Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90104
標題: Expression of Chimeric Bamboo mosaic virus Coat Protein by Potato Virus X vector
馬鈴薯X病毒載體表達竹嵌紋病毒重組外鞘蛋白
作者: 蘇峻暐
Chun-Wei Su
關鍵字: 馬鈴薯X病毒
植物病毒表現載體
口蹄疫
PVX
FMD
plant viral vector
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摘要: 口蹄疫為一世界性的疫病,在歐洲、英國及台灣都曾對畜產業造成嚴重損害。而今口蹄疫疫病控制使用去活化病毒疫苗的仍有活病毒汙染的風險,因此,安全性較高的次單位疫苗可視為一良好的開發方案。在過去研究發現,藉由奈米顆粒可以增進疫苗的效價,於此,實驗室過去發表了利用在竹嵌紋病毒 (Bamboo mosaic virus, BaMV) 為表現載體,外鞘蛋白於其N端融合上口蹄疫表面抗原VP1,構築了重組病毒BVP1,以植物病毒顆粒為奈米顆粒藉以刺激提高生物體產生抗體之效力。然BaMV的寄主範圍狹窄,多為竹科植物,使其在生產利用上受到了限制。馬鈴薯X病毒 (Potato virus X, PVX) 在過去已被廣泛使用於植物體中表達外源蛋白,而其寄主範圍廣泛包含了許多可食用植物,如:馬鈴薯、番茄、小黃瓜、波菜、豇豆、旱芹等,因此,本實驗中利用PVX病毒載體來表達FMDV VP1和BaMV CP重組病毒蛋白,期待藉此拓展病毒載體的可用寄主範圍,並且於疫苗生產時可以省去高成本的純化動作,簡單的萃取收集後便可直接投入使用。另外,在此實驗設計中,由於對PVX在植物中移動有關鍵性影響的PVX CP其Open reading frame (ORF) 被置換成了目標基因 (Gene of interest, GOI),在拓展寄主範圍的同時也去除了病毒的感染力和系統性移動的能力,因此此病毒載體具有很好的生物安全性。本研究中以PVX做為模板表現過去構築之重組竹嵌紋病毒鞘蛋白 (BVP1 CP) ,事實上,起初期望對PVX基因組以最少的修改來達到表現目的,因此優先構築了XΔCP-BVP1,然而於實際試驗後發現單純的XΔCP-BVP1載體無法達到表現需求,因此又構築了XΔTC-BVP1並與來自菸草蝕紋病毒 (Tobacco etch virus, TEV) 之沉默抑制子 (silencing suppressor) HcPro 蛋白共同接種。經Coomassie blue staining和Western blot確認了病毒載體經修改後可正常複製產生BVP1 CP,但於部分寄主植物中,其可能由於BVP1 CP表現量太低導致信號過弱而無法偵測,因此欲擴展載體的應用寄主範圍,其往後仍有許多需繼續研究及改良之空間。
Foot-and-mouth disease virus (FMDV) is a major plague in animal farming. The present vaccine used is produced by chemical inactivation which may has the risk of live virus contamination. Therefore, the subunit vaccine can be considering as a safer choice for future vaccine production. Accordingly, a Bamboo mosaic virus (BaMV) based viral vector expressing FMDV epitope - BVP1 recombinant virus had been established in previous study, expressing FMDV VP1 epitope by fusing it to the N-terminal of BaMV coat protein (CP). But since the BaMV host range is relatively narrow, that plants can be applied in vaccine production are still restrained. Potato virus X (PVX) has been widely used on foreign protein expressing in plants, it has a wide host range that includes several edible plants, such as potato, tomato, cucumber, spinach, cowpea and celery. Thus, we combined BaMV with PVX viral vector system to improve the applicability of the BVP1 CP viral expressing system. Expressing BVP1 CP via PVX viral vector in edible plants would allow the vaccine product put into use directly or simply with some extraction process, omitting the costly purification process. Furthermore, due to the PVX CP ORF replacement with our gene of interest (GOI), the systemic transport ability and infectious virion forming are eliminated. In fact, to minimize the modification on the PVX genome, we only constructed XΔCP-BVP1 originally. But after the test, we found that the protein yield from XΔCP-BVP1 was far from enough. In that case, we introduced the HcPro silencing suppressor from Tobacco etch virus (TEV) and compared the expression with another newly constructed XΔTC-BVP1. Though after the Coomassie blue staining and Western blot analysis, it is confirmed that BVP1 CP can express with both two strategy but BVP1 CP were still undetectable in some hosts, possibly due to the low expression. That is, before we can put this system into application, there's still a lot more research and study to do in the future.
URI: http://hdl.handle.net/11455/90104
文章公開時間: 2016-08-31
Appears in Collections:生物科技學研究所

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