類別:

Ｃ型肝炎病毒蛋白對細胞內甲型干擾素反應可能之影響

2021-06-13T04:28:57Z

標題: Ｃ型肝炎病毒蛋白對細胞內甲型干擾素反應可能之影響; The Possible Effects of HCV Proteins on Cellular IFN-alpha Responses 作者: Tsai-Yi Lu; 呂采宜摘要: C型肝炎病毒（HCV）的感染已經成為公共衛生以及傳染病學界的重要課題之一。根據世界衛生組織的統計，全世界約有百分之三的人為HCV的帶原者，其中高達百分之八十的人在二十到三十年之後會發生肝硬化，甚至最後死於HCV所引起的肝癌。目前僅有長效型的甲型干擾素（IFN-α）合併抗病毒藥物Ribavirin可治療HCV的感染，但是成效最多只有百分之五十至六十，而且隨著病毒基因型的不同也會有所差異。本研究主要從兩方面來探討造成HCV無法被IFN-α清除的分子機制。首先，我們確認HCV的NS3/4A、NS4B和NS5A蛋白質具有抑制IFN-α對下游JAK-STAT signaling活化的能力。在檢驗每個活化過程的步驟之後，發現NS4B可以阻止ISGF3 complex和DNA的結合，因而導致ISRE主導的基因轉錄無法被啟動。另一方面，我們也重新檢視core在ISRE活化過程當中的影響。在七個從病人血清裡分離出來、分屬基因型1b和2a的core clone當中，每個clone對於ISRE的活化和下游ISG的表現都有不同程度的影響。於是我們比對這七個core的胺基酸序列，並推測某些胺基酸的改變是造成此現象的原因。根據以上的結果，我們認為更多關於NS4B對抗IFN-α訊息傳遞所使用機制的研究，將有助於開發對抗HCV感染的新方法；同時我們也要指出，研究core胺基酸序列的改變與其如何影響JAK-STAT signaling的關係，將有助於釐清core在HCV對抗IFN-α時所扮演的角色。; Hepatitis C virus (HCV) infection is one of the major problems of public health worldwide. Approximate 3% of the world’s population are chronically infected and may gradually develop chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (HCC) in the subsequent 20 to 30 years. IFN-alpha is one of the cytokines produced by most animal cells to resist viral replication and is by far the most standard therapy for HCV infection. However, the overall achievement of eliminating HCV only reaches 50-60% and is significantly altered by the virus genotypes. Here we investigated the underlying mechanisms from two aspects. First, we investigate the mechanisms that NS3/4A, NS4B, or NS5A used to inhibit the IFN-alpha-induced responses. The signaling of ISRE activation was examined step by step. It was found that NS4B significantly reduced the DNA binding ability of ISGF3. Second, we examined the effects of core on the ISRE activity using seven clones derived from patient sera infected by HCV genotype 1b or 2a. Distinct properties on ISRE activity and expression of ISGs were demonstrated clone by clone, and some putative amino acid changes were proposed to be responsible for these obscure results. We thus suggested that HCV NS4B deserved more attention for the development of new anti-HCV strategies and when attempting on unraveling the character of core, one should beware of the influence caused by every single amino acid substitutions.

