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標題: 小麥胚芽萃取物中雙股RNA引起的蛋白轉譯抑制之研究
Study on dsRNA-dependent translation repression in wheat germ extract
作者: 連力緯
Lien, Li-Wei
關鍵字: dsRNA
translation repression
出版社: 生物化學研究所
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摘要: 當病毒入侵哺乳動物體內,細胞會進行先天免疫反應,抑制病毒蛋白合成。然而病毒也因應宿主發展對抗先天免疫反應機制。其中流感病毒轉譯NS1A蛋白,會影響(dsRNA -dependent protein kinase)PKR活性,協助病毒在宿主體內存活。 為了了解雙股RNA誘導轉譯抑制的機制,在植物系統中是否有類似的現象。以竹嵌紋病毒 ( Bamboo mosaic potexvirus, BaMV )和其攜帶之衛星核酸 (satellite RNA, satBaMV)為材料。不同衛星核酸可能會減輕病毒所引起的病徵;轉譯出的P20蛋白具有結合RNA的能力,猜測P20蛋白可能會結合dsRNA。所以我們想探討P20蛋白在小麥胚芽萃取物中是否也像NS1A蛋白,可以藉由結合雙股RNA去調控蛋白質轉譯。此外,在小麥胚芽萃取物裡,是否有小麥胚芽蛋白因子會結合雙股RNA和NS1A亦進行分析。以下為目前之實驗結果: (1)在in vitro條件下BaMV蛋白於小麥胚芽中之表現量確實會受到雙股RNA (Polyriboinosine-polyribocytidylic acid (poly(IC))所抑制。(2)在有沒有雙股RNA存在下,衛星核酸的質體存在都會提升BaMV蛋白於小麥胚芽中之表現量。(3)從poly IC親合層析管柱分析之實驗結果,獲得6個可能的雙股RNA結合蛋白。將篩選出的蛋白進行dsRNA-binding motif(dsRBM)序列分析比對,其中5個與dsRBM序列有30%的相似性。(4)純化GST-NS1蛋白發現含有太多雜質,不利於分析小麥胚芽蛋白因子結合NS1A之研究。
As the viruses invade mammalian cells, the cells execute the innate immune responses which suppress translation of the viruses. But the viruses antagonize the innate immune response mechanism in conformity with the development of hosts as well. Influenza virus translates NS1A protein which suppresses double strand RNA -dependent protein kinase (PKR) activation, helps the virus to live in host cells. We focus here on the suppression of translation mechanism by dsRNA to address if there is a similar phenomenon in the plant system. The Bamboo mosaic virus (BaMV) carries satellite RNA (satBaMV) and variants of satBaMV may attenuate the symptoms caused by the virus. SatBaMV encodes P20 protein which can bind to RNA. Therefore, if P20 protein can regulate the translation in wheat germ extract by sequestering dsRNA in a way similar to NS1A protein remains to be determined. In addition, factors in the wheat germ extract capable of binding with dsRNA or NS1A protein will be investigated. Some results are summarized below: First, BaMV protein translation in the wheat germ extract can be suppressed by dsRNA (polyriboinosine - polyribocytidylic acid, poly IC ) in vitro. Second, addition of satBaMV plasmid enhances BaMV protein translation in the wheat germ extract with or without the existence of dsRNA. Third, six possible dsRNA-binding proteins within wheat germ extract are recovered by poly IC affinity column. After sequence alignment with dsRNA-binding motif (dsRBM), five of them share an average of 30% similarity with dsRBM. Fourth, the result of NS1 protein associated factors is uncertain due to impurities problem.
其他識別: U0005-2308200813374500
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