TWI309238B - Protein and nucleic acid for glutathione-dependent formaldehyde dehydrogenase (gfd), alcohol dehydrogenase and s-nitrosoglutathione reductase from antrodia camphorata, manufacturing method and uses therefor - Google Patents

Description

1309238 七、指定代表圖： (一) 本案指定代表圖為： (二) 本代表圖之元件符賴單說明： 示發明特*的化學式 八本案若S^，請揭示最能頻 九、發明說明： ⑽以顯 【發明所屬之技術領域】 胺基酸及其核親。核 宿主細胞及製造方法， 本發明係關於一種樟芝GFD之蛋白質、 苷酸及可供製造該蛋白質之表現載體、 與含有該蛋白質之醫藥組成物。 【先前技術】 棒芝（Antrodiac鄉Aorata)又名牛樟足、牛棒兹報導指出 對肝腫瘤及子宮頸腫瘤的治療非常有效（1，弘+曰 J囚能在台灣 牛樟樹生長，故民間視樟芝為珍寳。因樟芝是菇類唯—能代謝牛 樟樹中大量抑菌性黃樟素而能生長的㈣，因有於他種兹類 的生理機能。樟芝在民間傳統療法上最主要應用於食品中毒，腹 瀉、下腹部疼痛、皮膚癢及肝癌等症狀《在眾多具有生理活性的 成分中，抗氧化物特別受到重視Π)，因此針對其抗氧化成分， 其抗氧化成分的分析，在深層培養中被詳細的研究，指出在樟芝 菌絲體的萃取液中，三帖類對於脂質的過氧化作用具有抑制的的 功能’三帖類與多酚化合物對於清除氧自由基扮演極重要的角 色。由於樟芝中含有大量且成分複雜的抗氧化成分，因此一系列 藉由提升抗氧化效果而達到保護正常細胞或毒殺癌化細胞的機 轉’也陸續做了研究（2-6)。以樟芝菌絲體甲醇粹取物在低劑量 下，對肝腫瘤細胞株Hep G2有很強的細胞毒殺作用，其次為水 13092381309238 VII. Designated representative map: (1) The representative representative of the case is as follows: (2) The component of the representative figure is a list of the following: If the invention is a special case, if the case is S^, please reveal the most energy. : (10) Ying [Technical field to which the invention belongs] Amino acid and its core. Nuclear host cell and method for producing the same, the present invention relates to a protein, a glycoside of Ganoderma lucidum GFD, an expression carrier for producing the protein, and a pharmaceutical composition containing the protein. [Prior Art] Antrodiac Aorata, also known as burdock and ox, reports that it is very effective in the treatment of liver tumors and cervical tumors. (1, Hong+曰J prisoners can grow in Taiwan burdock, so folks Zhizhi is a treasure. Because Antrodia is a kind of mushroom--can metabolize a large amount of bacteriostatic baicalin in burdock tree and can grow (4), because it has the physiological function of his species. Antrodia is the most important application in folk traditional therapy. Symptoms such as food poisoning, diarrhea, lower abdominal pain, itchy skin and liver cancer "In many physiologically active ingredients, antioxidants are particularly valued", so the antioxidant components are analyzed for their antioxidant components in deep layers. In the culture, detailed research has pointed out that in the extract of Agaricus blazeii mycelium, the three-poster has an inhibitory function on lipid peroxidation. 'Triple and polyphenol compounds play an important role in scavenging oxygen free radicals. Character. Since Antrodia sinensis contains a large number of complex antioxidant components, a series of mechanisms to protect normal cells or carcinogenic cells by increasing the antioxidant effect have also been studied (2-6). At low dose, the mycelium of Antrodia camphorata has a strong cytotoxic effect on hepatic tumor cell line Hep G2, followed by water 1309238

萃取部分，但發酵濾液則幾乎不具毒殺性。以C57BL/6* bALB/c 兩種小鼠進行試驗，結過顯示鼠血中TNF-α、IFN-t"和IL-2皆 明顯增加，而IL-4和IL-10則與控制組無明顯差異。此外，樟 芝菌絲體發酵過渡液藉由抗氧化物與清馀自由基的能力，有保護 其肝功能正常的效果。透過樟芝菌絲體萃取液的處理，可避免人 類正常的紅jk球細胞中的GSH與ATP的消耗，並對白血病 leukimia HL-60細胞產生細胞毒性，顯示樟芝菌絲.體可能具有 抗氧化物與抑制腫瘤的效應。樟芝對於肝功能的疾病療效十分良 好，其多醣體對於β型肝炎病毒有明顯的抑制效果。在已發表的 菇類多醣體中，樟芝為第一個被證實其多醣體對於Β型肝炎病毒 有直接抑制效果的物種。但樟芝子實體之研究少有報告，其療效 應高於菌絲體，故以新鮮樟芝子實體為材料。The fraction was extracted, but the fermentation filtrate was almost non-toxic. The mice were tested with C57BL/6* bALB/c. The results showed that TNF-α, IFN-t" and IL-2 were significantly increased in the blood of rats, while IL-4 and IL-10 were not associated with the control group. Significant differences. In addition, the mycelium fermentation transition solution has the effect of protecting normal liver function by virtue of its ability to resist oxidation and clear free radicals. Through the treatment of the mycelium extract of Antrodia camphorata, the consumption of GSH and ATP in human normal red jk cells can be avoided, and the cytotoxicity of leukemia leukimia HL-60 cells is shown, indicating that the mycelium may have antibiotics. Oxidation and the effect of inhibiting tumors. Antrodia camphorata has a good effect on liver function diseases, and its polysaccharide has obvious inhibitory effect on β-hepatitis virus. Among the published mushroom polysaccharides, Antrodia is the first species to be confirmed to have a direct inhibitory effect on the hepatitis virus. However, the research on the fruit body of Antrodia camphorata has rarely been reported, and its curative effect should be higher than that of mycelium.

