博碩士論文 952204025 詳細資訊




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姓名 賴穎慧(Ying-Hui Lai)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 以水稻懸浮培養細胞蛋白質生產系統生產mGMCSF
(Production of the mGMCSF in rice cell suspension culture)
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摘要(中) 自從1970年成功的利用大腸桿菌生產胰島素後,越來越多種醫藥蛋白質正開發中,除了微生物以外還有真菌、昆蟲、哺乳類動物…等生物都被作為表達系統。近年來,植物表達系統因為生產成本低,具有完整轉譯後修飾作用,且較無安全性顧慮等特性,已經被應用於生產許多不同的蛋白質,其中水稻細胞生產系統為重要的植物表達系統之一。為了讓我們的水稻表達系統生產更有效率,我們在目標蛋白質的N端附加上一段具分泌特性的信號肽,藉此將目標蛋白質運送至細胞外。我們選用了水稻蛋白質αAmy3、CIN1(cell wall invertase1)以及33kD的信號肽分別接上GFP作為報導基因以及mGMCSF作為我們的目標基因,並建立GFP以及mGMCSF分別融合三組信號肽的T1轉植水稻細胞系。利用西方點墨法偵測蛋白質在細胞中以及在液態培養基中的含量,並比較三種信號肽的分泌效率,分析的結果發現CIN1之信號肽在GFP和mGMCSF的轉植株中都有最佳的分泌效率,而33KD信號肽在轉植株中有次佳的分泌效率。另外,我們也得知Amy3啟動子中插入A1 intron片段比插入Ubi intron片段的具有更強表現mGMCSF 基因的能力。未來希望藉由集結這具有最佳分系效率的信號肽搭配最有效率的啟動子,以及最有效抑制懸浮培養系統中的蛋白水解酶的方式,發展一套高表現量的水稻細胞表達系統。
摘要(英) After the production of insulin by E. coli in 1970s, more and more therapeutical proteins were produced by different organisms, such as microorganism, insect and mammal. In the past few years, the plant expression system had been used to produce many kinds of recombinant proteins because of low cost, safety and post-translational modification. The rice cell production system is one of the most efficient plant expression systems. To improve the yield of the rice cell expression system, different signal peptides including αAmy3, CIN1 and 33kD have fused to the N-terminal of GFP or mGMCSF to explore the secretory efficiency of foreign proteins. Transgenic rice containing fusion proteins have been generated, and the intracellular and extracellular recombinant proteins were determined by western blotting to compare the secretory efficiency of three different signal peptides.. We found that CIN1 is the most efficient signal peptide for the secretion of both GFP and mGMCSF. On the other hand, we found that the addition of A1 intron in the 5’ end of fusion protein give rise to better secretory efficiency than the Ubi intron in the rice cell expression system. In the further, we would like to combine the most efficient signal peptide, the strong promoter and the inhibition of proteolysis to develop highly protein expression system in rice 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-----------------------------------------------------------------------------------------V
表目錄------------------------------------------------------------------------------------------VII
圖目錄------------------------------------------------------------------------------------------VII
壹、研究動機-------------------------------------------------------------------------------------1
貳、序論-------------------------------------------------------------------------------------------1
1. 基因工程(Genetic engineering)-----------------------------------------------------------1
2. 生技醫藥的發展(Biomedical drugs)-----------------------------------------------------1
3. 重組蛋白質表達系統(recombinant protein expression system)---------------------2
3.1 原核生物表達系統 ( prokaryotic system)-----------------------------------------------2
3.2 酵母菌表達系統(yeast system)-----------------------------------------------------------3
3.3昆蟲表達系統 (insect system)------------------------------------------------------------3
3.4 哺乳動物表達系統 (mammalian system)----------------------------------------------4
3.5 植物表達系統 (plant system)------------------------------------------------------------5
4 植物系統應用於醫療上的發展------------------------------------------------------------6
5. 農場製藥的進展-----------------------------------------------------------------------------6
5.1 啟動子的改良-------------------------------------------------------------------------------6
5.2. 內插子(intron)----------------------------------------------------------------------------------7
5.3. 信號肽(signal peptide)-------------------------------------------------------------------8
5.4. 水稻懸浮培養表達系統------------------------------------------------------------------9
5.5. 水稻中的信號肽-------------------------------------------------------------------------10
5.6. 重組蛋白質的純化與分析-------------------------------------------------------------11
6. 綠色螢光蛋白 (Green Fluorescent Protein)--------------------------------------------12
7. 顆粒細胞一巨噬細胞集落刺激因子(granulocyte-macrophage colony
stimulating factor,GM-CSF)-------------------------------------------------------------12
8. 植物表達系統生產的GM-CSF ---------------------------------------------------------13
9. 交接&實驗綱要----------------------------------------------------------------------------14
10. 實驗目的-----------------------------------------------------------------------------------14
參、研究方法與材料---------------------------------------------------------------------------15
