Development and Application of Mushroom Heterologous Gene Expression Systems

本研究成功建立了簡便且具有通用性之食用菇轉形策略，以發芽的擔孢子或是小片段的營養菌絲為材料，利用電穿孔的方式完成轉形。此方法免除原生質體的製備及物種的限制，完成測試並成功表現重組蛋白質之菌株包含金針菇、洋菇、鮑魚菇及香菇，轉形效率約為每微克質體可得5~50株轉形株。南方氏雜合分析結果顯示異源基因隨機插入染色體中，在無選殖壓力的環境下繼代培養數次仍維持穩定。以本研究所建構之pMush系列表現載體，可放置受測異源基因於載體中的多限制酶切位(multiple cloning site, MCS)以進行重組蛋白質表現測試。使用gpd啟動子及其第一個intron，可使金針菇、洋菇及鮑魚菇成功表現Egfp，表現量最高分別可占總可溶性蛋白質的2%、1%及0.5%。Egfp可在金針菇生活史中的每個階段表現，包括基原體、成熟之子實體、經減數分裂所生成之擔孢子及同核體。雙核菌株及其減數分裂所生成同核體之南方氏雜合分析訊號相符，顯示外來基因在減數分裂後仍能穩定存在。使用同源gpd啟動子及其第一個intron，香菇之GUS表現量最高可占總可溶性蛋白質的0.05%。以B型肝炎表面抗原為目標測試菇菌生產口服疫苗之可能性，發現野生型B型肝炎表面抗原基因序列遭受錯誤的剪輯，且有多個菇菌不常用之密碼子。經過序列修飾之B型肝炎表面抗原基因，可在香菇中順利生成重組蛋白質，於蛋白質C端外加內質網停留訊息胜肽HDEL，可使B型肝炎表面抗原表現量增加三倍，表現量占總可溶性蛋白質 1.31A simple and applicable mushroom transformation procedure based on electroporation of basidiospores or mycelial fragments was developed. This method eliminated the problem of protoplast preparation and the limitation of host specificity. Flammulina velutipes, Agaricus bisporus, Pleurotus ostreatus, and Lentinula edodes were transformed and expressed recombinant protein successfully; the transformation efficiency were 5~50 transformants per ug DNA. Southern analysis of transformants indicated that the integration of heterologous genes might occur randomly and were maintained stably after multiple rounds of subculture without selection pressure. Heterologous genes tested for recombinant proteins expression were cloned in the multiple cloning site of pMush-series expression vectors constructed in this study. Fused to the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter with its first intron, Egfp could be expressed successfully in F. velutipes, A. bisporus and P. ostreatus, and the highest expression of EGFP was 2%, 1%, and 0.5% of total soluble protein, respectively. Egfp was expressed during the whole life cycle of F. velutipes including primordia, mature fruiting bodies, basidiospores and monokaryons that produced from meiosis. Southern analysis of dikaryon and its green progeny showed all signals of progeny coincide to those of parental dikaryon. These results revealed that the integrated DNA was stable during meiosis. In the case of L. edodes, the highest expression of GUS was 0.05% of total soluble protein by using homologous gpd promoter and the 1st intron. Incorrect splicing and several uncommon codons were found when wild type Hepatitis B surface antigen gene was tested as oral vaccine produced in mushrooms. Modified Hepatitis B surface antigen gene could be expressed in L. edodes, and an ER retention signal (HDEL) fused to its C terminus can enhance the expression by threefold, as 1.31 ug/g of total soluble protein. The formation of hepatitis B virus-like particles with a diameter of 20 nm was revealed by transmission electron microscopy and the production of specific antibody was induced by oral immunization or injection in animal trials.摘 要 IBSTRACT II 目 錄 VI 目 錄 VII、總論 1、基因轉形 1、分子農場 6. 生產特用產物 6. 開發食用疫苗 8. 轉基因菇類於分子農場的應用 12、菇類與分子生物技術 16. 菇類概述 16. 菇類分子育種與分子農場 17、研究動機 19、菇菌基因轉形系統之建立與報導者基因之表現 22、前言 22. 宿主(host)細胞模式 22. 載體 25.1 酵母菌表達系統 25.2 絲狀真菌表達系統 27. 轉形策略 28.1 PEG法 29.2 REMI (restriction enzyme mediated integration)法 29.3 電穿孔法 29.4 土壤農桿菌法 30、材料與方法 32. 菌株與培養 32. 鮑魚菇gpd cloning 33.1 鮑魚菇部分甘油醛-3-磷酸脫氫酶選殖 33.2 鮑魚菇甘油醛-3-磷酸脫氫酶擴增選殖 33. 質體與建構 34.1 商用載體 34.2 轉形測試質體建構 36.3 pMush系列載體建構 37.4 GUS表現質體之建立 44.5 EGFP表現質體之建立 44.6 建構具有第一個gpd intron 的報導者表現載體 44. 轉形條件測試 47. 轉形材料改進 47. 轉形株鑑定 47.1 PCR 47.2 Southern hybridization 47. 報導者基因表現檢測 48.1 GUS 活性定性測試 48.2 GUS 活性定量測試 48.3 GUS 比活性測定 49.4 EGFP表現之螢光顯微鏡檢定與ELISA定量 49.5 EGFP西方雜合分析 49. 轉形株穩定性檢測及出菇測試 50、結果 51. 食用菇轉形系統測試、建立與報導者基因之表現 51.1 金針菇 56.2 香菇 63.3 鮑魚菇 67.4 洋菇 70、討論 72amp;#21442;、B型肝炎表面抗原基因於食用菇之表現研究 82、前言 82、材料與方法 86. 菌株與培養 86. HBsAg序列修正與基因合成 86. 質體與建構 88. 菌株轉形與轉形株挑選 92. RT-PCR檢測 92. HBsAg檢測 92. 以超高速離心分離HBsAg類病毒顆粒(virus like particle, VLP) 93. 以穿透式電子顯微鏡進行HBsAg VLP觀測 93. 以小鼠模式進行HBsAg專一性抗體生成測試 93、結果 94. HBsAg基因於金針菇與香菇之表現測試 94. 以質體改進與蛋白質融合策略測試HBsAg之表現 94. 金針菇轉形株Egfp-HBsAg之RT-PCR檢測 96. HBsAg基因序列修正與合成 99. mHBsAg基因於食用菇之表現 99.1 mHBsAg 在香菇之表現 101.2 mHBsAg-HDEL在香菇之表現 102.3 Intron-mHBsAg在香菇之表現 103.4 Intron-mHBsAg-HDEL在香菇之表現 103. 以超高速離心分離HBsAg VLP 105. 以穿透式電子顯微鏡進行HBsAg VLP觀測 106. 以小鼠模式進行HBsAg專一性抗體生成測試 108、討論 110、綜合討論 115、轉形系統 115、蛋白質表現限制因子 116、表現量分析 119、GMO管理 120、總結 122、未來工作與展望 123考文獻 12