博碩士論文 982204016 詳細資訊




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姓名 葉如芳(Ju-fang Yeh)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討αAmy3、OsCIN1與Os33KD信號肽在水稻懸浮培養細胞中的功能及特性
(Characterized analysis of αAmy3, OsCIN1 and Os33KD signal peptide in rice cell suspension culture)
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摘要(中) 生物工程技術的應用有效地提供了重組蛋白質的生產。由不同類型的寄主细胞所生產的重組蛋白質可能會有不同的生物活性和蛋白質的穩定度,尤其是醫藥蛋白等高價值蛋白質。若要生產具有完整生物活性的高價值重組蛋白質,則以具備完整蛋白質後修飾作用的哺乳類動物細胞和植物寄主细胞作為生產平台之優先考量。在過去幾年,植物分子農場被認作為是一個兼具便利和濟策略表達外源蛋白質生產平台,例如抗體,醫藥蛋白,細胞素等等。水稻細胞生產系統為一有效率的重組蛋白質生產系统。為了要改良重組蛋白質在水稻細胞生產系統中之分泌效率,三種不同的信號肽包括αAmy3,OsCIN1 和Os33KD 融合到GFP 和mGMCSF 基因之N 終端序列,藉由分析轉殖細胞內外的重組蛋白質累積量,以探討三種信號肽之分泌效率。包含有三種不同信號肽的GFP 轉殖細胞株被建立,並且細胞內蛋白質和細胞外蛋白質由西方點墨法或FLUOROSK ANASCENTFL
來偵測重組蛋白質的累積量,而每一種信號肽之分泌效率在各自獨立的穩定轉殖水稻懸浮細胞株中進行比較。實驗結果顯示,Os33KD 信號肽相對其他兩者而言在GFP 水稻細胞懸浮培養有糖中有最高的分泌效率,而αAmy3 和OsCIN1 信號肽在缺醣處理下顯示了在缺醣情況下其分泌效率有被提升的現象。因此我們發現,在有糖誘導表現量較強的Ubiquitin 啟動子,使用αAmy3 和OsCIN1 信號肽會有較佳的分泌比例的表現。藉由對信號肽更多的探討及研究,我們能夠知道不同的生長環境可能會影響信號肽之分泌功能,深入認識信號肽之特性能夠幫助我們在不同的生長條件下選擇不同的信號肽,以期在植物細胞生產平台能夠達到更大量的重組蛋白質之產量。
摘要(英) Application of biotechnical approach has provided to be useful in the production of
recombinant proteins. Recombinant proteins produced from different types of host
cell have various biological activities and protein stability. To produce bioactive
recombinant protein, mammal and plant host cells were used due to they have
post-translational modification. In the past few years, plant molecular farming is
considered as a convenient and economical production strategy to express foreign
proteins, such as antibody, pharmaceutical medicine, cytokine, and so on. The rice
cell production system is a useful recombinant protein production system. To
improve the secretion efficiency of recombinant proteins in rice expression system,
different signal peptides including αAmy3, OsCIN1 and Os33KD were fused to the
N-terminal of GFP and mGMCSF respectively to explore the secretary efficiency of
foreign proteins. Three different signal peptides fused to N-terminal GFP in
transgenic rice cell lines were generated, and the intracellular proteins and
extracellular proteins was detected by western blotting or FLUOROSKAN
ASCENTFL. Therefore, the secretion efficiency of each signal peptide can be
compared among different stable transformed rice suspension cell lines. The result
showed that the Os33KD signal peptide has the highest secretion ratio in GFP
transgenic rice cell suspension culture containing sucrose, but the αAmy3 and OsCIN1 signal peptides showed an increase in secretion during sugar starvation
treatment. The efficiencies of signal peptides that are able to secret more
recombinant proteins into culture medium is different according to the growth
condition, therefore we can choose the suitable signal peptide in rice suspension cell
bioreactor.
關鍵字(中) ★ 信號肽
★ 水稻懸浮培養細胞
關鍵字(英) ★ signal peptide
★ rice cell suspension culture
論文目次 圖目錄 IVV
表目錄 IVV
結果圖目錄 ........................................................................................................................... IVV
第一章 前人研究 ................................................................................................................ 1
一、 基因工程 ............................................................................................................ 1
二、 重組蛋白質在現今各領域之應用 .................................................................... 2
1. 在醫學研究方面: ........................................................................................ 2
2. 在農業方面: ................................................................................................ 3
3. 在工業及環境污染防治方面: .................................................................... 3
三、 重組蛋白質表達系統 ........................................................................................ 4
1. 大腸桿菌表達系統 ........................................................................................ 5
2. 酵母菌表達系統 ............................................................................................ 5
3. 昆蟲表達系統 ................................................................................................ 6
4. 哺乳類動物表達系統 .................................................................................... 7
5. 植物表達系統 ................................................................................................ 8
四、 詳述植物表達系統在現今各領域中之發展 .................................................... 9
五、 植物表達系統的改良策略 .............................................................................. 11
1. 啓動子的改良 (promoter) ......................................................................... 11
2. 內插子 (intron) ........................................................................................... 12
3. 信號肽 (signal peptide) .............................................................................. 12
4. 水稻懸浮培養系統 (rice cell suspension culture system) ....................... 13
第二章 研究目的及相關背景說明 .................................................................................. 15
一、 研究動機與目的 .............................................................................................. 15
二、 研究策略 .......................................................................................................... 15
1. 表達載體的構築 .......................................................................................... 15
2. 建立轉殖水稻細胞株 .................................................................................. 20
3. 重組蛋白質之定量定性分析 ...................................................................... 21
第三章 MATERIALS AND METHODS .......................................................................................... 22
1. Establishment of GFP and mGMCSF transgenic rice cell lines ................. 22
1.1 Rice callus induction ................................................................................... 22
1.2 Transformation competent Agrobacterium strain EHA105 .................... 22
1.3 Transformation by particle bombardment .............................................. 22
1.4 Extraction of rice genomic DNA and RNA .............................................. 23
1.5 Rice genomic PCR and RT-PCR ............................................................... 24
1.6 GUS staining ............................................................................................... 24
III
2. Analysis of expression recombinant protein ................................................ 25
2.1 Establish transgenic rice suspension culture system ............................... 25
2.2 Western blotting .......................................................................................... 25
2.3 GFP-specific detection by FLUOROSKAN ASCENTFL ....................... 26
第四章 結果 ............................................................................................................................ 27
一、 高效率信號肽融合GFP 表現量細胞之篩選與確認 .................................... 27
1. 建立信號肽融合之GFP 轉殖細胞 ............................................................ 27
2. 轉殖水稻細胞株信號肽融合GFP 蛋白質之分泌效率分析 .................... 28
3. 轉殖水稻細胞株信號肽融合GFP 蛋白質在有醣缺醣情況下分泌效率之
分析 30
二、 高效率信號肽融合mGMCSF 表現量細胞之篩選與確認 .......................... 32
1 建立信號肽融合之mGMCSF 轉殖細胞 .................................................. 32
2 轉殖水稻細胞株信號肽融合mGMCSF 蛋白質之分泌效率分析 .......... 33
第五章 討論 ............................................................................................................................ 35
第六章參考文獻 .................................................................................................................... 54
附錄一、實驗所使用之引子序列 .......................................................................................... 63
附錄二、實驗信號肽序列及其胺基酸組成 .......................................................................... 64
附錄三、研究材料配置 .......................................................................................................... 65
I Culture Medium ........................................................................................................ 65
II Analysis reagent ....................................................................................................... 69
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指導教授 陸重安(Chung-an Lu) 審核日期 2011-9-14
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