Establishment of stable heterologous gene expression platform by mating strategies in Flammulina velutipes

菇類分子農場 (Mushroom molecular pharming) 係以菇類作為生物反應器，生產醫藥用蛋白質或發展為口服疫苗，具有低基因汙染風險、操作簡單及成本便宜等優勢。過去本實驗室以農桿菌媒介轉形法得到多種菇類轉形株，亦發展不同分生策略以提升異源基因之表現，卻發現繼代間異源蛋白質表現量不穩定，推測原因可能係農桿菌媒介轉形法無法確保將異源基因送入所有雙核菌絲的兩個核內，造成繼代雙核菌絲轉形株時會混雜著不等比例的野生型菌絲、僅有一核嵌有異源基因及雙核皆有異源基因之菌絲，而無法獲得穩定表現異源蛋白質的轉形株。因此必須建立雙核菌絲所有核都含有異源基因之篩選方式，以利菇類生產醫藥用蛋白質及口服疫苗之開發。 本篇研究使用前人建構能表現增強型綠色螢光蛋白質 (Enhanced green fluorescent protein；eGFP) 之金針菇 Figpd-d3-2轉形株孢子，以流式細胞分選儀篩選能表現高綠色螢光強度之孢子，並將孢子萌發出來之單核菌絲進行交配，得到全部雙核菌絲之雙核都嵌有異源基因 egfp 的純淨轉形株。結果顯示流式細胞分選儀搭配交配策略可以得到表現異源蛋白質較穩定之轉形株，且 eGFP 表現量高於Figpd-d3-2。本研究是第一篇建立純淨菇類轉形株之研究。 未來可將此技術應用於腸病毒 Enterovirus 71 (EV 71) 及其他傳染性疾病之口服疫苗開發，因此本篇研究亦建構經序列刪減之金針菇 gpd-d1 啟動子表現腸病毒 EV 71 外鞘蛋白 VP1 基因之質體，並利用可於轉譯時斷裂的 2A 胜肽連接報導基因 egfp ，確保轉形株能同時表現 eGFP 和 VP 1，以供往後大量生產腸病毒菇類口服疫苗之應用。Mushroom molecular pharming, a novel biotechnology application using mushrooms as bioreactors to produce pharmaceutical proteins or oral vaccines, exhibits advantages such as less genetic pollution, simple operation and lower costs etc. In our laboratory, we have established Agropbacterium-mediated transformantion (AMT) in mushrooms and have tried to enhance heterologous gene expression by various molecular biology strategies. However, the previous studies show that the expression of heterologous proteins varies a lot between subcultures, indicating the mycelia are a mixture of transformants and wild type. Futhermore, it appears AMT does not ensure delivering heterologous genes to the two nuclei of dikaryotic mycelia and lead to transformants with unstable heterologous expression. Therefore, it’s critical to acquire the pure transformants which all nuclei contain heterologous genes for further studies. In order to establish the pure transformants of Flammulina velutipes in our study, we used Fluorescence-Activated Cell Sorter (FACS) to obtain the green fluorescent spores with high intensity from Figpd-d3-2 transformants and mated the different haploid mycelia germinating from spores. The result shows we have good chances to obtain the pure and stable transformants expressing heterologous proteins with higher intensity of enhanced green fluorescent protein (eGFP) expression than Figpd-d3-2 transformants. This study is the first report regarding the establishment of selecting pure transformants. In the future, the platform of selecting pure transformants can be applied in oral vaccines of enterovirus 71 or other infectious diseases. We also constructed the plasmid which contains glyceraldehyde-3-phosphate dehydrogenase promoter d1 (gpd-d1) , VP1 as target gene, egfp as report gene, and 2A peptide which can cleave in translation to ensure transformants expressing eGFP and VP1 simultaneously. The selection of pure and stable VP1 transformants is undergoing