臺灣博碩士論文加值系統

高血脂症會增加高血壓病人發生心臟病及腦中風的機率，因此有效抑制血脂的升高，為一大重要課題。活性胜肽Val-Val-Tyr-Pro (VVYP)，可抑制腸道吸收三酸甘油脂的主要因子，可經由降低三酸甘油脂濃度達到降血脂的作用。本研究利用基因工程改造大豆貯藏性蛋白Glycinin Gy5與Gy1基因，使之帶有降血脂機能性活性胜肽VVYP之胺基酸序列，即可因食用此具功能性的大豆，藉由腸胃道內酵素分解利用後，達到抑制血脂濃度的目的，因而可開發大豆成為大量生產此活性胜肽之生物工廠。首先選殖Gy5與Gy1作為攜載體，並進一步完成Gy5帶有五套與十套VVYP活性胜肽之載體構築。另外為了要提高外來基因Gy5-VVYP/Gy1-VVYP表現量，嘗試抑制原有的貯藏性蛋白質之表現，因此針對Glycinin group I 之Gy2/Gy3基因，進行靜默化處理。目前已完成RNAi載體構築，正進行基因轉殖。本研究進行Gy5與Gy1抗體的製備。利用西方轉漬法分析發現，Gy5抗體除了對本身具有良好辨識能力，但對Gy1辨識能力較差。經由不同品種大豆進行西方轉漬法分析結果可知，Gy5抗體與Gy1抗體對於Glycinin group I及group II之貯藏蛋白質具有不同的辨識能力，可供未來轉殖植株偵測之用。
利用植物生產醫藥用藥物、抗體與疫苗具有相當多的優點。相較於微生物與細胞培養系統之一般常用的技術，植物具有較低成本與危險性之優勢。本研究的目的在於利用轉殖水稻生產醫藥工業用酵素N-acylamino acid racemase, (NAAAR)，其功能為可將N-acety-D-homopheylalanine (NAc-D-HPA)轉換成N-acety-L-homopheylalanine (NAc-L-HPA)，進一步搭配L-aminoacylase (LAA)，可以生成高血壓抑制物ACEI之重要前驅物L-HPA。本研究利用水稻胚特有基因啟動子Ose705、持續表現型CaMV35S啟動子及水稻肌動蛋白啟動子(actin)啟動NAAAR基因之表現。以抗生素篩選、PCR與西方轉漬法分析後，獲得三株705-NAAAR、兩個35S-NAAAR以及兩個Act-NAAAR轉殖株之同質品系。經由Ninhydrin呈色法初步鑑定其活性，結果顯示由水稻萃取之NAAAR具有將NAc-D-HPA轉換成NAc-L-HPA之能力。並進一步以高效能液相層析儀(HPLC)搭配Chirobiotic T 管柱，證明於移動相methanol/ 0.01 M ammonium acetate=70/30，流速= 0.5 mL/min，可將NAc-D-HPA轉換成NAc-L-HPA。轉殖水稻所生產之NAAAR其最適反應時間為2小時、最適反應溫度為50℃。綜合以上結果，可證實來自Deinococcus radiodurans BCRC12827菌株之NAAAR可於水稻中表現，因此利用轉殖水稻作為生物反應器，可以生產醫藥工業用酵素NAAAR。

Hyperlipidemia can cause the heart disease and the brain stroke. Effective suppression of the amount of cholesterol in blood will help to reduce their potential risks. The bioactive peptides─Val-Val-Tyr-Pro (VVYP) is one of the well known effective materials to suppress the triglyceride absorption of intestinal tract, and to reduce the blood cholesterol. In this study, we engineered transgenic soybean plants to carry bioactive peptides VVYP. The strategy included the construction of soybean storage protein Glycinin Gy5 and Gy1 genes containing VVYP and using intestine protease to release the bioactive peptides when ingested. The constructs of Gy5 containing 5 copies of VVYP or 10 copies of VVYP were complete. In principle, the soybean may contain the fixed amount of storage protein in seeds. In order to increase the engineered VVYP-containing recombinant proteins in transgenic soybeans, using RNA interference approach to suppress the expression of endogenous soybean storage protein can be effective. The RNAi vectors had been constructed and agrobacterium-mediated transformation is ongoing. In addition, this study also prepares antibodies against Gy5 and Gy1 proteins respectively. The western hybridization results revealed that Gy5 and Gy1 antibodies can differentially recognize acidic and basic chains of different groups of glycinin, providing an useful tool to identify the engineered proteins in transgenic soybeans.
Comparing to current microbial and mammalian cell culture systems, plants have many advantages as bioreactors for the production of pharmaceuticals, antibodies and vaccines, such as the less cost of production and no risk of contamination by animal pathogens. The L-homophenylalanine (L-HPA) is a chiral and unnatural amino acid. It was used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. In this study, we engineered transgenic rice plants to produce the pharmaceutical enzyme N-acylamino acid racemase (NAAAR) in seeds. Seven homozygous transgenic rice plants (three 705-NAAAR, two 35S-NAAAR, and two Act-NAAAR) were obtained and confirmed by using hygromycin selection, PCR analysis, Southern blot and western blot analysis. NAAAR combining with L-aminoacylase (LAA) can convert N-acetyl-D-homophenylalanine to L-HPA. The activity assay were performed with high performance liquid chromatography (HPLC) using a chirobiotic T column. The optimal separation condition of NAc-D-HPA and NAc-L-HPA was methanol/ 0.01 M ammonium acetate=70/30 as mobile phase at a flow-rate of 0.5 mL/min. The reaction time and temperature optima of the NAAAR were 2 hrs and 50℃, respectively. This study confirmed that the rice-based-production of NAAAR combining with LAA can be used for the production of L-HPA.