Glutathione-dependent formaldehyde dehydrogenase (GFD) ’ 是屬於 alcohol dehydrogenase3(ADH3)，主要是分解甲 輕（formaldehyde)、酒儉及還原被硝基化之物 (S-nitrososylated proteins)，對生物體解毒維持健康扮演重 要角色（7-15)。 GFD可防止甲搭和蛋白質及核酸等反應而傷害到生物体在 植物亦能由GFD而代謝甲醛⑻，有很多生理功能其中一種 是信息傳遞，其能傳遞信息是靠蛋白質進行硝基化 (S-nitrosoylation)與否而達到調控代謝性酵素、結構性蛋白或轉 譯因子。近年的研究報告發現咖，亦具有還原硝基化物 (s-nitr0S0Sylati0n)，特別是 s—nitr〇s〇giutath〇ne(GsN〇)， 1309238 因此可協助守護生物免受nitrosative stress (11)，在真菌 (fungus)中尚未有報告，特別是台灣的樟芝。GDF亦能代謝較高 濃度的酒精（7)，故來自台灣的樟芝GFD，兼具三種去毒功能（去 除曱醛、酒精及還原被硝基化之物），是第一次發現。 GFD相關基因常因來源不同使其蛋白質的穩定性和活性不 同，但樟芝子實體GFD基因產物所知甚少，即使其它生物，亦是 近幾年的報告，因此值得將樟芝子實體GFD基因作有系統的研 究。 以採自鹿谷之新鮮樟芝子實體為材料，抽取RNA，合成cDNA。 藉由EST序列資訊設計引子，用樟芝子實體cDNA為模板，以PCR 增生GFD MA並進行篩選，篩得樟芝子實體GFD之片段DNA，再 進行YRACE和TRACE，最後得到全長GFD之cDNA，繼之與表現 型載体連接，植入酵母菌進行大量生產並純化。GFD基因產物為 蛋白質，分子量40 kDa，而如何植入真核細胞是另一大考驗， 因此在N端分別接上幾個胺酸（Tat)，則Tat-GFD產物將容易植 入真核細胞以發揮其功能。值得應用於健康食品，或添加在燙 傷藥膏中以分解NO造成之傷害。 【發明内容】 本發明係提供一種樟芝GFD之蛋白質與核苷酸，及製造該蛋 白質之載體、宿主細胞及方法，與含該蛋白質之組成物。 本發明係由新鮮樟芝子實體之選殖並選擇表現GFD。該蛋 白可去除甲醛、酒精及還原被硝基化之物並藉此值得應用於健 康食品，或添加在烫傷藥膏中。 1309238 本發明提供一種分離核苷酸分子，包含編碼具SEQ ID N0:1 所示樟芝GFD胺基酸序列之核苷酸序列。本文所使用之語詞"核苦 酸分子"意指包括DNA分子（如cDNA或基因組DNA)、RNA分子（如 mRNA)、使用核苷酸同類物所產生的DNA或RNA同類物及其衍生 物、片段及同源物。核苷酸分子可為單股或雙股’但以雙股DNA 較佳。本文所使用之語詞"分離核苷酸"分子意為分離自存於天然 來源之其他核苷酸者。 根據本發明，核苷酸可僅包含編碼樟芝GFD生物活性之部分片 段。本文所使用之語詞”片段"意指編碼仍具生物活性之樟芝GFD 片段之核苷酸序列部分。 根據本發明之一具體實施例中，本發明分離核苷酸分子具有 SEQ ID N0:1之核苷酸序列或其簡併序列（擺動假說，Wobble hypothesis)。在另一具體實施例，本發明分離核苷酸分子包括 如SEQ ID NO: 1所示之核苷酸序列，其全長為1373，轉譯區有1134 bp，可以轉譯出378個胺基酸。本發明核苷酸分子與其他來源的 序列比較，有高的相似性。本發明核酸所編碼之樟芝GFD可分解 曱醛、酒精及還原被硝基化之物，提供了生物體抵抗毒性（甲 醛、酒精及N0)所帶來的破壞與傷害。如熟習該項技術者所認同 者，基於基因密碼的簡併性，可製得許多編碼本發明樟芝GFD蛋 白質之核苷酸。因此，針對一已被鑑定之特定胺基酸序列，熟習 該項技術者可在不改變樟芝GFD蛋白質之胺基酸序列之情況下， 藉簡單修飾一或多個密碼而製出各種不同核苷酸。 根據本發明，編碼樟芝GFD蛋白質之核苷酸可使用標準雜交及 選殖技術來分離。特別的，本發明核苷酸可使用標準選殖及篩選 技術’自新鮮樟芝子實體之cDNA庫分離出來。本發明核苷酸之放 1309238 大可根據PCR放大技術，使用cDNA、mRNA或基因組DNA作為模板及 適當的寡核苷酸引子而得。經放大之核苷酸可選殖至適當載體並 藉DNA序列分析來鑑定。 表現載體及宿主系統 本發明亦提供一表現載體，包含本發明之核苷酸。本發明表 現載體包含一編碼如SEQ ID N0:1所示之蛋白質核苷酸序列。 本文所使用之語詞"表現載體"係可直接表現連接至其上之基 因梭苷酸分子。載體為可自行複製及表現連接於其上之核苷酸 者。一般言之，可用於重組DNA技術之表現載體通常為"載體”形 式，一般為環狀雙股之MA，在其為載體形式時並未融合至染色 體。 本發明編碼G F D蛋白質或其功能相似物之核苷酸序列可*** 適當之表現載體中以表現具生物活性之GFD蛋白質。該表現載體 需含有***編碼序列之轉錄及轉譯等必要元件。根據本發明，熟 習技藝人士可利用熟知之方法構築含有編碼GFD蛋白質及適當轉 錄及轉譯控制元件之表現載體。此等方法包括體外重組MA技 術5合成技術及體内基因重組技術等。 本發明另一目的係提供包含表現載體之宿主細胞，該載體包 含編碼GFD蛋白質之核苷酸序列。本文所使用之語詞”宿主細胞" 為可經載體（如質體）感染之宿主細胞。根據本發明，許多宿主系 統可用於包含且表現編碼GFD蛋白質之序列。此等宿主系統包括 但不限於微生物（如以重組質體或表現載體轉形之細菌）、酵母菌 (如以酵母菌表現載體轉形之酵母菌）、或動物細胞系統。本發明 之宿主細胞為大腸桿菌及酵母菌。本發明將GFD蛋白質之cDNA在 大腸桿菌或酵母菌系統表現，確實能表現出具有活性的重組蛋白 1309238 質，經親和性管柱進行快速純化後，獲得純的gfd蛋白質。 根據本發明之一具體實施例：本發明樟芝GFD係以pYEX-Sl 為表現型載體之構築；設計N端含限制酶及N9RI片段（5，GGAATTCG ATG TCC ACA GTA GGA AAA CC 3’）與C端引子含6 His-tag and ibo RI (5, GGAATTCCTA GTG GTG GTG GTG GTG GTG AGA CAT GTC GAC GAC ACA GC 3，），以GFD蛋白質之cDNA為模板與引子進行PCR得到 之DNA。將該DNA送入大腸桿菌中進行篩選，抽取之質體DNA係使 用限制酶及τσ RI處理，所得片段與相同限制酶處理過之pYEX-S 1 接合，送入酵母菌yeast ’並以介質YPD medium (1% yeast extract, 2% peptone, 2% glucose)在30°C 下培養5 D (170 rpm)，打破菌 體’以10, OOOxg離心5 min，收集上清液後’經親和性管柱進行 快速純化後。電泳後作蛋白質染色，經10% SDS-PAGE，於40 kDa 處可看到明顯色帶，此即為GFD蛋白質。為方便GFD蛋白質穿過細 胞膜’可在前端接上含有九個胺基酸之Tat，並將接上Tat之GFD 蛋白質DNA送入酵母菌中進行表現。 組成物 本發明亦包括一種醫藥組成物，包含本發明之GFD。除本發明 蛋白質外，該醫藥組成物可含有適當之醫藥上可接受之載劑。 本發明之醫藥組成物可以本技藝已知之方式（例如藉常用方 法）製造，該常用方法包括混合、溶解、製粒、製錠、磨細、乳 化、包膠、包陷及/或冷凍乾燥等步驟。 本發明之醫藥組成物可藉任何途徑投藥，此等途徑包括（但不 限於）口服、靜脈内、肌内、動脈内、穿皮、皮下、腹膜腔内、 鼻内或腸道投與。 皇J性 1309238 本發明樟之來源之樟芝GFD蛋白f，其活性皆比其來源之㈣ 蛋白質為t。因此本發明樟芝來源之樟^gfd蛋白質在醫學、食 品、學術研究等方面更具利用價值及貢獻。 t發明KiFD蛋白質可分解甲链、酒精及還原被石肖基化之 物，提供生物體抵抗（甲輕、酒精及N〇)所帶來的破壞與傷害。本 發明GFD蛋白質應用於健康食品，添加於烫傷藥膏可加速傷口 癒合，在整形外科之皮膚移植及人造皮移植應用中，可藉由gfd ^白質防止移植皮膚之組織壞死’進而加速移植皮膚之缝合。 【實施方式】 下列實例進一步說明本發明，但非欲限制本發明之範圍， 任何熟悉該項技術者所知之替代和改變，均仍涵蓋於本發明 之範圍中’而不偏離本發明之精神和目的。 搜乏子實體總RNA之拙肽 取4 g樟芝子實體以液態氪急速冷凍，於研妹中磨成粉末後取 出’馬上與20 mL的溶解緩衝液lysis buffer(Novalgen’s Straight’s mRNA Isolation System)於30 mL 離心管中混合， 置於4°C下5 min後以9, OOOxg在4°C離心15 min，離心後取上面水 溶液層，大約有20 mL。將水溶液吸至50 mL falcon中加入3 mg magnetight particles 混勻，置於4 °C下以磁座需住 magnetight particles，以 1.5 mL wash buffer 洗蘇三次，在 60°〇以0.5‘水洗離即為抓人（10叩），加入0.1¥〇1.3 113〇(11111〇 acetatp 和 0.8 vol.isopropanol 置於-75°C 20 min，隨後以 12, 000 rpm在4°C下離心15 min ’以70% ethanol共清洗三次’乾 11 '1309238 燥後儲存於-70°C備用。 樟芝 5’-RACE-Ready cDNA與 3’-RACE-Ready cMA之合成： Based on BD Biosciences Clontech5s SMART RACE cDNA Amplification Kit. 5’-RACE-Ready cDNA : 將下列所有反應溶液加入於1. 5 mL的微量試管中並在冰上完 成：Glutathione-dependent formaldehyde dehydrogenase (GFD) ' belongs to alcohol dehydrogenase 3 (ADH3), which mainly decomposes formaldehyde, wine cellar and S-nitrososylated proteins, and plays a role in detoxification and maintenance of organisms. Important role (7-15). GFD can prevent the reaction of necry and protein and nucleic acid and damage the organism. In plants, it can also metabolize formaldehyde by GFD. (8) There are many physiological functions. One of them is information transmission, and the information can be nitrated by protein. -nitrosoylation) or not to regulate metabolic enzymes, structural proteins or translation factors. In recent years, the research report found that coffee also has a reduced nitrite (s-nitr0S0Sylati0n), especially s-nitr〇s〇giutath〇ne (GsN〇), 1309238, thus helping to protect organisms from nitrosative stress (11). There have been no reports of fungi (fungus), especially Taiwan's Antrodia. GDF can also metabolize higher concentrations of alcohol (7), so Ganoderma lucidum GFD from Taiwan has three detoxification functions (to remove furfural, alcohol and reduce nitrated substances), which is the first discovery. GFD-related genes often differ in their stability and activity depending on the source, but little is known about the GFD gene product of the A. camphorata fruit body. Even other organisms are reported in recent years, so it is worthwhile to have the Ganoderma lucidum fruiting body GFD. Genes are systematically studied. The RNA was extracted and the cDNA was synthesized by using the fresh amaranth fruit body from Lugu. The EST sequence information was used to design primers, and the cDNA of G. sinensis was amplified by PCR and screened to obtain the DNA of the GMD fragment of Ganoderma lucidum. Then the YRACE and TRACE were obtained, and the cDNA of full-length GFD was obtained. This is followed by attachment to a phenotypic vector, implantation of yeast for mass production and purification. The GFD gene product is a protein with a molecular weight of 40 kDa. How to implant eukaryotic cells is another test. Therefore, if several amino acids (Tat) are attached to the N-terminus, the Tat-GFD product will be easily implanted into eukaryotic cells. To play its function. It is worth applying to healthy foods or added to the scalding cream to break down the damage caused by NO. SUMMARY OF THE INVENTION The present invention provides a protein and a nucleotide of Ganoderma lucidum GFD, a vector for producing the protein, a host cell and method, and a composition containing the protein. The present invention is selected from the fresh A. camphora fruiting body and selected to express GFD. The protein removes formaldehyde, alcohol and reduces the nitrated material and is therefore worthy of being applied to health foods or added to scalding creams. 1309238 The invention provides an isolated nucleotide molecule comprising a nucleotide sequence encoding a G. sinensis GFD amino acid sequence having the SEQ ID NO: 1. The term "nucleotide molecule" as used herein, is meant to include DNA molecules (such as cDNA or genomic DNA), RNA molecules (such as mRNA), DNA or RNA congeners produced using nucleotide congeners, and derivatives thereof. Substances, fragments and homologs. The nucleotide molecule may be single-stranded or double-stranded but is preferably double-stranded DNA. As used herein, the term "isolated nucleotide" molecule means isolated from other nucleotides of natural origin. According to the present invention, the nucleotide may comprise only a portion of the fragment encoding the biological activity of Ganoderma lucidum GFD. The phrase "fragment" as used herein refers to a portion of a nucleotide sequence encoding a still biologically active Antrodia campanum GFD fragment. According to a particular embodiment of the invention, the isolated nucleotide molecule of the invention has SEQ ID NO: a nucleotide sequence of 1 or a degenerate sequence thereof (Wobble hypothesis). In another specific embodiment, the isolated nucleotide molecule of the present invention comprises the nucleotide