Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23769
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dc.contributor.advisor張邦彥zh_TW
dc.contributor.advisorBan-Yang Changen_US
dc.contributor.author林宇星zh_TW
dc.contributor.authorLin, Yu-Hsingen_US
dc.date2001zh_TW
dc.date.accessioned2014-06-06T07:21:08Z-
dc.date.available2014-06-06T07:21:08Z-
dc.identifier.urihttp://hdl.handle.net/11455/23769-
dc.description.abstract本研究最終目的在探討竹嵌紋病毒 (BaMV) 之TGBp2蛋白在染病之植物細胞中存在的位置,以及它的生化特性。利用胺基酸序列之比對分析,推測此蛋白可能屬於一種膜蛋白,它含有2個疏水性的transmembrane helices,而這2個transmembrane helices,由35個胺基酸所形成的loop來連結。為了確定TGBp2屬於膜蛋白,以及進行TGBp2之膜特性研究,我們首先大量生產TGBp2蛋白,並製備其抗體。藉由差異性離心對細胞成分之劃分、Tricine SDS-PAGE之技巧、以及敏感較高的Chemiluminenscence reagent進行Western blot之分析,我們可以穩定地偵測到TGBp2主要存在感染竹嵌紋病毒白蔾 (Chenopodium quinoa) 葉片細胞之膜狀成分中。此蛋白在白藜葉片接種竹嵌紋病毒3天後開始出現,隨後含量逐漸累積,至第11天此蛋白含量隨即開始衰減。為了要證實TGBp2蛋白的確是與膜結合以及研究TGBp2蛋白的生化特性,製備proteoliposome上具有生化活性之TGBp2蛋白樣品對於未來進一步的研究工作是必要的。接著,我利用體外蛋白質重新摺疊及proteoliposome重組的方法,希望能構築出在proteoliposome上具有生化活性之TGBp2蛋白。實驗結果顯示,大腸桿菌大量表現的TGBp2蛋白經變性處理後,於含有Triton X-100或n-octyl-β-D-glucopyranoside等兩種界面活性劑之緩衝液中進行重新摺疊時,僅有少部分的TGBp2蛋白呈現可溶性的狀態,顯示TGBp2蛋白本身非常容易聚結,並且不易和界面活性劑形成micelle。將進行二次重新摺疊純化之可溶性TGBp2蛋白與phospholipid加以混合,利用疏水性吸附材 (Bio-bead) 、或透析方式移除界面活性劑,進而以Ficoll gradient分析TGBp2和phospholipid之分佈,發現此兩成分在離心場下,位移位置是重疊的。這種結果顯示,TGBp2蛋白可能存在於重組完成之proteoliposome上。不過利用上述2種不同界面活性劑進行proteoliposome的體外重組,所形成的proteoliposome在電子顯微鏡觀察的型態上、以及在Ficoll gradient分布的均勻度上均有所差異。這種TGBp2蛋白與liposome在體外 (in vitro) 重組所形成的proteoliposome應該可以作為研究竹嵌紋病毒TGBp2之生化特性、以及研究三種轉移蛋白相互作用的材料。zh_TW
dc.description.abstractThe final goal of this study is to locate the TGBp2 protein in the BaMV infected-tissues and to analyze the biochemical properties of this protein. Amino acid sequence alignment analysis has revealed that TGBp2 is a transmembrane protein with two transmembrane helices interrupted by a loop with 35 amino acids in length. Taking advantage of the techniques of differential centrifugation, Tricine SDS-PAGE, and a sensitive chemiluminscence immunodetection system, we have been able to detect TGBp2 mainly in the membrane fraction of the BaMV-infected leaves of Chenopodium quinoa reproducibly. The content of TGBp2 started to accumulate in the leaves 3 days postinoculation, gradual increased up to the 11th day postinoculation, and diminished gradually thereafter. In order to confirm the association of TGBp2 with membrane and to prepare sample for studying the biochemical property of TGBp2 in the future, preparation of proteoliposome containing TGBp2 are necessary. In vitro protein refolding and in vitro proteoliposome reconstitution were thus performed. Two detergents, Triton X-100 and n-octyl-b-D-glucopyranoside, were incorporated into the refolding and the reconstitution buffers. Only a small portion of TGBp2 in the refolded TGBp2 sample became soluble, implicating that TGBp2 was prone to aggregation by itself and was difficult to form micelle with