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標題: | 神經壞死病毒外套蛋白與核醣體L7a在石斑魚腦細胞之交互作用 Interaction of nervous necrosis virus coat protein with ribosomal protein L7a in grouper brain cells |
作者: | Pei-Ru Wang 王珮儒 |
指導教授: | 張繼堯(Chi-Yao Chang) |
關鍵字: | 神經壞死病毒,外套蛋白,核醣體蛋白 RPL7a,病毒鋪覆蛋白試驗,類病毒顆粒,親和性管柱, Nervous necrosis virus ( NNV),coat protein,ribosomal protein RPL7a,VOPBA,virus-like particle,affinity column, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 神經壞死病毒( Nervous necrosis virus, NNV)是感染超過 50 種重要養殖海水魚類幼魚嚴重災情的致病病毒,此病毒近幾十年來危害全世界魚苗養殖場,造成養殖業重大損失。為了瞭解 NNV 與宿主細胞間的交互作用,本實驗室利用 NNV 類病毒顆粒( Virus-like particle, VLP)固定在樹脂上,作為親和性管柱,並把石斑魚腦細胞(GB cell)蛋白質萃取液加入管柱,沖提出的產物以銀染( Silver staining)及病毒鋪覆蛋白試驗( Virus overlay protein binding assay, VOPBA)及 LC-MS/MS 進行分析,推測在 30 kDa 上的條帶為核醣體蛋白 L7a。為了進一步了解 NNV 外套蛋白與核醣體蛋白 L7a 間的交互作用,先用大腸桿菌表現出重組 L7a,再做 VOPBA 確認L7a 與 NNV 外套蛋白有交互作用,且兩者結合的區域在核醣體蛋白 RPL7a 的 1-85 a.a.。用免疫螢光染色發現核醣體蛋白 L7a 與 NNV 外套蛋白在轉殖後 15 小時,重組表現的 NNV 外套蛋白與核醣體蛋白 L7a 共同分布在核仁;而轉殖重組核醣體蛋白 RPL7a 後再以 NNV 感染則發現原本聚集在核仁的核醣體蛋白 RPL7a 會離開核
仁,並散布在整個細胞質。另外病毒感染 GB 細胞後收集不同時間點的細胞總蛋白,發現核醣體蛋白 RPL7a 在感染後並無明顯變化,但以 RNAi 降低核醣體 RPL7a轉錄量卻可促進神經壞死病毒複製。此研究證明 GB 細胞的核醣體蛋白 RPL7a 會與神經壞死病毒外套蛋白結合,在病毒感染 GB 細胞後改變核醣體蛋白 RPL7a 在GB 細胞的分布區域,而降低核醣體 L7a 可能會促進神經壞死病毒增殖。 Nervous necrosis virus ( NNV) is a positive sense single-stranded RNA virus, it infects more than 50 species of marine fish and causes significant economic losses. To investigate the interaction of NNV coat protein and grouper brain ( GB) cell, we set up the NNV virus-like particle-coupled affinity column. By LC-MS/MS analysis, we define the 30 kDa band in SDS-polyacrylamide gel is ribosomal protein L7a (RPL7a). The interaction between NNV coat protein and RPL7a were further confirmed by virus-overlay protein binding assay ( VOPBA), and the binding region of RPL7a against NNV coat protein was defined to 1-85 a.a. By cotransfection of expression constructs which encoded NNV coat protein and RPL7a with HA or Myc tags, we find that both NNV coat protein and RPL7a colocalized in nucleolus 15 hr after cotransfection . With NNV infection, recombinant RPL7a was found to translocate from nucleolus to cytoplasm. The protein level of RPL7a in GB cell was constant during NNV infection by Western analysis. However, the amount of RNA2 in NNV infected GB cells was increase slightly after RPL7a knockdown by RNAi. Taken together, these results showed that the interaction between NNV coat protein and RPL7a relocated RPL7a from nucleolus to cytoplasm and influenced the propogation of NNV. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70761 |
DOI: | 10.6342/NTU201802281 |
全文授權: | 有償授權 |
顯示於系所單位: | 漁業科學研究所 |
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