sequence set forth in SEQ ID NO: 1, the full length thereof The translation region is 1134 bp, and 378 amino acids can be translated. The nucleotide molecules of the present invention have high similarity to the sequences of other sources. The Ganoderma lucidum GFD encoded by the nucleic acid of the present invention can decompose furfural. , alcohol and reduction of nitrated substances, providing damage and damage caused by the organism's resistance to toxicity (formaldehyde, alcohol and N0). As recognized by those skilled in the art, based on the degeneracy of genetic code, Numerous nucleotides encoding the Ganoderma lucidum GFD protein of the present invention can be prepared. Therefore, for a specific amino acid sequence that has been identified, those skilled in the art can change the amino acid sequence of the Ganoderma lucidum GFD protein without changing the amino acid sequence. Next, Various nucleotides are simply modified to produce a variety of different nucleotides. According to the present invention, nucleotides encoding the Ganoderma lucidum GFD protein can be isolated using standard hybridization and colonization techniques. In particular, the nucleotides of the present invention can be used. Standard selection and screening techniques are isolated from the cDNA library of fresh A. sinensis fruiting bodies. The nucleotides of the present invention 1309238 can be used according to PCR amplification techniques, using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotides. The amplified nucleotides can be selected for selection into appropriate vectors and identified by DNA sequence analysis. Expression Vectors and Host Systems The present invention also provides a performance vector comprising the nucleotides of the present invention. A protein nucleotide sequence encoding the sequence of SEQ ID NO: 1. The term "expression vector" as used herein directly expresses the fucuric acid molecule attached thereto. The vector is self-replicating and expressing. The nucleotides to which the nucleotides are attached. In general, the expression vectors that can be used in recombinant DNA technology are usually in the form of a "carrier", generally a ring-shaped double-stranded MA, in which Not when fused to form chromosomes. The nucleotide sequence of the present invention encoding a G F D protein or a functional analog thereof can be inserted into a suitable expression vector to express a biologically active GFD protein. The expression vector needs to contain necessary elements such as transcription and translation of the inserted coding sequence. In accordance with the present invention, a skilled artisan can construct a performance vector containing a protein encoding a GFD protein and appropriate transcription and translation control elements using well-known methods. These methods include in vitro recombinant MA technology 5 synthesis technology and in vivo gene recombination technology. Another object of the invention is to provide a host cell comprising an expression vector comprising a nucleotide sequence encoding a GFD protein. The term "host cell" as used herein is a host cell that can be infected by a vector, such as a plastid. According to the present invention, a number of host systems can be used to contain and represent sequences encoding a GFD protein. Such host systems include, but are not limited to, A microorganism (such as a bacterium transformed with a recombinant plastid or a expression vector), a yeast (such as a yeast transformed with a yeast expression vector), or an animal cell system. The host cell of the present invention is Escherichia coli and yeast. The invention expresses the cDNA of the GFD protein in the Escherichia coli or the yeast system, and can indeed exhibit the active recombinant protein 1309238, and obtains the pure gfd protein after rapid purification by the affinity column. According to one embodiment of the present invention Example: The Ganoderma lucidum GFD line of the present invention is constructed by using pYEX-Sl as a phenotype vector; the N-terminal restriction enzyme and the N9RI fragment (5, GGAATTCG ATG TCC ACA GTA GGA AAA CC 3') and the C-terminal primer containing 6 His- are designed. Tag and ibo RI (5, GGAATTCCTA GTG GTG GTG GTG GTG GTG AGA CAT GTC GAC GAC ACA GC 3,), DNA obtained by PCR using cDNA of GFD protein as a template and primer The DNA was sent to E. coli for screening, and the extracted plastid DNA was treated with restriction enzyme and τσ RI, and the resulting fragment was ligated with pYEX-S 1 treated with the same restriction enzyme, and sent to yeast yeast' YPD medium (1% yeast extract, 2% peptone, 2% glucose) was cultured at 30 °C for 5 D (170 rpm), and the cells were disrupted by centrifugation at 10, OOOxg for 5 min, and the supernatant was collected for 'affinity'. The column was subjected to rapid purification. After electrophoresis, protein staining was performed. After 10% SDS-PAGE, a clear band was observed at 40 kDa, which is the GFD protein. In order to facilitate the passage of GFD protein through the cell membrane, it can be connected at the front end. The Tat containing nine amino acids is expressed by feeding the GAD protein DNA of Tat to the yeast. Composition The present invention also includes a pharmaceutical composition comprising the GFD of the present invention. The pharmaceutical composition may contain a suitable pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may be made in a manner known in the art (e.g., by conventional methods) including mixing, dissolving, granulating, tableting, and grinding. Fine, emulsified, Steps of gelatinization, entrapment and/or freeze-drying. The pharmaceutical composition of the present invention can be administered by any route including, but not limited to, oral, intravenous, intramuscular, intraarterial, transdermal, subcutaneous, peritoneal. Intracavitary, intranasal or intestinal administration. Emperor J 1309238 The Ganoderma lucidum GFD protein f of the present invention is more active than its source (iv) protein t. Therefore, the g^gfd protein of the present invention is more valuable and useful in medicine, food, and academic research. The invention of KiFD protein can decompose the methyl chain, alcohol and reduce the osmolality, providing damage and damage caused by the organism's resistance (A light, alcohol and N〇). The GFD protein of the invention is applied to health foods, and the addition of the scald ointment can accelerate wound healing. In the application of plastic surgery and artificial skin transplantation of plastic surgery, the tissue necrosis of the transplanted skin can be prevented by gfd white matter, thereby accelerating the transplantation of the skin. Stitching. The following examples further illustrate the invention, but are not intended to limit the scope of the invention, and any alternatives and modifications known to those skilled in the art are still within the scope of the invention without departing from the spirit of the invention. And purpose. Search for the total RNA of the total RNA of the living body. Take 4 g of the scorpion scorpion body and freeze it in a liquid state. After grinding it into a powder, remove it and immediately extract it with 20 mL of lysis buffer (Novalgen's Straight's mRNA Isolation System). Mix in a 30 mL centrifuge tube, place at 4 ° C for 5 min, centrifuge at 9, OOO xg for 15 min at 4 ° C, and centrifuge to remove the upper aqueous layer, approximately 20 mL. Aspirate the aqueous solution into 50 mL falcon and add 3 mg of magnetight particles. Mix at 4 °C to hold the magnetight particles in the magnetic holder, wash the suicide three times with 1.5 mL wash buffer, and wash at 60 ° 〇 with 0.5' water. Grab the person (10叩), add 0.1¥〇1.3 113〇(11111〇acetatp and 0.8 vol.isopropanol to -75°C for 20 min, then centrifuge at 12°C for 15 min at 4°C to 70% The ethanol was washed three times 'dry 11 '1309238. After drying, it was stored at -70 ° C for use. The synthesis of 5'-RACE-Ready cDNA and 3'-RACE-Ready cMA: Based on BD Biosciences Clontech5s SMART RACE cDNA Amplification Kit. 5'-RACE-Ready cDNA: All of the following reaction solutions were added to a 1.5 mL microtube and completed on ice:

mRNA (ug/uL) (1 ug) 1 uLmRNA (ug/uL) (1 ug) 1 uL

5’-CDS primer 1 uL5’-CDS primer 1 uL

Smart II A Oligo 1 uL.Smart II A Oligo 1 uL.

H20 2 uLH20 2 uL

總計 5 uLTotal 5 uL

3 5-RACE-Ready cDNA: mRNA (ug/uL) (1 ug) 1 uL3 5-RACE-Ready cDNA: mRNA (ug/uL) (1 ug) 1 uL

3 ’ - CD S primerA 1 uL3 ’ - CD S primerA 1 uL

H2 〇 3 uLH2 〇 3 uL

總計 5 uL 均勻混合反應溶液，並稍微離心後置於72°C恆溫水浴槽中2 min。作用後將離心管置於冰上2 min。加入2uL 5X first-strand buffer > 1 uL 20 mM DTT * 1 uL 10 mM dNTP mix > 1 uL powerscript reverse transcriptase 於42°C 恆溫水浴槽中 1. 5 h。後加入200 uL tricine-EDTA buffer於72°C恆溫水浴槽中7 min後儲存於 12 *1309238 -70°C備用。 樟芝GFD基因的選殖 彖 藉由樟芝EST序列資訊設計引子，用樟芝5’-RACE-ReadycDNA 與3’-RACE-Ready cDNA為模板，以PCR增生GFD相關基因DNA並進 行篩選，得樟芝GFD基因之片段DNA，再進行YRACE與3’-RACE，最 後得到全長GFD之cDNA。 GFD cDNA之表現 經由PCR、膠體之製備及電泳、次選殖、在酵母菌表現型載體 (pYEX-Sl)之構築、及蛋白質的誘發與樣品的電泳分析等程式達 成。 PCR程式 將下列反應試料加至0. 5mL微量試管中，依序加入下列試劑：A total of 5 uL of the reaction solution was uniformly mixed, and centrifuged slightly and placed in a 72 ° C constant temperature water bath for 2 min. After the action, the tube was placed on ice for 2 min. Add 2uL 5X first-strand buffer > 1 uL 20 mM DTT * 1 uL 10 mM dNTP mix > 1 uL powerscript reverse transcriptase in a 42 ° C constant temperature water bath 1. 5 h. After adding 200 uL tricine-EDTA buffer in a 72 ° C constant temperature water bath for 7 min, store at 12 * 1309238 -70 ° C for use. The selection of the GHD gene of Antrodia camphorata was carried out by using the EST sequence information of Antrodia camphorata, using the 5'-RACE-Ready cDNA and 3'-RACE-Ready cDNA as templates to PCR-proliferate GFD-related gene DNA and screen it. The DNA of the GHD gene fragment was further subjected to YRACE and 3'-RACE, and finally the full-length GFD cDNA was obtained. The expression of GFD cDNA was achieved by PCR, colloid preparation, electrophoresis, secondary selection, construction of yeast phenotype vector (pYEX-Sl), and induction of proteins and electrophoresis analysis of samples. PCR program The following reaction samples were added to 0.5 mL microtubes, and the following reagents were added in sequence:

模板 ΜΑ 0. 2 ug 5’ -正義引子 10. pno 1 3'-反義引子 10 p m o 1 OX Taq DNA聚合酶緩衝液 5 uL 15 mM MgCh 6uL Taq DNA 聚合酶 2.5 units 10 mM dNTP 1.5 uL 加入無菌水至總計 50 uL 加入50~100 uL礦物油，進行25 cycles of PCR(94 °Cfor 30 13 *1309238 min, 50°Cfor 3 min, 72°Cfor 1.5 min) PCR反應結束後，可得 DM產物，經回收後可用來進行下一步的實驗，例如進行DNA重 組，分析其序列θ 膠體之製備及電泳 所用之膠體為1. 0%瓊脂醣膠，倒入IX TAE缓衝液以能完 全覆蓋住膠。取15 uL PCR產物，加上其1/10體積的追蹤染劑， 注入孔中，另外也注入6 uL之1 kb DNA標記用來瞭解DM樣品的 大小，接著以100伏特的電壓進行電泳，待DNA染料移動至膠之 2/3處，即可中止電泳。將完成泳之膠以溴乙唆（EtBr)染色15 min 後，放入水中退染，之後即可置於紫外光源箱下觀察。 次選殖 1. PCR產物輿載體接合輿宿主細胞韓形 取1 uL PCR產物，加入1 uL鹽溶液，1 uL的無菌水與0.5 uL的 pCR4之後，於室溫下反應5 min以進行連接。取前述已接合好妁 重組DNA，加入18 uL TOPO 10活性細胞中，置於冰上30 min，後 置於42°0的水浴中3〇56(：進行熱休克後立即置於冰上2 111丨11，加入 150 uL SOC培養基，在37°C下振盪培養60 min，將其均勻塗布 在含有50ug/mL抗生素ampicillin的LB瓊脂聽的培養皿，在37 °C培養12-16 h之後，挑選50-80個單一菌株於新的ampicillin 之LB瓊脂醣上，待進一步實驗。 2. 菌體之轉印與放射性探針之製備 1309238 將轉印紙覆於培養皿表面，並以針頭紮洞作定位之後，取出 轉印紙，此時培養m上的菌落轉印到轉印紙上，轉印工作完成。 準備一淺盤，上覆3 mm濾紙，倒入變性溶液（0. 5 M NaoH，1. 5 Μ NaCl)，倒入量以能充分濕潤3 mm濾紙，將轉印紙以吸附菌面朝 上，放在濕潤之濾紙之上7 min，接著取另一淺盤以中和溶液（1 MTris-HCl, 1 M NaCl，pH7. 5)充分濕潤濾、紙之上7 min。重覆中 和液步驟之後，將轉印紙置於烘箱中，以60-70°C烘乾。之後， 將轉印紙放於紫外交聯儀機器中，進行網狀連結15 sec，待進一 步以放射性探針來篩選。 放射性探針之製備Template ΜΑ 0. 2 ug 5' - sense primer 10. pno 1 3'-antisense primer 10 pmo 1 OX Taq DNA polymerase buffer 5 uL 15 mM MgCh 6uL Taq DNA polymerase 2.5 units 10 mM dNTP 1.5 uL Add sterility Water to a total of 50 uL Add 50~100 uL of mineral oil for 25 cycles of PCR (94 °C for 30 13 *1309238 min, 50 °C for 3 min, 72 °C for 1.5 min) After the PCR reaction is completed, the DM product can be obtained. After recovery, it can be used for the next experiment, for example, DNA recombination, analysis of the sequence θ colloid preparation and electrophoresis gel used is 1.0% agarose gel, poured into IX TAE buffer to completely cover the gel. Take 15 uL of PCR product, add 1/10 volume of trace dye, inject into the well, and inject 6 uL of 1 kb DNA marker to understand the size of DM sample, then carry out electrophoresis at 100 volts. The DNA dye is moved to 2/3 of the gel to stop the electrophoresis. The finished gel was stained with bromine (EtBr) for 15 min, then de-stained in water, and then placed under a UV light source box for observation. Secondary selection 1. PCR product 舆 vector 舆 host cell Han shape Take 1 uL of PCR product, add 1 uL of saline solution, 1 uL of sterile water and 0.5 uL of pCR4, then react at room temperature for 5 min to connect. The recombinant DNA was ligated, added to 18 uL of TOPO 10 active cells, placed on ice for 30 min, and placed in a 42 ° 0 water bath for 3 〇 56 (: immediately after heat shock, placed on ice 2 111丨11, 150 uL SOC medium was added, shake cultured at 37 ° C for 60 min, and evenly spread on an LB agar plate containing 50 ug/mL antibiotic ampicillin, and cultured at 37 ° C for 12-16 h, then selected 50-80 single strains on the new ampicillin LB agarose for further experimentation 2. Transfer of the bacterial cells and preparation of the radioactive probe 1309238 Transfer the transfer paper to the surface of the culture dish and position it with a needle After that, the transfer paper is taken out, and the colony on the culture m is transferred to the transfer paper, and the transfer operation is completed. Prepare a shallow tray, overlay the 3 mm filter paper, and pour the denatured solution (0.5 M NaoH, 1.5) Μ NaCl), pour the amount to fully wet the 3 mm filter paper, place the transfer paper with the adsorption side facing up, place it on the wet filter paper for 7 min, then take another tray to neutralize the solution (1 MTris-HCl , 1 M NaCl, pH 7. 5) Fully wet the filter, 7 min on the paper. After repeating the neutralization step, After the printed paper placed in an oven dried at 60-70 ° C., Placed on the transfer paper UV crosslinking meter machine, for connecting the mesh 15 sec, to be further screened to a radioactive probe. The radioactive probe was prepared