鼠模式中高脂飲食、肥胖與脂質調控基因之表現

2021-06-13T05:52:02Z

標題: 鼠模式中高脂飲食、肥胖與脂質調控基因之表現; High-fat diets, obesity and lipid regulated gene expressions in rodent model 作者: Shan-Ching Hsu; 許珊菁摘要: 過氧化體增殖劑活化受器 (peroxisome proliferators-activated receptor, PPAR)，是固醇核受器的成員。PPAR有α、β、γ三種isoform，其中PPARα與PPARγ分別對脂肪代謝、運輸、生合成、儲存與血糖恆定具有調節作用。脂肪酸是PPARα ligands，脂肪酸的濃度、種類與結構透過PPARα調節脂肪代謝有其不同的影響。本論文研究，主要目的在探討飲食油脂、肥胖、PPAR及脂質代謝相關基因表現之關聯性。已知18:1 (n-9) 是PPARα ligands，能活化PPARα傳訊途徑。本論文研究採用富含高18:1 (n-9) 的紅花籽油作為實驗用油。第一部份，將含5%、20%或30%紅花籽油實驗飼料 (5S、20S、30S) 分別餵食 Wistar大鼠13週或C57BL/6J小鼠22週。發現此兩種齧齒類對此二種高油脂飲食反應不同：30S使大鼠肥胖，但20S使小鼠肥胖，30S對小鼠則否。高脂飲食雖有增進PPARα下游基因表現之趨勢，但以肥胖鼠（30S大鼠與20S小鼠）肝中PPARα與其下游相關基因的表現增高最明顯。推測高脂飲食確實可能因提供較多PPARα ligands，而使 PPARα下游基因表現增加。但造成肥胖後，因PPARα本身的表現也增加，使得整個PPARα傳訊途徑的增加最為明顯。此外，血清leptin濃度反應脂肪量，但不能反應攝食量。而30S造成Wistar大鼠肥胖與20S造成C57BL/6J小鼠肥胖，均同時呈現明顯之血糖與血脂異常現象。第二部份實驗探討餵食不同飽和度油脂是否會因體內脂肪酸作為PPARα ligands的種類不同，進而影響PPARα傳訊途徑。將含5B (4% 奶油與1%紅花籽油)、30B (29%奶油與1%紅花籽油) 、5S (5%紅花籽油) 與30S (30%紅花籽油) 飼料分別餵食大鼠13週後，可見30B組鼠之葡萄糖耐受度顯著劣於對照組，且血清三酸甘油酯濃度亦顯著較30S組高。此時將30B組隨機分成二組，一組維持原飼料，另一組則更換為30%紅花籽油 (30B/S)，繼續飼養。至30B/S組葡萄糖耐受度顯著改善，即進行動物犧牲，共歷時15週。研究結果顯示不論飽和或不飽和之高油脂飲食均造成鼠肥胖，且伴有肝臟PPARα傳訊途徑表現增加，但此表現增加量在30B組低於30S組，顯示不飽和油脂對PPARα下游基因表現之促進活性比飽和油脂強。有趣的是，在脂肪組織觀察到30S組比30B組有較低的lipoprotein lipase, adipocyte P2、fatty acid synthase、acetyl-CoA carboxylase與sterol regulatory element binding protein -1c的mRNA表現，且腹脂重量亦較低。在肝臟更觀察到30S組比30B組有較高的acyl-CoA oxidase (ACO), carnitine palmitoyl-transferance 1A, fatty acid binding protein (FABP) 及 mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase的mRNA表現。暗示不飽和度高之紅花籽油使鼠脂肪組織中有較低之脂肪生合成與肝臟中有較高的脂肪酸氧化。在30S組的肝臟與脂肪組織中皆發現有顯著較高的18:2 (n-6) 與20:4 (n-6) 含量，約為30B組的2倍。這些結果指出高不飽和油脂飲食抑制體內生脂作用及脂肪堆積或許是與組織中有較高的18:2 (n-6) 與20:4 (n-6) 含量有關。此外，血清leptin濃度與肝臟PPARα mRNA呈正相關。肥胖鼠具有hyperleptinemia特徵，且同時具有顯著較高的肝臟PPARα與其下游基因表現。是否leptin會促進肝臟PPARα之表現? C57BL/6J小鼠經腹腔注射recombinant mouse leptin (1 mg/kg body weight/day)，長期14天與短期1.5小時。腹腔注射leptin會降低小鼠攝食量，因此增加一組對飼育 (pair feeding) 組 (PF組)，控制其攝食量與leptin組相同。leptin投予14天，小鼠肝臟中PPARα、ACO與FABP的mRNA表現顯著較PF組增加 (P < 0.05)。實驗證明leptin濃度調控PPARα並增加其下游基因表現。本論文建立一個飲食誘發肥胖與胰島素抗性的動物模式且提供了支持性的證據：飲食脂肪酸之質與量，可能透過影響體內脂肪酸組成，而藉由PPAR與SREBP等轉錄因子調控其下游基因的表現，來影響脂質代謝與生合成。另外，肥胖鼠呈現hyperleptinemia及肝臟PPARα與其下游基因表現增加之現象。由於外源性投予leptin也會增加PPARα轉錄因子本身的表現，推測高脂飲食造成肥胖，促使脂肪組織釋出leptin，進而增進肝臟PPARα表現。而高不飽和油脂飲食提供不飽和脂肪酸作為PPARα之ligands，可更有效提高PPARα傳訊途徑，增進脂肪酸氧化，降低血脂、肝脂與腹脂。; Fatty acids are known to regulate lipid metabolism at the level of gene expression. Peroxisome proliferator activated receptors (PPAR) is the first transcription factor identified as a mediator of fatty acid-regulated gene expression. PPARs regulate the metabolism, transportation, and storage of fat and glucose homeostasis. Common fatty acids are ligands of PPARs. High-fat diets provide more fatty acids that presumably could enhance lipid catabolism through an up-regulation of PPARα signaling. However, high intake of fat could also lead to obesity. This study is aimed at examining the mRNA expressions of PPARα and related genes involved in lipid metabolism in high-fat diets feeding and the resulted obesity.The first experiment examined the hepatic mRNA expression of PPARα and some of its target genes in Wistar rats and C57BL/6J mice fed 2 levels (20% or 30% wt/wt, the 20S or 30S diets) of high oleic acid-rich safflower oil (ORSO) diets until