臺灣博碩士論文加值系統

中文摘要
本研究的目的係將人類血清蛋白(human serum albumin, HSA)轉殖於阿拉伯芥中表現。近二十年來，由於生物技術及基因轉殖技術的蓬勃發展，使許多科學家將具有醫療用途的蛋白質表現於植物體中。而從植物生產醫藥用品不僅生產成本相對地較低，最重要的可減少使用從人體或動物細胞萃取醫藥用品受到未知病毒污染的潛在危險性。吾人為了使人類血清蛋白能夠表現於阿拉伯芥細胞內、細胞外，及可為人體腸道細胞吸收，分別構築三種表現載體：pHSA-1、pHSA-2及pHSA-3。pHSA-1含prepro HSA基因；pHSA-2則含成熟之HSA，並在其N-端接上一段signal peptide以利分泌於胞外；pHSA-3除了成熟之HSA外，亦於N-端接上霍亂毒素B次單元(CTB)及signal peptide，而所接上之CTB可幫助HSA於腸道細胞之吸收。我們分別將pHSA-1、pHSA-2及pHSA-3轉形入農桿菌品系LBA4404，然後將LBA4404 (pHSA-1)、LBA4404 (pHSA-2)及LBA4404 (pHSA-3)轉殖入阿拉伯芥中。我們共得pHSA-1轉殖種子1669顆、pHSA-2轉殖種子2268顆及pHSA-3轉殖種子3124顆。經種植於含hygromycin 15mg/l的1/2 MS培養基後，篩選三週後轉殖HSA-1植株僅70棵存活，轉殖HSA-2植株僅59棵存活，轉殖HSA-3植株僅126棵存活，我們將這些生長情況較佳的植株轉移到不含hygromycin的1/2 MS培養基中培養兩週後，再由培養基中移出種植於培植土中繼續長大。吾人選取生長情況良好的基因轉殖株共45棵；其中轉殖HSA-1有6棵，轉殖HSA-2有2棵，轉殖HSA-3有37棵。抽取其genomic DNA，並以PCR增殖與南方吸漬法進行偵測。結果顯示PCR增殖並未觀察到有HSA DNA片段被增殖出，而南方吸漬法也未找到具有轉殖HSA基因的植株。

Abstract
The goal of this study is to produce human serum albumin (HSA) in Arabidopsis. For the past two decades, many scientists used biotechnology to produce biopharmaceutical proteins in transgenic plants. Using plants as a factory for producing biopharmaceutical proteins may avoid pollution of unknown viruses concomitance with the extraction of proteins from human or animals by traditional extraction methods. In addition, the cost for producing biopharmaceutical proteins in crops on an agricultural scale may be less than that by traditional methods. To make an edible HSA, we constructed the following vector: pHSA-1, pHSA-2, pHSA-3. The pHSA-1 contains the preproHSA gene in pCAMBIA1302, a disarmed Ti vector. The pHSA-2 contains a signal peptide on the N-terminus of the mature HSA. The signal peptide comes from the gene encoding thaumatin-like proteins, a pathogenesis-related protein from oats. The pHSA-3 contains a cholera toxin B subunit (CTB) on the N-terminus of the mature HSA. These three clones were transformed into Agrobacterium strain LBA4404. We then introduced the different clones of HSA gene into Arabidopsis by Agrobacterium-mediated transformation. A total of 1669, 2268, and 3124 transgenic seeds were obtained from Arabidopsis transformed with LBA4404 (pHSA-1), LBA4404 (pHSA-2), and LBA4404 (pHSA-3), respectively. After grown in medium containing hygromycin 15 mg/l for 3 weeks, 70, 59, and 126 plants transformed with HSA-1, HSA-2, and HSA-3, respectively, were survived from selection. These plants were then cultured in MS medium without hygromycin for another 2 weeks. The plants were then transferred to pots and incubated in growth chambers till the plants grew up. We chose 45 putative transgenic plants for PCR and southern blot analyses. Of these 45 plants, 6, 2, and 37 plants come from plants transformed with HSA-1, HSA-2, and HSA-3, respectively. However, no signal was detected by PCR nor southern blot analyses.

目錄
中文摘要.......................I
Abstract......................II
目錄..........................IV
一、研究背景及目的.............1
二、研究材料與方法............12
三、結果......................24
四、討論......................28
五、參考文獻..................31
圖表..........................37
附錄封面......................53
附錄中文摘要..................54
附錄Abstract..................55
一、附錄研究背景及目的........56
二、附錄研究材料與方法........61
三、附錄結果與討論............69
四、附錄參考文獻..............72
附錄圖表......................74

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