寡核苦酸（3 pmole/uL) 0· 5 uL 10X T4聚核普酸酶缓衝液 3. 0 uL 聚核普酸酶 （12 unit) 1. 5 uL [r-32p] ATP 3. 5 uL H2O 21. 5 uL 總計 30. 0 uL 混合均勻，置於37°C下反應30 min後。即完成放射性探針之 製備，將放射性探針加入雜交溶液中，用來篩選所要的目標基因。 3.目標基因DNA之選殖 取出前述之轉印紙，加入以1.5X SSC及0.1% SDS配製成的洗 劑，在44°C下震搖30 mi η，倒掉洗劑，加入不含放射性探針的雜 15 1309238 交溶液（5X SSC，5X Denhardt，0.5% SDS，100 ug/mL sperm DNA)，此一過程稱之為預雜交。倒出雜交溶液，加入含有放射性 探針之雜交溶液，置於44°C水浴震搖16 h以上，倒出含有放射性 探針之雜交溶液’用前述洗劑，在44°C震搖15 min，重覆此一步 驟2-4次。壓乾轉印紙，進行自動放射顯影，沖片後，挑出正反 應株。 4.細菌晳體之純化與檢視 挑出正反應株養於含ampicillin(50 ug/mL)之LB，於37°C下 培養6-8 h。以10, 〇〇〇xg離心3min，取得菌體沈澱物，加入200 uL 再懸浮溶液（50 mM Tris，pH 7. 5, 10 mM EDTA)振盪，使菌體懸 浮狀態。加入200 uL溶裂液（0. 2 M NaOH, 1% SDS)，輕輕的混勻’ 使菌體能被完全溶掉，質體能溶離出來。加入200 uL中和液（1.32 Μ醋酸卸），而後以12, OOOxg離心10 min，取上清液，注於0.5 mL 離心管（含DNA吸附膜），以12, 000xg離心lmin，使DNA能吸附於 膜上，之後加入mL的洗滌溶液，以12, 000xg離心2 min後。 再以12, 000xg離心2 min以去除殘留的酒精，最後加入50 uL的熱 水（60°C )靜置30 sec後，以12, 000xg離心2 min，收集離心下來 的質體DNA作質體之限制反應與電泳分析。即取MA以及適量的 限制酶，加上10 X reactin缓衡液，最後加無菌水使體積為 uL，在適當的溫度下反應2 h，以1%瓊脂醣膠電泳分析，以確定 嵌^入DNA的大小。 16 1309238 5.序列之判譫 利用ABI PRISM 377-96 DNA定序儀進行自動定序。 在酵母菌表現裀盤體之構築：Oligonucleotide (3 pmole/uL) 0· 5 uL 10X T4 Polynucleotidase Buffer 3. 0 uL Polynucleotidase (12 unit) 1. 5 uL [r-32p] ATP 3. 5 uL H2O 21. 5 uL Total 30. 0 uL Mix well and place at 37 ° C for 30 min. That is, the preparation of the radioactive probe is completed, and the radioactive probe is added to the hybridization solution to screen the desired target gene. 3. Selection of target gene DNA The above transfer paper was taken out, and a lotion prepared with 1.5X SSC and 0.1% SDS was added, and shaken at 44 ° C for 30 mi η, the lotion was poured off, and no radioactivity was added. The probe was mixed with 15 1309238 (5X SSC, 5X Denhardt, 0.5% SDS, 100 ug/mL sperm DNA), a process called pre-hybridization. Pour the hybridization solution, add the hybridization solution containing the radioactive probe, shake it in a water bath at 44 ° C for more than 16 h, and pour out the hybridization solution containing the radioactive probe' with the above-mentioned lotion and shake at 44 ° C for 15 min. Repeat this step 2-4 times. The transfer paper is dried and subjected to automatic radiographic development. After the film is punched, the positive reaction strain is picked. 4. Purification and examination of bacterial clears The positive reaction strains were picked up in LB containing ampicillin (50 ug/mL) and cultured at 37 ° C for 6-8 h. After centrifugation at 10, 〇〇〇xg for 3 min, the bacterial cell pellet was obtained, and 200 uL of a resuspension solution (50 mM Tris, pH 7.5, 10 mM EDTA) was added to shake, and the cells were suspended. Add 200 uL of lysate (0.2 M NaOH, 1% SDS) and mix gently to make the cells completely dissolved and the plastids can be dissolved. Add 200 uL of neutralizing solution (1.32 Μ acetic acid unloading), then centrifuge at 12, OOOxg for 10 min, take the supernatant, and inject into 0.5 mL centrifuge tube (containing DNA adsorption membrane), centrifuge at 12,000 xg for 1 min to make DNA Adsorbed on the membrane, then added to the mL wash solution and centrifuged at 12,000 xg for 2 min. Centrifuge at 12,000 xg for 2 min to remove residual alcohol. Finally, add 50 uL of hot water (60 ° C) for 30 sec, centrifuge at 12,000 xg for 2 min, and collect the centrifuged plastid DNA for plastids. Limit reaction and electrophoresis analysis. Take MA and an appropriate amount of restriction enzyme, add 10 X reactin, and finally add sterile water to make the volume uL, react at appropriate temperature for 2 h, analyze with 1% agarose gel to determine the inlay The size of the DNA. 16 1309238 5. Sequence determination 自动 Automated sequencing using the ABI PRISM 377-96 DNA Sequencer. The construction of yeast in the yeast body:

表現載體為pYEX-Sl，GFD係以pYEX-Sl為表現型載體之構築； 設計N端含限制酶及^ RI片段（5，GGAATTCG ATG TCC ACA GTA GGA AAA CC)與C端引子含6 His-tag and 价σ RI (5’ GGAATTCCTA GTG GTG GTG GTG GTG GTG AGA CAT GTC GAC GAC ACA GC 3，），以GFD 蛋白質之cDNA為模板與引子進行PCR得到之DNA。將該DNA送入大 腸桿菌中進行篩選，抽取之質體DNA係使用限制酶及RI處理， 所得片段與相同限制酶處理過之pYEX-Sl接合，送入yeast，並以 YPD medium (1% yeast extract, 2% peptone, 2% glucose)在The expression vector was pYEX-Sl, and the GFD line was constructed with pYEX-Sl as the phenotype vector; the N-terminal restriction enzyme and the RI fragment (5, GGAATTCG ATG TCC ACA GTA GGA AAA CC) and the C-terminal primer contained 6 His- Tag and valence σ RI (5' GGAATTCCTA GTG GTG GTG GTG GTG GTG ATG CAT GTC GAC GAC ACA GC 3,), DNA obtained by PCR using the cDNA of GFD protein as a template and primer. The DNA was sent to E. coli for screening, and the extracted plastid DNA was treated with restriction enzyme and RI. The resulting fragment was ligated with the same restriction enzyme-treated pYEX-S1, and sent to yeast, and YPD medium (1% yeast). Extract, 2% peptone, 2% glucose) at