1. 轉植株的建構、篩選及培養-------------------------------------------------------------15
2. 水稻轉植株的再生-------------------------------------------------------------------------15
3. 轉殖基因的確認----------------------------------------------------------------------------15
4. 基因組DNA之PCR篩選----------------------------------------------------------------15
5. 自水稻未成熟的胚誘導出癒傷組織(callus)----------------------------------------16
6. 建立水稻懸浮細胞培養系統 -----------------------------------------------------------16
7. 蛋白質的收集-------------------------------------------------------------------------------16
8. 蛋白質的定量 (Bradford, 1976) -------------------------------------------------------16
9. 蛋白質的濃縮 (SDS-PAGE sample) ---------------------------------------------------17
10. 西方墨點法分析(Western blot)-------------------------------------------------------17
11. 呈色反應-----------------------------------------------------------------------------------17
12. 酵素免疫吸附分析法(ELISA)-------------------------------------------------------18
13. RNA的抽取--------------------------------------------------------------------------------18
14. RNA 純化 (去除DNA污染)-------------------------------------------------------18
15. 反轉錄PCR (Reverse-transcription PCR) --------------------------------------------19
肆、結果-----------------------------------------------------------------------------------------20
第一部分
1. 信號肽融合GFP轉殖株的表現分析--------------------------------------------------20
1.1 轉植株子代的取得及水稻細胞懸浮培養系統的建立-----------------------------20
1.2.T0代GFP轉殖水稻細胞系的蛋白質表現分析--------------------------------------20
1.3 T2代GFP轉殖水稻細胞系的蛋白質表現分析--------------------------------------20
1.4 T2代GFP轉殖水稻細胞系的RNA表現分析----------------------------------------20
第二部分
2. 信號肽融合mGMCSF之轉殖株的表現分析-----------------------------------------26
2.1 建立子代植株與轉殖表現的篩選及水稻細胞懸浮培養系統的建立-----------26
2.2 T0代mGMCSF轉殖水稻細胞系的蛋白質表現分析-------------------------------26
2.3 T1代mGMCSF轉殖水稻細胞系的蛋白質表現分析-------------------------------26
2.4 T1代mGMCSF轉殖水稻細胞系的RNA表現分析--------------------------------27
第三部分
3. 轉植株αAmy3(P)-(In)-αAmy3(SP)-mGMCSF的表現分析------------------------35
3.1 T1代轉殖水稻細胞系的獲得與水稻細胞懸浮培養系統的建立-----------------35
3.2 T1代αAmy3(P)-(In)-αAmy3(SP)-mGMCSF轉殖水稻細胞系蛋白質表現分析35
3.3 T1代αAmy3(P)-(In)-αAmy3(SP)-mGMCSF轉殖水稻細胞系RNA表現分析-35
3.4 αAmy3(P)-A1(In)-αAmy3(SP)-mGMCSF在缺糖五天中蛋白質與RNA表現36
伍、討論-----------------------------------------------------------------------------------------44
陸、參考文獻------------------------------------------------------------------------------------46
柒、附錄------------------------------------------------------------------------------------------51
附錄一 質體建構圖(Ubi::GFP) ------------------------------------------------------------51
附錄二 質體建構圖(Ubi::mGMCSF) -----------------------------------------------------52
附錄三 質體建構圖(αAmy3::mGMCSF) -----------------------------------------------53
附錄四 本研究所使用之引子序列---------------------------------------------------------54
附錄五 信號序列及其氨基酸組成---------------------------------------------------------55
附錄六 mGMCSF的醣化作用預測--------------------------------------------------------56
附錄七 研究材料配製------------------------------------------------------------------------57
附錄八 重組蛋白質mGM-CSF相關研究-------------------------------------------------66
表目錄
表一 醫藥蛋白表達系統之比較-------------------------------------------------------------8
表二 生產GM-CSF蛋白質之表達系統比較------------------------------------------------13
圖目錄
附圖一、信號肽攜帶蛋白質與胞器模上受器反應機制-----------------------------------9
附圖二、蛋白質N端信號肽序列之性質分析------------------------------------------------11
圖一、T0代GFP轉殖水稻細胞系的蛋白質表現分析--------------------------------------22
圖二、T2代GFP轉殖水稻細胞系在懸浮培養中蛋白質的表現-------------------------23
圖三、T2代GFP轉殖水稻細胞系在懸浮培養中蛋白質的表現-------------------------24
圖四、T2代GFP轉殖水稻細胞系在懸浮培養中RNA的表現----------------------------25
圖五、再生技術流程圖-----------------------------------------------------------------------------28
圖六、T1代mGMCSF轉殖水稻細胞系細胞內的蛋白質表現---------------------------29
圖七、T0代mGMCSF轉殖水稻細胞系細胞內蛋白質mGMCSF的表現------------30
圖八、T1代mGMCSF轉殖水稻細胞系的蛋白質表現分析------------------------------31
圖九、T1代mGMCSF轉殖水稻細胞系的蛋白質表現分析------------------------------32
圖十、T1代mGMCSF轉殖水稻細胞系的蛋白質表現分析------------------------------33
圖十一、T1代mGMCSF轉殖水稻細胞系的RNA表現分析---------------------------34
圖十二、轉植株αAmy3(P)-(In)-αAmy3(SP)-mGMCSF在有糖缺糖下的蛋白質
表現分析---------------------------------------------------------------------------------37
圖十三、T1代αAmy3(P)-(In)-αAmy3(SP)-mGMCSF的蛋白質表現分析------------38
圖十四、T1代αAmy3(P)-(In)-αAmy3(SP)-mGMCSF的蛋白質表現分析------------39
圖十五、T1代αAmy3(P)-(In)-αAmy3(SP)-mGMCSF缺糖三天蛋白質表現分析---40
圖十六、T1代αAmy3(P)-(In)-αAmy3(SP)-mGMCSF缺糖三天的RNA表現分析--41
圖十七、αAmy3(P)-A1(In)-αAmy3(SP)-mGMCSF在缺糖五天中蛋白質表現分析42
圖十八、αAmy3(P)-A1(In)-αAmy3(SP)-mGMCSF在缺糖五天中RNA表現分析--43
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指導教授 陸重安(Chung-An Lu) 審核日期 2009-1-19
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