detergents. After mixing the soluble portion of TGBp2 with phospholipid, and removal of the detergent molecules with Bio-bead or a dialysis apparatus, the reconstituted sample was then fractionated with a ficoll gradient. Co-migration of TGBp2 with phospholipid in the gradient was observed, suggesting that TGBp2 was associated with proteoliposome. However, the shape of proteoliposome as examined by electron microscopy and the distribution of proteoliposome in the ficoll gradient were different with different detergents. The proteoliposome thus constructed can be of use in our future biochemical study of TGBp2 as well as in the study of mutual interaction among the three movements proteins of bamboo mosaic potexvirus.en_US
dc.description.tableofcontents中文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧1 Abstract‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧3 前言‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧5 材料與方法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧11 一、pCT14質體的構築‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧11 二、TGBp2蛋白之大量表現‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧11 三、TGBp2蛋白的回收‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧12 四、BaMV染感白藜葉片細胞膜成分之劃分‧‧‧‧‧‧‧‧‧‧12 五、TGBp2蛋白之重新摺疊‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧13 六、可溶性及不可溶性TGBp2蛋白之Western blot分析‧‧‧‧‧14 七、Lipid-detergent mixed micelles之製備‧‧‧‧‧‧‧‧‧‧‧14 八、二次重新摺疊製備純化之可溶性TGBp2蛋白‧‧‧‧‧‧‧15 九、含有TGBp2蛋白之proteoliposome的製備‧‧‧‧‧‧‧‧15 十、利用分析型電子顯微鏡 (analytical electron microscope, AEM)觀察liposome‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧16 十一、Ficoll gradient分析proteoliposome的之分布‧‧‧‧‧‧‧16 十二、Phospholipid含量之訂定‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧17 結果‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧18 一、大腸桿菌TGBp2蛋白之大量表現‧‧‧‧‧‧‧‧‧‧‧‧18 二、健葉及感染BaMV病葉細胞膜狀成分之劃分‧‧‧‧‧‧‧18 三、TGBp2蛋白在感染BaMV病葉中含量之消長‧‧‧‧‧‧‧19 四、TGBp2蛋白之重新摺疊及水溶性狀態之形成‧‧‧‧‧‧‧20 五、二次重新摺疊製備純化之可溶性TGBp2蛋白‧‧‧‧‧‧‧21 六、Liposome在分析型電子顯微鏡 (analytical electron microscope, AEM) 下觀察之型態‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧21 七、phosphate assay定phospholipid的含量‧‧‧‧‧‧‧‧‧‧22 討論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧24 Reference‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧28 附圖及說明‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧35 附錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧54zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物化學研究所zh_TW
dc.subjectbamboo mosaic virusen_US
dc.subject竹嵌紋病毒zh_TW
dc.subjectrefoldingen_US
dc.subjectphospholipiden_US
dc.subjectproteoliposomeen_US
dc.subjectTGBp2en_US
dc.subject重新摺疊zh_TW
dc.subject磷脂質zh_TW
dc.subject蛋白脂微體zh_TW
dc.subject三重疊基因第二轉譯蛋白zh_TW
dc.title竹嵌紋病毒三重疊基因區第二轉譯蛋白膜結合特性之研究zh_TW
dc.titleThe membrane-associated property of the triple gene block protein 2 of bamboo mosaic virusen_US
dc.typeThesis and Dissertationzh_TW
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