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標題: 利用免疫共沉澱探討竹嵌紋病毒移動複合體相關蛋白之間的交互作用
Identification and Functional Analyses of the Components Interacting with BaMV Movement Complex By Co-immunoprecipitation
作者: 黃奇賢
Huang, Chi-Hsian
關鍵字: Bamboo mosaic virus
movement protein
出版社: 生物科技學研究所
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摘要: 竹嵌紋病毒( Bamboo mosaic virus , BaMV )為Flexiviridae科potexvirus 屬的單股正極性RNA病毒,基因體具有五個轉譯架構 ( open reading frame),其中三重疊轉譯區蛋白組 ( Triple-gene-block protein , TGBps 1-3 ) 與病毒外鞘蛋白對病毒在細胞間移動有關。目前已知potexvirus RNA於寄主細胞中會與外鞘蛋白及TGBp1構成移動複合體,並由細胞原生質絲移動到鄰近細胞,然而TGBp2及TGBp3是如何協助BaMV完成細胞間移動的詳細機制至今尚未明瞭。由於前人利用酵母菌雙雜交篩選的結果無法明確得知TGBp2及TGBp3是否會和移動複合體中任何蛋白質相互作用。本實驗使用BaMV外鞘蛋白的抗體進行免疫共沉澱搭配西方轉漬法偵測分析,希望能初步探討鞘蛋白、TGBp1、TGBp2和TGBp3之間是否具有交互作用,並同時尋找其他和鞘蛋白交互作用之因子。BaMV鞘蛋白和TGBp1具有交互作用,但目前實驗仍未證實BaMV鞘蛋白和TGBp2有相互作用情形。 此外,將c-myc 胜肽融合於TGBp2 N端及TGBp3 C端,轉殖此兩段融合基因於衛星核酸載體表現系統,並建立的基因轉殖菸草,與非轉殖菸草同時進行BaMV接種,分別利用抗TGBp2和抗c-myc 的血清進行西方轉漬法偵測TGBp2轉殖菸草,在轉殖菸草較非轉殖菸草明顯測得TGBp2訊號,証實N端帶有c-myc的TGBp2在接種BaMV後會大量表現。因此可利用抗c-myc的血清取代抗TGBp2血清偵測轉殖菸草中TGBp2的表現,但仍無法藉由免疫共沉澱測得BaMV外鞘蛋白和TGBp2有相互作用。同樣的,利用抗c-myc 的血清進行西方轉漬法偵測TGBp3轉殖菸草,可測得大量帶有c-myc的TGBp3。轉基因植物大量表現的融合蛋白c-myc TGBp2及TGBp3-c-myc是否具有生物功能幫助移動複合體移動,仍有待進一步的實驗加以證實。
Bamboo mosaic virus ( BaMV ) is a member of the protexvirus genus in the family Flexiviridae. BaMV contains a single-strand RNA genome which consists of five open reading frames ( ORFs ). BaMV encoded triple gene block ( TGB ) proteins participate in the virus cell to cell movement and spread in the host. It has been known that potexvirus RNA , coat protein, and TGBp1 could form complexes to facilitate the translocation to neighboring cells through plasmodesmata. However , the roles of movement proteins TGBp2 and TGBp3 in the movement complex are not clear yet. Previous yeast-two hybrid experiments could not prove the interaction among movement complex and TGB proteins. We try to re-examine the interaction of TGBp2 and TGBp3 in movement complex and to find any components interact with them by co-immunoprecipitation with BaMV coat protein antibody. The results show that TGBp1 but not TGBp2 could interact with coat protein. Furthermore, we used BaMV TGBp2 transgenic Nicotiana benthamiana plants in which the transgene TGBp2 was fused with c-myc tag at N-terminal and constructed in satBaMV expression system. The accumulation level of TGBp2 in transgenic lines was amplified by inoculating with BaMV, compared to nontransgenic plants, probed with either c-myc or TGBp2 antiserum. Accordingly, we use c-myc antiserum to replace TGBp2 antiserum for detecting of c-myc-TGBp2. Transgenic Nicotiana benthamiana expressing c-myc tagged TGBp3 was also constructed as TGBp2 lines, except c-myc tag was fused at C terminal. By co-immunoprecipitation , we still gain no evidence to show that the c-myc-TGBp2 or TGBp3-c-myc could interact with coat protein. The biological functions of those c-myc tagged proteins to complement the virus movement will be proven by experiments in the future.
其他識別: U0005-2207201010260300
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