animals showed significantly higher body weight (13 wk for rats and 22 wk for mice) than those of control groups fed a 5% ORSO diet. At the end of these respective feeding periods, only the 30% SFO fed rats and the 20% SFO fed mice among the two high fat fed groups showed significantly higher body weight, white adipose tissue weight, serum leptin concentration and the mRNA expression of PPARα (P < 0.05) compared to the respective control group. Despite of the elevated acyl-CoA oxidase (ACO, a PPARα target gene) protein and activity in both of the two high fat fed groups, the mRNA expression level of most PPARα target genes examined correlated mainly to PPARα mRNA levels and not to fat intake or liver lipid levels. The second experiment compared a high ORSO diet and a high butter diet for their effect on adipose mass and expressions of genes regulated by PPAR and SREPB-1c. Four groups of Wistar rats were respectively fed 30S (30% ORSO), 5S (5% ORSO), 30B (29% butter plus 1% ORSO), or 5B (4% butter plus 1% ORSO) diets for 15 weeks. Compared to the 30B group, the 30S group had significantly less retroperitoneal adipose (RWAT) mass and lower mRNA expressions of lipoprotein lipase, adipocyte P2, fatty acid synthase and sterol regulatory element protein-1c (SREBP-1c) in the RWAT, higher mRNA expressions of ACO, carnitine palmitoyl-transferance 1A, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase and fatty acid binding protein (FABP) in the liver (P < 0.05). The 30% fat diets significantly increased mRNA expressions of PPARα and SREBP-1c in the liver, decreased expressions of SREBP-1c in the RWAT (P < 0.05). The linoleic and arachidonic acids content in liver and RWAT lipids of the 30S group was more than 2 fold those of the 30B group (P < 0.05). These results implied that the smaller RWAT mass in rats fed the high ORSO diet might be related to the effect of higher tissue linoleic and arachidonic acids that might increase the expressions of fatty acid catabolic genes through the activation of PPARα in the liver and reduce the expression of lipid storage and lipogenic gene expressions through the suppression of SREBP-1c in the RWAT.The third experiment examines the role of hyperleptinemia in the higher expressions of heaptic PPARα and some of its target genes. Leptin was administrated i.p. to C57BL/6J mice for 14 days at the dose of 1 mg/kg/day-1. A group of mice were pair-fed (the PF group) to the leptin treated mice. The leptin treated mice showed significantly higher mRNA expressions of PPARα, ACO and FABP in the liver relative compared to the PF group (P < 0.05).In conclusion, the roles of lipid-regulated transcription factors, PPARα and SREBP-1c, in the regulation of fat metabolism and storage were demonstrated by comparing the obesitogenesis of a high ORSO diet and a high butter diet. With the accumulation of more unsaturated fatty acid in tissues, the less obesitogenic high ORSO diet resulted in higher expressions of fatty acid catabolic genes through the activation of PPARα in the liver and lower expression of lipid storage and lipogenic gene expressions through the suppression of SREBP-1c in the RWAT. It is also demonstrated that the hyperleptinemia in obesity might further up-regulated the hepatic PPARα mRNA expression.

