基之水稻細胞生產重組mGMCSF 31
4-2-2 利用已減少植物特有Xylose與Fucose醣基水稻細胞生產重組mGMCSF蛋白 31
4-3 植物特有醣基角色的探討 32
4-3-1 水稻醣蛋白持續缺乏植物特有醣基不影響懸浮細胞的生長 32
4-3-2干擾植物特有醣基的添加減緩水稻懸浮細胞降解澱粉的能力 32
4-3-3干擾植物特有醣基的添加影響水稻植株型態 33
4-4建立Amy 3 promoter: XT RNAi (pAmy-XTi) 誘導系統表達載體 33
4-4-1 確認 RNAi 與目標載體片段 33
4-4-2構築LR重組所需的Entry vector: pENT-XTi 33
4-4-3 確認pAmy-XTi 載體 34
4-5建立Amy-XTi誘導靜默基因轉殖水稻系統 34
4-5-1 確認Amy-XTi轉殖水稻細胞之基因重組片段 34
4-5-2 分析Amy-XTi轉殖細胞中醣基轉移酶基因表現情形 34
4-6 建立Amy 3 promoter: triple RNAi (pAmy-tri) 誘導系統表達載體 35
4-6-1 構築LR重組所需Entry vector：pENT-tri 35
4-6-2 構築pAmy-tri 載體 35
4-7 建立Amy-tri誘導靜默基因轉殖水稻系統 35
4-7-1 確認Amy-tri轉殖水稻細胞之重組基因片段 35
4-7-2 分析Amy-tri轉殖細胞中醣基轉移酶基因缺糖誘導降低表現情形 36
4-8 建立Amy-tri水稻懸浮細胞培養系統 36
4-8-1 分析Amy-tri懸浮轉殖細胞中醣蛋白表現情形 36
4-8-2 Amy-tri懸浮細胞中醣基轉移酶基因的表現情形 37
4-8-3 懸浮細胞中醣蛋白接上醣基的情形 37
4-9 懸浮細胞的胞外分泌蛋白 37
4-9-1 大部分胞外分泌蛋白為醣蛋白 37
4-9-2在胞外培養液中的蛋白質 37
4-9-3 分析胞外培養液中之醣蛋白 38
4-10可誘導醣基化後修飾的轉殖水稻懸浮細胞不影響降解澱粉的能力 38
五.討論 39
5-1 在水稻中表現mGMCSF重組蛋白 39
5-2 在水稻中”人類化”醣蛋白修飾系統表現mGMCSF 40
5-2-1 單一使醣蛋白上植物特有醣基xylose降低之轉殖細胞株 40
5-2-2 使醣蛋白上植物特有醣基xylose與fucose降低之轉殖細胞株 40
5-3蛋白質醣基化的正確性影響植物細胞的生理功能 41
5-4 構築可誘導性改變蛋白質醣基化的RNAi載體 41
5-5 建立可誘導式人類化醣基修飾的RNAi轉殖水稻 42
5-5-1 Amy-XTi 42
5-5-2 Amy-tri 42
5-6 植物醣基化與逆境的關係 43
5-7 醣蛋白上醣基分析 43
5-8 可誘導之”人類化”醣基化水稻懸浮細胞內RNAi的表現情形 44
5-9 可誘導之”人類化”水稻懸浮細胞RNAi對於胞外蛋白質的影響 44
六.結論與未來展望 46
6-1 結論 46
6-2 未來展望 47
七.參考文獻 48
八.附錄 96
8-1 前培養培養基成分 96
8-2 水稻組織培養基成分 96
8-3 分析試劑 102
8-4 本研究所構築DNA載體的定序分析：pAmy-XTi 108
8-5 本研究所構築DNA載體的定序分析：pAmy-tri 109
8-6 農桿菌感染水稻細胞流程 111
8-7 pAmy-XTi表現質體的構築策略圖 112
8-8 pAmy-tri表現質體的構築策略圖 114

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