30°C下培養5 D (170 rpm)，打破菌體’以i〇,〇〇〇xg離心5 min ’ 收集上清液後，經親和性管柱進行快速純化後。電泳後作蛋白質 染色，經10%SDS-PAGE，於40 kDa處可看到明顯色帶，此即為GFD 蛋白質。更佳的，為方便GFD蛋白質穿過細胞膜，可在前端接上 含有九個胺基酸之Tat，並將接上Tat之GFD蛋白質DNA送入酵母 菌中進行表現。 將其轉殖入酵母菌[trp一ada…ura—]作為表現宿主。pYEX-Sl (皆先以及RI處理連接），取前述已接合好的重組洲人，加入酵 母菌活性細胞中’篩選挑出正反應株以進行蛋白質表現。 在酵母菌棘形 17 1309238 1.製備轉形細， ⑴方法1 :選取單-菌落並完全的在YPD培養皿上劃線，在 28-32。〇培養8〜96h，從細菌培養基表層刮取菌，加入Yeastern 商品scos混合液至no a使試管中細菌濃度達5 χΐ〇7個/試 管。 ⑵方法2 :選取單-新鮮菌落加入w〇此γρ])培養液中於 28〜32 t：搖晃培養20, he將培養液加人ι〇 ι〇〇虹則培 養液中靜置8〜24h ’以2000 x g離心1〇山，去掉上清夜，以 無菌水清洗兩次去除殘餘的YPD培養液，將沈澱物混合擾摔入 110-115 μι SCOS混合液直至細菌濃度達5 χ1〇7個/試管。 2.製備SCOS混釦饬 在1. 5 mL試管中力认100 A SC0S緩衝夜，加入5 A i Μ術 (保存於-2G，加人5 ss DNA (保存於—抓），加入質 體DNA (不超過5μΙ〇。 3. 製備乾式遠擇性斑基m 完全乾燥的培養皿在Yeastern，s沉卯轉形系統中可提供良好 的轉形反應，因此建議在將培養皿叠置前先將無蓋培養I至於無 菌操作臺内lh左右，待其完全乾燥。 4. SCOS轉里 (1)將scos混合液與酵母菌懸浮細胞混合攪拌製成sc〇s轉形混 合液。 18 1309238 (2) 將試管加蓋，置於45. 5°C 10〜60 min。 (3) 將轉形混合液直接塗敷於乾燥培養皿[YNBD (1_ 7 g yeast nitrogen base, 5 g ammonium sulfate, 20 g glucose, 15 g agarose in 1 L water) containing 20 ug Trp/mL]，此程式 20 sec内完成，將培養皿置於28-32 °C2~4天。 蛋白質表現 取單_;*菌落（1〜2 111111)於10瓜]^¥1^人.1'11)(1.7 8 76381；1111：1'〇忌611 base, 5 g asparagine, 20 g glucose in one L) containing 20 ug Trp/mL培養液，此容器使用125 mL燒瓶，並於30 °C培養箱中 以250 rpm震盪培養過夜，次日再加入5 mL培養液使ODeoo達到〇丄 時並繼續以30 °C、250 rpm震盪培養72 h後，再加入10 mL γΡΐ) 並繼續以30°C、250 rpm震盥培養48 h。 將所有菌液以5000xg、4°C離心10 min，收取菌加入〇 5 〇· •呂坡 璃球及2 niL PBS(pH8. 0)，打破菌體，以10, 〇〇〇xg離心5 min , "故 集上清液後，再加入2 mL緩衝液，重覆此一萃取步驟，最後用2虹 緩衝液再萃取一次，一共收集6 mL上清液，此為粗蛋白質樣品 待進一步的電泳，電泳之膠體大小為1〇 cmx8cmx〇. 75匪。 1.電泳勝體的匍借 將鋁板與玻板拭淨後，和隔條組合好，放入製膠模型. '’再 依下表所列之用量配製膠體溶液：分離凝膠，混勻並注入模型 中，再加入適當的水壓平，約經過1 h，膠體形成後，再注入堆 19 1309238 積凝膠，並插上槽梳，待成膠即可。5 D (170 rpm) was incubated at 30 ° C, and the cells were disrupted by centrifugation at 〇〇〇 xg for 5 min. The supernatant was collected and rapidly purified by affinity column. After electrophoresis, the protein was stained. After 10% SDS-PAGE, a clear band was observed at 40 kDa, which is the GFD protein. More preferably, in order to facilitate the passage of the GFD protein through the cell membrane, a Tat containing nine amino acids may be attached to the front end, and the GAD protein DNA attached to Tat may be sent to the yeast for expression. It was transferred into yeast [trp-ada...ura-] as a performance host. pYEX-Sl (both first and RI treatments are connected), and the above-mentioned joined recombinant humans are added to the yeast active cells to screen out positive reaction strains for protein expression. In the yeast spines 17 1309238 1. Prepare the fine shape, (1) Method 1: Select the single-colony and completely streak the YPD dish at 28-32. The sputum was cultured for 8 to 96 hours, and the bacteria were scraped from the surface of the bacterial culture medium, and the Yeastern commercial scos mixture was added to no a to make the bacterial concentration in the test tube reach 5 χΐ〇 7 / test tube. (2) Method 2: Select single-fresh colonies to add w〇 to this γρ]) culture solution at 28~32 t: shake culture 20, and add the culture solution to human ι〇ι〇〇 rainbow and let the culture solution stand for 8~24h. 'After centrifugation at 2000 xg, remove the supernatant, wash twice with sterile water to remove the residual YPD medium, and mix the sediment into the 110-115 μm SCOS mixture until the bacterial concentration reaches 5 χ1〇7/ test tube. 2. Prepare the SCOS mixed sputum in a 1.5 mL tube to recognize 100 A SC0S buffer night, add 5 A i Μ ( (stored in -2G, add 5 ss DNA (save in - grab), add plastid DNA (Not more than 5μΙ〇. 3. Preparation of dry-type selective plaques. Completely dry culture dishes provide good transformation in the Yeastern, s-converting system, so it is recommended that the dishes be stacked before they are stacked. No cover culture I until about 1h in the aseptic processing station, until it is completely dry. 4. SCOS transfer (1) Mix the scos mixture with the yeast suspension cells to make a sc〇s transformation mixture. 18 1309238 (2) The test tube was capped and placed at 45. 5 ° C for 10 to 60 min. (3) The transfer mixture was directly applied to a dry culture dish [YNBD (1-7 g yeast nitrogen base, 5 g ammonium sulfate, 20 g glucose , 15 g agarose in 1 L water) containing 20 ug Trp/mL], this program is completed within 20 sec, and the culture dish is placed at 28-32 °C for 2-4 days. Protein performance takes _; * colony (1~2 111111) in 10 melon] ^ ¥ 1 ^ person. 1 '11) (1.7 8 76381; 1111:1 ' jealous 611 base, 5 g asparagine, 20 g glucose in one L) containing 20 ug Trp / mL The container was incubated with a 125 mL flask and shaken at 250 rpm in a 30 °C incubator. The next day, 5 mL of the culture solution was added to allow ODeoo to reach 〇丄 and continue to incubate at 30 °C, 250 rpm. After h, add 10 mL of γΡΐ) and continue to culture for 48 h at 30 ° C and 250 rpm. Centrifuge all the bacteria at 5000xg, 4°C for 10 min, add the bacteria to the 〇5 〇·• Lupo glass ball and 2 niL PBS (pH 8.0), break the cells, centrifuge at 10, 〇〇〇xg for 5 min. , " Therefore, after the supernatant is added, add 2 mL of buffer, repeat this extraction step, and finally extract again with 2 rainbow buffer, and collect a total of 6 mL of supernatant, which is a crude protein sample to be further Electrophoresis, electrophoresis gel size is 1〇cmx8cmx〇. 75匪. 1. After electrophoresis, the aluminum plate and the glass plate are wiped clean, and the spacer is combined with the spacer to put into the rubber molding model. '' Then prepare the colloidal solution according to the dosages listed in the table below: separate the gel, mix and Inject into the model, and then add appropriate water flattening. After about 1 hour, after the colloid is formed, inject 193130938 of the gel and insert the slot comb to form the glue.