鼠尿 alpha-CEHC Sulfate 純化鑑定與 Conjugated alpha-CEHC 分析方法之重建及活化 PPAR alpha 傳訊途徑對維生素 E 代謝生成 alpha-CEHC 之影響

2021-06-15T00:14:29Z

標題: 鼠尿 alpha-CEHC Sulfate 純化鑑定與 Conjugated alpha-CEHC 分析方法之重建及活化 PPAR alpha 傳訊途徑對維生素 E 代謝生成 alpha-CEHC 之影響; Isolation and Identification of alpha-CEHC Sulfate in Rat Urine and an Improved Method for the Determination of Conjugated alpha-CEHC and Effects of PPAR alpha Activation on the Metabolism of Vitamin E to alpha-CEHC 作者: Yi-Jen Li; 李亦臻摘要: 近年發現 2,5,7,8-tetramethyl-2-(2’-carboxyethyl)-6-hydroxychroman (alpha-CEHC) 為維生素 E 側鏈被切短後的水溶性代謝產物。目前推測其側鏈截短之代謝途徑牽涉到 omega-hydroxylation 及支鏈 beta-oxidation。由於催化 omega-hydroxylation 之酵素 cytochrome P450 4A1 (CYP4A1) 及過氧化體 beta-oxidation 的酵素 acyl-CoA oxidase 1 (ACO1) 均為受 peroxisome proliferator-activated receptor alpha (PPARalpha) 調控之下游基因。為探討 PPARalpha 對維生素 E 代謝生成 alpha-CEHC 之影響效應，本研究以大鼠之動物模式，投予 PPARalpha 活化劑 clofibrate 及 perfluorodecanoic acid (PFDA) 誘發 PPARalpha 標的基因表現，觀察活化 PPARalpha 傳訊途徑對鼠尿中維生素 E 代謝產物 alpha-CEHC 排出量之影響。 alpha-CEHC 於體內會與 glucuronic acid 或 sulfate 等結合，形成alpha-CEHC conjugate，隨尿液排出體外。文獻分析樣品 alpha-CEHC之方法，多用 beta-glucuronidase 酵素水解 alpha-CEHC conjugates，再以 high performance liquid chromatography with electrochemical detector (HPLC-ECD) 定量之。然而，本實驗發現鼠尿經 HCl 強酸處理後，HPLC-ECD 所測得之 alpha-CEHC 含量會明顯竄升，以 beta-glucuronidase 酵素處理者則否。為確認鼠尿 alpha-CEHC conjugate之結構，我們將鼠尿進行純化分離，經化學結構鑑定確認此HCl releasable alpha-CEHC conjugate為 6-O-sulfated alpha-CEHC (alpha-CEHC sulfate)。另外，我們亦重建酸水解萃取分析尿液 conjugated alpha-CEHC 之方法：將含抗壞血酸之樣品，以 6 N HCl 於 60度C 反應 1 小時進行酸加熱水解，之後以乙醚萃取，續以 HPLC-ECD 進行 alpha-CEHC 定量分析。在熱酸水解過程中，以抗壞血酸作為抗氧化劑可以有效地保護 alpha-CEHC不被破壞。此分析方法的優點為快速、靈敏及回收率佳。動物實驗共分三部分進行。實驗一採雙因子變因設計，分別給予大鼠餵食含 50 mg/kg all-rac-alpha-tocopheryl acetate (alpha-TA) (L 組)、50 mg/kg alpha-TA + 0.5% clofibrate ( LC 組)、500 mg/kg alpha-TA (H 組) 或 500 mg/kg alpha-TA + 0.5% clofibrate ( HC 組) 之試驗飼料為期一週，其間並收集尿液。結果顯示 clofibrate 處理會造成大鼠肝臟及血清 alpha-tocopherol (alpha-TOH) 含量明顯下降及肝臟中 PPARalpha 下游基因 ACO1、CYP4A1 及 D-bifunctional protein (D-BF) 之酵素活性、蛋白質或 m-RNA 表現量顯著增加。尿液 alpha-CEHC 排出量方面，LC 組老鼠之每日 alpha-CEHC 排出量顯著高於 L 組老鼠 (p<0.05)，HC 組卻稍低於 H 組。若將 alpha-CEHC 排出量以佔每日飲食維生素 E 攝入量的比例來表示，可以發現LC 組老鼠之 alpha-CEHC 排出量顯著高於 L 組 (p<0.05)，HC 組老鼠之 alpha-CEHC 排出量略高於 H 組，但組間無統計差異。延續實驗一之結果，實驗二改以管餵方式提供正常劑量之維生素 E (5 mg alpha-TA/kg B.W./day)，觀察給予含 0 (C 組)、0.1 % (0.1P 組)、0.25% (0.25P 組)、0.5% (0.5P 組) 或 1 % (1P 組) 之 clofibrate 試驗飼料一週，對維生素E代謝之影響。結果顯示，clofibrate 處理會造成 PPARalpha 標的基因 ACO1、CYP4A1及參與支鏈脂肪酸代謝之 ACO2 及 D-BF 等之酵素活性、蛋白質或 mRNA 表現量顯著增加，且具劑量效應之趨勢。尿液中每日 alpha-CEHC 排出量亦隨 clofibrate 處理劑量增加而上升。且 ACO1、CYP4A1、ACO2 及 D-BF之表現與尿液alpha-CEHC 排出量呈顯著之正相關性 (r=0.30-0.46, p<0.05)。實驗三則在正常維生素 E 劑量 (50 mg alpha-TA/kg diet) 試驗飼料下，每日以腹腔方式給予 0、0.5、1、2.5、5 或 10 mg/kg B.W. 之 PFDA，誘發 PPARalpha 傳訊途徑，觀察不同類型之 PPARalpha 活化劑對維生素E代謝之影響。