Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13702
標題: 羊傳染性化膿性病毒E3L基因之選殖表現及功能分析
Cloning, expression and functional assay of the E3L gene of orf virus
作者: 周佳季
Chou, Chia-Chi
關鍵字: 羊傳染性
Orf virus
化膿性
病毒
E3L
TAP
出版社: 獸醫學系暨研究所
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摘要: 傳染性化膿性皮膚炎 (contagious pustular dermatitis) 又叫做Orf,可發生在全世界的山羊與綿羊,尤其是在年幼動物。Orf會在嘴唇、口鼻周圍以及口腔黏膜產生增生性的病變。Orf病毒屬於副痘病毒屬,痘病毒科。病毒顆粒為卵圓形,基因體為線狀的雙股DNA。Vaccinia 病毒的 E3L 基因其 C 端包含了 DRBM (dsRNA binding motifs) 存在著一個雙倍體可結合 dsRNA,而抑制細胞中抗病毒的double-stranded RNA (dsRNA)-dependent protein kinase (PKR) 蛋白。因此,為了探討 orf 病毒的 E3L 蛋白是否具有結合dsRNA的活性。本實驗中我們以PCR的方式增幅出E3L全長基因,並將其轉接至原核表現載體pET24a。將建構好的重組質體轉型至E. coli BL21 (DE3) 中,再以IPTG誘導表現E3L重組蛋白。表現出的重組蛋白以西方墨點法與質譜儀 (mass spectrometry) 鑑定確定為orf病毒的E3L蛋白。接著,以親和性樹脂管柱純化出E3L蛋白。純化後的E3L蛋白免疫小鼠製造多株抗體 (polyclonal antibody)。再以 electrophoretic mobility shift assays (EMSA) 分析其與dsRNA結合能力。此外,我們利用串聯式親和性樹脂管柱純化策略 (tandem affinity purification, TAP) 以及免疫沉澱 (immunoprecipitation, IP) 技術在真核表現系統當中尋找E3L相關作用蛋白 (E3L-interacting proteins)。經過 mass spectrometry確認蛋白質身份,在免疫沉澱實驗當中所發現的細胞性60S ribosomal protein L12 很有可能為E3L所作用之蛋白質。
Orf, also known as contagious ecthyma, contagious pustular dermatitis, Scabby mouth and sore mouth, occurs worldwide in sheep and goats. Orf virus is a member of the Parapoxvirus genus of Poxviridae. The virus particles are ovoid and the viral genome is a linear double-stranded DNA. The E3L gene of vaccinia virus (the prototype virus of poxvirus) encodes a dsRNA-binding protein that can inhibit activation of the cellular antiviral protein PKR. To study the function of E3L of orf virus, we amplified the E3L gene of the Nantou strain orf virus by polymerase chain reaction (PCR) and expressed the E3L protein in E. coli strain BL21(DE3). The authenticity of recombinant E3L protein was verified with western blotting and mass spectrometry analysis. The purified rE3L was used as antigen to produce polyclonal antibody in mice. The dsRNA-binding property of the rE3L was examined with electrophoretic mobility shift assays (EMSA). Furthermore, we searched for E3L-interacting proteins in mammalian cells by means of tandem affinity purification (TAP) and immunoprecipitation (IP) followed by mass spectrometry. A cellular 60S ribosomal protein L12 was found to be associated with our recombinant E3L protein by immunoprecipitation.
URI: http://hdl.handle.net/11455/13702
其他識別: U0005-0408200904084900
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