取適量樣品與樣品載入缓衝液混勻後注入膠體孔洞中，以100 伏特電壓進行電泳，native-PAGE約需65 min，SDS-PAGE約需130 min後中止電泳，取下膠體，進行Coomassie blue染色：將膠體 浸於染劑中，均勻搖動30 min，移去染劑，用水清洗一次後，加 入退染劑，退染12 h即可。 UFD之純化 因 C 端帶有 6 個 His，以 Ni2+-nitrilotriacetic acid Sepharose superflow來進行親和性管柱純化如下：3 mL的樹脂 管柱，用5 vol.的PBS平衡，之後將6 mL的粗蛋白質樣品注入並 收集流下來的液體約6 mL。再以3 vol. PBS含20 mM imidazole 洗管柱，繼之以1.5 mL PBS含100 mM imidazole沖提，共收集6 個部分，其中有2値部分共3 mL有收集到蛋白貧，因為含有 imidazole，所以需經過透析除去。 透析液：將樣品約3 mL裝入透析膜中，以透析夾夾好，放入 含200 mL 1/3 PBScontaining 1 mM DTT and 5%glycerol透析液 的燒杯中，於4°C下透析4h以上，重覆此透析步驟即可分裝待進 一步分析。 5.GFD之蛋白質含晉 先製作蛋白質標準曲線（BSA standard curve):即分別取蛋 20 1309238 白質標準品（BSA，0_ 5 mg/mL)卜2、3、4、5、6 uL於其所對應之 滅菌水中，使最後體積達800 uL，之後分別加入呈色劑200 uL 室溫下反應5 min，測ODmnm並依吸光值求出蛋白質標準曲線。將 採收之樣品依相同方法測得吸光值即可由標準曲線求得蛋白質 含量。 6.活性測定 GFD活染：（1)首先將完成電泳之膠體，浸泡於70 mM potassium phosphate buffer (pH8.0), 0.5 M potassium chloride, 4.8 mM formaldehyde and ImM reduced GSH的水溶 液中，25°C搖動5 min後，分別加入不同量NAD、nitroblue tetrazolium、phenazine methosulfate 使最終濃度為 1.2 mM, 0. 4 mg/mL、0· 03 mg/mL，膠體於37°C搖動1 h後，以去離子水清 洗膠體後，呈現紫色帶之區域為具有GFD活性之處。 GSN0R活染：（2)將完成電泳之膠體，浸泡於0. 1 Μ potassium phosphate buffer (pH 8.0),2 mM NADH 水溶液中， 置於冰上15 min後，去除excess buffer，膠體覆上含有3 mMGSNO 的滤紙15 min後，除去爐紙，膠體在ultraviolet light上可觀 看NADH fluorescence消失的情形。 GFD 活性測定：（3)依據 Uotila and Koivusalo (13)作部分 修改，在 100 uL 反應液中：〇. 25〜1 pg GFD，100 mM potassium phosphate buffer (pH 8. 0), 1 mM HMGSH [GSH and formaldehyde 21 1309238 (1:1)在 25°C 混合 5 min 使產生 HMGSH]，2 mM NAD·在 340 nm 測NAD被還原的量以換算GFD之活性（12-13)。 7. GFD性質 熱穩定性 分別取適量GFD於1.5 mL試管中，各5管，分別於50 °C加 熱 0、2、4、8、16 min 後，置於冰上，進行 10% native-gel 電 泳，分別作蛋白質染色（2 ug)及活性測試（2 ug)。結果顯示此 甚為安定，在50 °C加熱其活性減少一半所需時間為5 min。 pH的影響 分別取適量液，於1. 5 mL試管中，共6管，加入不同pH值缓 衝液：0. 2 Μ檸檬酸鈉緩衝液（pH 2. 3、or 5. 4)，0. 2 M Tris-Hcl 緩衝液（pH 7. 8、or 9.0)，0.2 M甘胺酸NaOH 緩衝液（pH 10. 4 or 11.2)，在37 °C下反應lh後，置於冰上，分別進行10% nat i ve-ge 1電泳後作蛋白質染色（2 ug)及活性測試（2 ug)。結果 顯示此甚為安定，顯示以酸鹼處理很安定，尤其至pH7. 8〜pHll 處理lh對活性並沒有影響。 SDS的影響 分別取適量液，於1.5 mL試管中，共5管，分別加入不同量 的20 % SDS，使SDS最終濃度為0、1、2、3、4%，在37 °C下反應 lh後，分別進行10% native-gel電泳後作蛋白質染色（2 ug)及活 性測試（2 ug)。結果顯示SDS的處理不安定。 22 1309238 咪唑的影響 分別取適量GFD’於1>5 mL試管中，共5管，分別加入不同量 的咪唑，使咪唑最終濃度為0、0.2、0_4、〇_8、K6 M，在37。〇 下反應lh後，分別進行10% native-gel電泳後作蛋白質染色（2 ug) 及活性測試（2 ug)。結果顯示以咪唑的處理亦很安定，必須高至 0.8 Μ時對活性才有抑制（30%)。 香白酸的影響 分別取適量GFD ’於1.5 mL試管中，共4管，分別加入相當 GFD量1/20的胰蛋白酶或胰凝乳蛋白酶於Tri-HC1緩衝液（pH 8 · 5 含 2 0 niM CaC I2)分別在 37 C 下反應 i〇、20、40 min，分 別進行10% native-gel電泳後作蛋白質染色（2 ug)及活性測試 (2 ug)。結果顯示以胰蛋白酶的處理40 min候，活性不變。 動力學 GFD 之動力學結果：The Lineweaver-Burke plot of the velocity (1/V。)對 1/HMGSH 為 1^=^.0.615 mM 和 Vmax= 128 μΜ/min，the. velocity (1/V。）對 1/NAD+為 Km= 1.105 mM 和 \^眶=131 μΜ/min。Mix the appropriate amount of sample with the sample loading buffer and inject into the colloidal pores. Electrophoresis is carried out at 100 volts. Native-PAGE is about 65 minutes. After SDS-PAGE takes about 130 minutes, the electrophoresis is stopped and the colloid is removed for Coomassie blue. Dyeing: Dip the colloid into the dyeing agent, shake it evenly for 30 min, remove the dyeing agent, wash it once with water, add the anti-dyeing agent, and de-stain for 12 h. The purification of UFD was carried out with 6 Hiss at the C-terminus and the affinity column purification with Ni2+-nitrilotriacetic acid Sepharose superflow was as follows: 3 mL resin column, equilibrated with 5 vol. PBS, then 6 mL of crude protein The sample was injected and collected about 6 mL of liquid flowing down. The column was washed with 3 vol. PBS containing 20 mM imidazole, followed by 1.5 mL PBS containing 100 mM imidazole. A total of 6 fractions were collected, 2 of which were collected in a total of 3 mL, and the protein was poor because it contained imidazole. So it needs to be removed by dialysis. Dialysate: Approximately 3 mL of the sample was placed in the dialysis membrane, clamped with a dialysis clamp, placed in a beaker containing 200 mL of 1/3 PBS containing 1 mM DTT and 5% glycerol dialysate, and dialyzed at 4 ° C for more than 4 h. Repeat this dialysis step to separate for further analysis. 5. The protein of GFD contains BSA standard curve: that is, take egg 20 1309238 white matter standard (BSA, 0_ 5 mg/mL), 2, 3, 4, 5, 6 uL Corresponding to the sterilized water, the final volume is up to 800 uL, and then reacted with a coloring agent of 200 uL for 5 min at room temperature, and the ODmnm is measured and the protein standard curve is determined according to the absorbance. The protein content can be determined from the standard curve by measuring the absorbance value of the harvested sample by the same method. 6. Activity measurement GFD live dyeing: (1) First, the electrophoresis colloid was immersed in an aqueous solution of 70 mM potassium phosphate buffer (pH 8.0), 0.5 M potassium chloride, 4.8 mM formaldehyde and 1 mM reduced GSH at 25 ° C. After shaking for 5 min, different amounts of NAD, nitroblue tetrazolium and phenazine methosulfate were added to make the final concentration of 1.2 mM, 0.4 mg/mL, 0·03 mg/mL, and the colloid was shaken at 37 ° C for 1 h to deionize. After washing the colloid with water, the area showing the purple band is where GFD is active. GSN0R live dyeing: (2) The electrophoresis colloid was immersed in 0.1 Μ potassium phosphate buffer (pH 8.0), 2 mM NADH aqueous solution, placed on ice for 15 min, the excess buffer was removed, and the colloidal coating contained 3 After 15 minutes of the filter paper of mMGSNO, the furnace paper was removed, and the colloid was observed on the ultraviolet light to disappear the NADH fluorescence. GFD activity assay: (3) Partial modification according to Uotila and Koivusalo (13) in 100 uL of reaction solution: 25. 25~1 pg GFD, 100 mM potassium phosphate buffer (pH 8. 0), 1 mM HMGSH [GSH And formaldehyde 21 1309238 (1:1) Mixing for 5 min at 25 °C to produce HMGSH], 2 mM NAD· The amount of NAD reduced at 340 nm to convert the activity of GFD (12-13). 7. GFD properties Thermal stability Take appropriate amount of GFD in 1.5 mL tubes, 5 tubes each, heat at 50 °C for 0, 2, 4, 8, 16 min, then place on ice for 10% native-gel Electrophoresis, protein staining (2 ug) and activity test (2 ug). The results showed that this was very stable, and the time required to reduce the activity by half at 50 °C was 5 min. The effect of pH was determined by taking an appropriate amount of liquid in a 1.5 mL tube, a total of 6 tubes, and adding different pH buffers: 0.2 Μ sodium citrate buffer (pH 2. 3, or 5. 4), 0. 2 M Tris-Hcl buffer (pH 7.8, or 9.0), 0.2 M glycine NaOH buffer (pH 10. 4 or 11.2), reacted at 37 ° C for 1 h, placed on ice, separately 10% nat i ve-ge 1 was subjected to protein staining (2 ug) and activity test (2 ug) after electrophoresis. The results showed that this was very stable, showing that the treatment with acid and alkali was very stable, especially to pH 7. 8~pHll treatment of lh had no effect on the activity. The effect of SDS was determined by taking appropriate amount of liquid in a 1.5 mL tube, and a total of 5 tubes were added with different amounts of 20% SDS to make the final concentration of SDS 0, 1, 2, 3, 4%, and react at 37 ° C for 1 h. After that, 10% native-gel electrophoresis was performed for protein staining (2 ug) and activity test (2 ug). The results show that the processing of SDS is not stable. 22 1309238 Effect of imidazole Take appropriate amount of GFD' in 1>5 mL tube, a total of 5 tubes, respectively add different amounts of imidazole, so that the final concentration of imidazole is 0, 0.2, 0_4, 〇_8, K6 M, at 37. After 1 hour of sputum reaction, protein staining (2 ug) and activity test (2 ug) were performed after 10% native-gel electrophoresis. The results showed that the treatment with imidazole was also very stable and must be inhibited (30%) when it was as high as 0.8 。. The effect of leucovoric acid was determined by taking appropriate amount of GFD' in a 1.5 mL tube, a total of 4 tubes, and adding 1/20 of the amount of GFD to trypsin or chymotrypsin in Tri-HC1 buffer (pH 8 · 5 contains 2 0 niM CaC I2) was reacted at 37 C for i〇, 20, and 40 min, respectively, and subjected to 10% native-gel electrophoresis for protein staining (2 ug) and activity test (2 ug). The results showed that the activity was unchanged after 40 min treatment with trypsin. Kinetics of kinetic GFD: The Lineweaver-Burke plot of the velocity (1/V.) for 1/HMGSH is 1^=^.0.615 mM and Vmax=128 μΜ/min, the. velocity (1/V. ) for 1/NAD+ is Km = 1.105 mM and \^眶 = 131 μΜ/min.

