食用菇異源基因表現系統之建立及其應用

Development and Application of Mushroom Heterologous Gene Expression Systems

郭俊毅(Chun-Yi Kuo)

指導教授 ： 黃慶璨

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.03237

摘要

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 1.31

關鍵字

gpd啟動子 ； 異源基因表現 ； 基因轉形 ； 食用菇 ； 電穿孔 ； 綠色螢光蛋白質 ； B型肝炎表面抗原 ； 口服疫苗

並列摘要

A 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.

並列關鍵字

gpd promoter ； heterologous expression ； transformation ； mushroom ； electroporation ； EGFP ； Hepatitis B surface antigen ； oral vaccine

參考文獻

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