結果顯示，PFDA 同樣會造成 PPARalpha 下游基因 ACO1、CYP4A1等之酵素活性、蛋白質或 m-RNA 表現量顯著增加，且具劑量效應。尿液中每日 alpha-CEHC 排出量亦隨 PFDA 處理劑量增加而上升。PPARalpha 下游基因 ACO1、CYP4A1之表現與尿液alpha-CEHC 排出量呈顯著之正相關 (r=0.40-0.56, p<0.05)。至於，目前文獻中提及可能參與維生素 E 代謝之相關蛋白質，如 CYP3A、CYP4F等，於本實驗中其 mRNA或蛋白質表現量與尿液 alpha-CEHC 排出量無關聯性，甚至呈負相關。因此，本研究之結果初步證實利用 PPARalpha 活化劑 clofibrate 及 PFDA 誘發 PPARalpha 標的基因的表現，會促進體內維生素 E代謝，增加尿液中 alpha-CEHC 的排出量。活化 PPARalpha 傳訊途徑可能會影響體內維生素 E 的代謝。; 2,5,7,8-tetramethyl-2-(2’-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the metabolite of alpha-tocopherol (alpha-TOH) with a shortened side chain but an intact hydroxychroman structure, has been identified in the urine. Pathway of the metabolism involves omega-hydroxylation of phytyl side chain and the following beta-oxidation. omega-Hydroxylation is known to be catalyzed by cytochrome P450 enzymes (CYPs), of which CYP3A and CYP4F is the most likely candidates. The enzymes which are responsible for the omega-oxidation (CYP4A1) and peroxisomal beta-oxdiation (acyl-CoA oxidase 1, ACO1) of fatty acid are transcriptionally regulated by peroxisome proliferator activated receptor alpha (PPARalpha). In order to investigate effects of PPARalpha activation on the vitamin E metabolism, Wistar rats were treated with PPARalpha activators - clofibrate and perfluorodecanoic acid (PFDA) and urinary alpha-CEHC was monitored in this study. alpha-CEHC was known to be conjugated with glucuronic acid or sulfate. Various CEHCs in biological specimen were mostly measured by high performance liquid chromatography with electrochemical detector (HPLC-ECD) preceded by beta-glucuronidase hydrolysis. In an attempt to analyze alpha-CEHC in rat urine accordingly, it observed that enzyme hydrolysis was relatively inefficient in releasing alpha-CEHC compared to high concentrations of HCl. The HCl releasable alpha-CEHC conjugate was therefore isolated and chemically identified as 6-O-sulfated alpha-CEHC (alpha-CEHC sulfate). Using the synthetic alpha-CEHC sulfate standard, it was found that sulfatase could not hydrolyze to a significant extent. On the other hand, pretreatment with HCl at 60。C in the presence of ascorbate, followed by a one-step ether extraction not only hydrolyzed the sulfate conjugate completely but also extracted alpha-CEHC with high recovery. The inclusion of ascorbate minimized the conversion of alpha-CEHC to alpha-tocopheronolactone in the HCl pretreatment. A complete procedure for the quantitative analysis of alpha-CEHC including HCl hydrolysis, ether extraction and reverse phase isocratic HPLC-ECD was thus established. A total of three rat experiments were conducted to examine the effects of PPARalpha activators on urinary alpha-CEHC excretion. In Experiment 