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Int J. Biol Sci 2006, 2, 117-124· 24 1309238 (13) Uotila, L.; Koivusalo, M. Fonnaldehyde dehydrogenase. Methods Enzymol. 1981, 77, 314-320. (14) Sanghani, P. C.; Robinson, H.; Bennett-Lovsey, R.; Hurley, T.D.; Bosron, W.F. Structure-function relationships in human Class III alcohol dehydrogenase (formaldehyde dehydrogenase). Chemico-Biological Interactions 2Q93,143-144,195-200. (15) Sakamotoa, A·; Uedab, M·; Morikawaa, H. Arabidopsis glutathione-dependent formaldehyde dehydrogenase is an S-nitrosoglutathione reductase. FEBS Letters 2002, 515,20-24. 25Ca(2+)-NO-cGMP pathway·i v. 2003, 73, 2769-2783· (3) Hseu, YC; Yang, HL; Lai, YC; Lin, J. G; Chen, G W.; Chang, YH Induction of apoptosis by Antrodia camphorata in human premyelocytic leukemia HL-60 cells. NutK Cancer 2004, 489 189-197. (4) Hsu, Y· L·; Kuo, Y· C·; Kuo, P· L.; Ng ,L' T.; Kuo, Υ· H.; Lin, C· C. Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines. Cancer Lett. 2005,227,77-89. (5) Shen K. Anti-inflanimatory activity of the extracts from mycelia of Antrodia camphorata cultured with water-soluble ftactions from five different Cinnamomum species, YC; Chou, CJ; Wang, YH; Chen, CF; Chou, Y C.; Lu? Μ. K. Anti-inflanimatory activity of the extracts from mycelia of Antrodia camphorata cultured with water-soluble ftactions from five different Cinnamomum species . FEMS Microbiol. Lett 2004, 231, 137-143. (6) Song, TY; Yen, GC Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCU-induced hepatic toxicity in rats. J, Agric. Food Chem. 2003, 51,1571-1577 (7) Hoog,J_ 0·; Hedberg,JJ; Stro Mberg, P.; Svensson, S. Mammalian alcohol dehydrogenase- functional and structural implications. J. Biomed. Sci 2001, 8, 71-76. (8) Fliegmann, J.; Sandermann, H. Jr. Maize glutathione-dependent formaldehyde Dehdrogenase cDNA: Plant Mol Biol 1997, 34, 843-854. (9) Hess, DT; Matsumoto, A.; Kim? S. 0.; Marshall, Η. E.; Stamler, JS Protein S-nitrosylation: purview and Nat Rev Mol Cell Biol 2005, 6,150-166. (10) Liu, L.; Hausladen, A.; Zeng, M.; Que, L.; Heitman, J.; Stamler, JS A metabolic enzyme for S- Nitrosothiol conserved from bacteria to humans. Nature 2001, 410, 490-494. (11) Liiis L_; Yan, Y_; Zeng, M·; Zhang, J·; Hanes, M·A.; Aheam, G·; McMahon, T. J.; Dickfeld, T.; Marshall, Η. E.; Que, L. G; Stamler, JS Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell 2004, 116, 617-628. Godoy, L.; GonzMez-Duarte, R.; Albalat, R. S-nitrosogluthathione reductase activity of amphioxus ADH3: insights into the nitric oxide me Tabolism. Int J. Biol Sci 2006, 2, 117-124· 24 1309238 (13) Uotila, L.; Koivusalo, M. Fonnaldehyde dehydrogenase. Methods Enzymol. 1981, 77, 314-320. (14) Sanghani, PC; Robinson, H.; Bennett-Lovsey, R.; Hurley, TD; Bosron, WF Structure-function relationships in human Class III alcohol dehydrogenase (formaldehyde dehydrogenase). Chemico-Biological Interactions 2Q93, 143-144, 195-200. (15) Sakamotoa , A·; Uedab, M·; Morikawaa, H. Arabidopsis glutathione-dependent formaldehyde dehydrogenase is an S-nitrosoglutathione reductase. FEBS Letters 2002, 515,20-24. 25

Claims (1)

13092381309238 、申請專 ;ί '〜種選白 ，. ---一J ^選自棒芝同時具有去甲醛、酒精及還原硝基化之 、（glutathi〇ne_dependent formaldehyde 炫 e h V* _ r〇genase’ GFD)，其胺基酸序列係如SEQ IDN0:2所示。 ' 之同時具有去甲醛、酒精及還原硝基化之 苷質（GFD)之核苷酸，其包含一核苷酸序列，該核 序歹J係如SEQ Π) N〇:1所示，可用以編碼如申請專 麵固第i項所述 、抱姓 J疋之樟芝GFD之胺基酸序列。 據申請專利範圍 社 固第2項所述之樟芝GFD之分離核 核酸係在酵母菌中進行表現及量 甘酸，其中該分離 化 4 :::現载體，其包含如申請專利範圍第3項之嫩序列。 、1製造：其包含如申請專利範圍第4項之載體。 之蛋白質表狀條件MU、胞，柯細由雜紐分和 b)自該宿主細胞培養液中回收該蛋白質。, apply for special; ί '~ kind of white, . --- a J ^ selected from the stick with formaldehyde, alcohol and reduced nitration, (glutathi〇ne_dependent formaldehyde eh eh V* _ r〇genase' GFD The amino acid sequence thereof is shown in SEQ ID NO: 2. a nucleotide having a de-formaldehyde, an alcohol, and a reduced nitration glucoside (GFD), which comprises a nucleotide sequence, which is represented by SEQ Π) N〇:1, available The amino acid sequence of Ganoderma lucidum GFD, which is described in the application for the specific terminology, and the surname J. According to the patent application scope, the isolated nuclear nucleic acid system of the Ganoderma lucidum GFD described in the second paragraph of the patent application is performed in the yeast and the amount of the sweet acid, wherein the isolated 4::: present vector contains the third patent as claimed in the patent application. The tender sequence of the item. 1, Manufacturing: It contains a carrier as in claim 4 of the scope of the patent application. The protein phenotypic conditions MU, cytosolic, cytosolic nucleus and b) recover the protein from the host cell culture broth.