金針菇多基因表現系統之建立及其於腸病毒71型類病毒顆粒生產之應用

Abstract

菇類分子農場是利用菇類作為生物反應器,以生產醫藥用或特用蛋白質,如 抗體及酵素等,其中又以開發口服疫苗(edible vaccine)最受矚目。本實驗室已建 立穩定的菇類農桿菌媒介轉形系統表現異源蛋白質。然而,實際應用與發展仍然 受限於異源基因表現量低與無法同時生產特用蛋白質複合體兩大問題。本研究成 功建立金針菇多基因表現平台,利用病毒來源之 2A 短鏈胜肽(2A peptides)可在 共轉譯時期經由 2A 胜肽媒介截切(2A-mediated cleavage)分離上下游基因產物 的特性,建構單基因多拷貝數系統以直接提高異源蛋白質表現量;以及多基因單 至多拷貝數系統以同時表現多個目標基因。具體完成目標如下:(1) 建構菇類雙 基因表現系統探討 2A 胜肽媒介截切應用於菇類的可行性。利用 2A 短鏈胜肽聯 結潮黴素篩選基因(hph)及綠色螢光蛋白質基因(egfp),並探討綠色螢光蛋白質基 因位於 2A 短鏈胜肽上下游表現量差異。萃取經農桿菌媒介轉形成功之金針菇轉 形株菌絲體總可溶蛋白質,以西方墨點分析結果證實 2A 短鏈胜肽可在金針菇轉 形株中媒介截切,以專一性單株抗體可偵測到正確大小約 27 kDa 的綠色螢光蛋 白質。酵素連結免疫分析結果顯示,當綠色螢光蛋白質基因位於 2A 短鏈胜肽上 游,因直接被啟動子驅動使表現量優於位於 2A 短鏈胜肽下游。 (2) 建構單基因 多拷貝數表現系統,利用 2A 短鏈胜肽串接一至四個綠色螢光蛋白質基因等不同 表現載體探討異源基因表現量。以南方氏墨點法及即時定量 PCR 確定嵌入金針 菇轉形株染色體之 egfp 拷貝數後以螢光顯微鏡與酵素連結免疫分析,結果顯示 金針菇轉形株可激發綠色螢光,螢光量與表現載體所帶之 egfp 拷貝數成正比, 以 3 倍基因拷貝數表現螢光量最強。(3) 建構多基因單至多拷貝數系統,並應用 於開發菇類腸病毒 71 型口服疫苗。腸病毒 71 型為感染幼兒的重要病原體,重 症病患致死率高,目前疫苗生產操作繁複、成本昂貴不易推廣,因此開發腸病毒71 型口服疫苗為當務之急。本研究建構一系列腸病毒 71 型類病毒顆粒表現載體, 利用 2A 短鏈胜肽聯結腸病毒外鞘蛋白質 P1 及 3C 蛋白質截切酶等基因,以洋 菇 gpd 啟動子或適當刪減之金針菇 gpd-d1 啟動子轉錄,經 2A 短鏈胜肽媒介截 切作用於共轉譯時期分離 P1 和 3C 後,3C 蛋白質截切酶接著將病毒外鞘蛋白質 分割成 VP1、VP2、VP3、VP4 等 4 個病毒次單元,可於胞內組裝成類病毒顆 粒。為提升生產病毒次單元的效率,利用單點突變法將 3C 蛋白質截切酶的第 52 胺基酸由 lysine 突變成 arginine,以避免宿主細胞 SUMO 蛋白質修飾化 (sumoylation)而提高轉錄活性。挑取經農桿菌媒介轉形後可穩定繼代的金針菇轉 形株,以南方氏墨點法分析顯示異源基因嵌入宿主染色體拷貝數介於 1-2 個。續 以西方墨點法經專一性單株抗體辨識,可偵測病毒次單元 VP1 及 VP2 訊號;各 病毒次單元之部分胺基酸序列亦可成功經液相層析質譜儀探測比對,即 2A 短鏈 胜肽及 3C 蛋白質截切酶皆具有功能可順利將 P1 外鞘蛋白質截切成四種病毒次 單元。進一步以蛋白質複合體熱穩定性分析法測試最適類病毒顆粒組裝緩衝液環 境後,經蔗糖連續梯度法純化及戊二醛固定蛋白質,利用穿透式電子顯微鏡取得 類病毒顆粒 2 維影像,並以電腦影像分析技術成功模擬類病毒顆粒之 3 維立體 結構,證實病毒次單元可於金針菇菌絲體內自行組裝成完整類病毒顆粒。本研究 成果期望為後續菇類口服疫苗發展奠定基石。

A reliable Agrobacterium tumefaciens-mediated transformation (ATMT) for the basidiomycete Flammulina velutipes, or commonly knows as enoki mushroom, has been established in our lab previously. However, the enhancement of protein production and expression of multi-target genes remain a big challenge. The 2A peptides mediated cleavage is emerging as a highly effective and novel tool for co-expression of polyproteins. In this study, a feasible polycistronic expression system was developed in F. velutipes. The several objectives have been achieved including: (1) Establishment of polycistronic expression system in enoki mushroom F. velutipes using selectable marker, hph, and reporter gene, egfp, linked by 2A peptides from porcine teschovirus-1. After ATMT, the F. velutipes tramsformants carrying antibiotic resistant capability expressed EGFP as evidenced by fluorescent microscopy and western blotting. Position effect of transgene expression was also investigated by locating egfp at upstream or downstream of 2A peptides. The EGFP amount of F. velutipes transformants assayed by ELISA showed that transgene at upstream of 2A peptides, which was directly driven by the promoter, was expressed more than that at downstream. (2) Enhancement of transgene expression using tandem repeated egfp linked by 2A peptides. The results of western blotting, ELISA and fluorescent microscopy showed that heterologous protein expression in the transgenic F. velutipes was notably enhanced via 2A peptide-mediated cleavage to co-express up to three copies of single gene. (3) Establishment of an EV71 virus-like particles (VLPs) expression system. The genes of EV71 P1 structural protein and 3C protease were constructed in the same transcript linked by 2A peptides. After P1 and 3C were separated via 2A peptides mediated cleavage in co-translational level, 3C protease can proteolytically cleavage P1 into four subunits, VP1, VP2, VP3, and VP4. These four subunits automatically assemble to form VLPs within transgenic mycelia. To increase the efficiency of subunits production, a site-directed mutation K52R of 3C protease was made to prevent sumoylation from host cell. After ATMT and F. velutipes transformants selection, the crude extraction of EV71 VLPs from transgenic mycelia were purified and fixed by GraFix method. The subunits VP1 and VP2 were detected using specific monoclonal antibodies. The partial peptide sequences of VP1 to VP4 were assigned by LC-MS/MS and mascot. These results demonstrated that both of 2A peptides and 3C protease are functional and successfully produce the EV71 VLPs in transgenic F. velutipes via poly-heterologous gene expression system. Moreover, the 2D images of EV71 VLPs can be observed under TEM, and their 3D structure was reconstructed de novo from the TEM-images of class III. This study is the first report regarding the establishment of mushroom polycistronic system. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.