1, rats were fed diets containing 50 mg/kg all-rac-alpha-tocopheryl acetate (alpha-TA) (L), 50 mg/kg alpha-TA + 0.5% clofibrate ( LC ), 500 mg/kg alpha-TA (H) or 500 mg/kg alpha-TA + 0.5%clofibrate (HC) for 1 week, and the urine was collected for alpha-CEHC analysis. PPARalpha target genes including CYP4A1, ACO1 and D-BF is induced significantly by clofibrate revealed by the expression of enzyme activity, protein or mRNA. Clofibrate treatment resulted in a significant decrease of the alpha-TOH content in serum and liver. The urinary alpha-CEHC content of LC group is significantly higher than that of the L group (p<0.05). The ratio of urinary alpha-CEHC to dietary vitamin E intake of the LC group is also significantly higher than the L group. However, no significant difference between H and HC group was found. In Experiment 2, rats were fed vitamin E devoid AIN-76 modified diets containing 0 (C), 0.1 (0.1CF), 0.25 (0.25CF), 0.5 (0.5CF), 1 (1CF) % clofibrate and were i.p. injected with 5 mg alpha-TA/kg B.W. daily for 1 week. Expressions of PPARalpha target genes, namely, CYP4A1, ACO1, ACO2 and D-BF that participated in the metabolism of fatty acid were all increased significantly and does-dependently by the clofibrate treatment as revealed by the of enzyme activity, protein or mRNA expression. The urinary alpha-CEHC excretion of all clofibrate treated groups were also increased does-dependently (p<0.05). Again, there were significantly positive correlations between the urinary alpha-CEHC and the expression of CYP450, CYP4A1 and ACO1 (r=0.40-0.56, p<0.05). In Experiment 3, another PPARalpha activator PFDA was used. All of the 6 groups of rats were fed the AIN-76 modified diet containing 50 mg/kg alpha-TA and respectively tube-fed vehicle (C) or 0.5 (0.5P), 1(1P), 2.5 (2.5P), 5 (5P) or 10 (10P) mg/kg body weight of PFDA daily for 1 week. PPARalpha target genes - CYP4A1 and ACO1 expression in the liver also increased significantly and does-dependently by PFDA as revealed by enzyme activity, protein or mRNA expression (p<0.05). The urinary alpha-CEHC content of all PFDA treated groups also increased does-dependently (p<0.05). Positive correlations between the urinary alpha-CEHC and the expression of CYP4A1 and ACO1 were again observed (r=0.42-0.50, p<0.05). However, CYP3A and CYP4F which has been considered to catalyze vitamin E catabolism to alpha-CEHC showed no correlation with urinary alpha-CEHC in this study (p>0.05). In conclusion, this study demonstrates that PPARalpha activation is associated with an increased urinary alpha-CEHC excretion. The activation of PPARalpha signal pathway may enhance the vitamin E catabolism through up-regulation of some of its target genes (ex. CYP4A1 and ACO1).