中文摘要……………………………………………………………………...………….i
英文摘要…………………………………………...………………………………........ii
目錄…………………………………………...…………………………………….…..iv
圖表目錄…………………………………………...………………..…….…………....vi
縮寫字對照表……………………………………………………………..……..……viii

前言…………………………………………...………………………………...……….1
第一章 利用大豆大量生產活性胜肽VVYP之研究
前人研究
ㄧ、活性胜肽的發展……………………………………….…….………………...6
二、活性胜肽之吸收與利用………………………………….….………………...7
三、大豆貯藏性蛋白質之研究…………………………………………….……....8
四、以植物或動物表現活性胜肽之相關研究…………..……………...………..10
五、基因轉殖植物之應用…………………………..…………………………….10
材料與方法
一、儀器及設備……………………………………………………...……....……13
二、藥品……………………………….......................……………………………13
三、試驗材料……………………………………………………………...…...….13
四、大豆貯藏性蛋白cDNA之選殖………………………………………....……13
五、表現載體之構築………………………………...……………………………14
六、農桿菌轉殖(agrobacterium-mediated transformation)…………...………...….16
七、蛋白質之大量表現及其抗體製備……………………………...……………17
八、大豆貯藏性蛋白質之分析……………………………...………...………….19
結果
一、大豆貯藏性蛋白Gy5與Gy1之cDNA基因選殖……………….….………..20
二、表現活性胜肽之大豆轉殖載體構築…………………………….…………..21
三、利用能產生雙股RNA之構築抑制Gy2及Gy3基因表現………………......22
四、大豆貯藏性蛋白質Gy1及Gy5之大量表現及其抗體製備………….……..23
五、利用西方轉漬法分析在E. coli中表現之大豆貯藏性蛋白Gy5及Gy1.....24
六、大豆貯藏性蛋白β-conglycinin及glycinin之SDS-PAGE分析……….….24
七、利用西方轉漬法分析大豆不同品種貯藏性蛋白質glycinin中不同族群
acidic及basic片段之分佈情形……………………………..………………....25
討論
一、利用大豆生產活性胜肽之構想、策略及表現載體構築……………………..26
二、抑制大豆內生性貯藏性蛋白基因表現之RNAi 策略及載體構築………..27
三、大豆貯藏性蛋白及帶有VVYP外源蛋白之偵測及分析……………….……28
參考文獻………………………………………………………………………...……..29
表………………………………………………………………………………...……..35
圖………………………………………………………………………………...……..36
附錄…………………………………………………………………………...………..59

第二章 利用轉基因水稻表現醫藥工業用酵素N-acylamino acid racemase之研究
前人研究
ㄧ、腎素-血管收縮素-醛固酮系統(renin-angiotensin-aldosterone system,
RAAS)………………………………………………………………………….63
二、血管收縮素轉化酶抑制劑的發展……………...…..…………………….........64
三、L-homophenylanine (L-HPA)之合成法……………………………………....65
四、N醯化胺基酸消旋酶(N-acylamino acid racemase, NAAAR)之相關
研究………………………………………………………………….......…..65
五、生物反應器………………………………………………..………………….66
六、啟動子之選擇……..………………………………………………………….67
材料與方法
一、儀器及設備……………….…………..…………………………………..……69
二、藥品及材料…………………………………………………………….………69
三、水稻基因組DNA之萃取……………..………………………………..……69
四、NAAAR蛋白質於E. coli菌體中之大量表現與純化…….………70
五、純化轉基因水稻穀粒之NAAAR目標蛋白………….……..…………….70
六、轉殖基因水稻生產NAAAR目標蛋白之酵素活性分析………....………71
結果
一、同質轉殖水稻品系之篩選….…………………………………………..73
二、轉殖基因水稻生產NAAAR目標蛋白之純化……...…………..…..75
三、轉殖基因水稻生產NAAAR目標蛋白之酵素活性分析……………..75
討論
一、同質轉殖水稻品系之篩選……………….…….………………………..77
二、轉殖基因水稻生產NAAAR目標蛋白之純化….……………………..78
三、轉殖基因水稻生產NAAAR目標蛋白之酵素活性分析…......………78
參考文獻……………………………………………………………………...………..80
表…………………………………………………………………………………...…..84
圖………………………………………………………………………………...……..85

王升陽、徐麗芬、楊寧蓀 (2005). 植物基因轉殖在醫藥上之應用. P.219-229. 植
物基因轉殖之原理與應用. 植物生物技術教學資源中心主編 2005年5
月再版

王月華、孫鵬凱、余淑美 (2005). 植物基因轉殖技術之研發與展望. P.7-23 植物
基因轉殖之原理與應用. 植物生物技術教學資源中心主編 2005年5月
再版
陳明豐 (2004). 高血壓防治手冊—高血壓偵測、控制與治療流程指引.行政院衛
生署國民健康局台灣內科醫學會2004年3月出版
陳建仁、游山林、白其卉、蘇大成、曾慶孝、簡國龍、黃麗卿 (2003).台灣地區
高血糖、高血脂、高血壓盛行率調查期末報告.P.58-83. 行政院衛生署國
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生署國民健康局台灣內科醫學會2004年3月出版
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李寬志 (2000) 轉基因水稻生產D-hydantoinase之研究，中興大學分子生物學研
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張繼中 (2002) 轉殖水稻生產豬乳鐵蛋白之研究：同質品系篩選、蛋白質純化及
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涂倉榮 (2000) Ose705及Ose719基因啟動子之分析，中興大學分子生物學研究所
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曾志偉 (2005) 利用轉基因水稻表現醫療工業用酵素L-N-Carbamoylase以生產
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