香葉草醇10-羥基酶之異源表現之研究

2021-06-13T00:44:50Z

標題: 香葉草醇10-羥基酶之異源表現之研究; Research on Heterogeneous Expression of Geraniol 10- Hydroxylase 作者: Yi-Chia Lin; 林毅佳摘要: 本研究的目標為建構一個微生物表現系統進行香葉草醇10-羥基酶 (G10H) 的異源表現。柚皮素 (naringenin) 可經G10H轉化成具有抗氧化以及抗腫瘤增殖活性的藥物聖草酚 (eriodictyol) 因而提高其經濟價值，使得大量表現G10H酵素具有工業生產的潛力。本研究中嗜甲醇酵母菌P. pastoris以及大腸桿菌E. coli 被選作表現的宿主，其分別使用pPICZaA以及pQE-30 Xa作為表現質體進行表現。在嗜甲醇酵母菌X33以及SMD1168品系表現G10H的研究結果顯示，無法以SDS- PAGE測得G10H。由於當初建構表現質體時為了顧及酵素折疊構形的正確性以及確保酵素的完整活性，沒有將6 x His-tag接於G10H上。現階段的結果顯示所建構的轉殖嗜甲醇酵母菌沒有達成表現G10H的目的。進一步重新建構表現質體以使用像是組胺酸標定 (His-tag) 的抗體偵測系統能較敏感和專一性地測定目標蛋白。而轉殖宿主能進一步以抗生素梯度篩選以取得高拷貝數的基因轉殖宿主。以大腸桿菌作為原核表現系統也於本研究中進行探討。大腸桿菌JM109以及M15轉殖株中均可以抗體標定偵測到外源蛋白的表現。宿主有效調控噬菌體T5啟動子是影響G10H的誘導表現量的重要因子。M15轉殖株儘管過量表現lacI蛋白，噬菌體T5啟動子於未添加誘導劑的情況下仍然會表現下游基因，因而持續性表現外源蛋白。這現象對於宿主細胞造成代謝上的負擔。大量表現外源蛋白只能在M15品系的轉殖株破菌後的不可溶蛋白劃分中測得。高效能液相層析儀分析G10H酵素活性的研究發現，目前的分析系統無法在嗜甲醇酵母菌或是大腸桿菌的轉殖株破菌抽出物中成功將柚皮素轉化生成聖草酚。本研究目前找到的大腸桿菌M15轉殖株的最適生長以及誘導的溫度為25oC，而最適合的誘導條件為以0.01 mM IPTG 進行誘導9個小時。; The aim of this research was to establish microbial hosts expression of economically valuable protein geraniol 10-hydroxylase (G10H). Naringenin was one of the substrates of G10H, which could be catalyzed into eriodictyol. Eriodictyol served as an antioxidant and was found to have antiproliferation activity against tumor cells. Pichia pastoris and Escherichia coli were chosen as expression hosts of G10H by using pPICZaA and pQE-30 Xa as expression vector respectively. During expression of G10H in P. pastoris host strains X33 and SMD1168, the expression level could not reach a level to be confirmed by SDS-PAGE. An antibiotic gradient during transformant selection to obtain high copy number transgenic strains and a more sensitive and specific detection system such as using His-tag detection were needed. Procaryotic expression system using E. coli stains JM109 and M15 were also examined. A tight regulation of the phage T5 promoter was an important factor for high induction expression of G10H. M15 transgenic strains had a leakage in phage T5 promoter inhibition even when lacI was overexpressing. The constitutive expression of foreign protein was presumed to cause a metabolic burden. A high level accumulation of foreign proteins could only be achieved in insoluble protein fractions of M15 transformants. High performance liquid chromatography analysis of G10H enzyme activity in vitro failed to detect eriodictyol production for all P. pastoris and E. coli transfomants. The results so far indicated that the optimum incubation and induction temperature for M15 transformants was 25oC, and 0.01 mM IPTG induction for 9 h was the most adequent condition for G10H expression.