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研究生:羅珮瑄
研究生(外文):Pei-shuan Lo
論文名稱:竹嵌紋病毒外鞘蛋白的多形現象及次單位相互作用之分析
論文名稱(外文):Polymorphism and interaction of coat protein subunits of Bamboo mosaic virus
指導教授:徐堯煇
指導教授(外文):Yau-Heiu Hsu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:59
中文關鍵詞:竹嵌紋病毒外鞘蛋白
外文關鍵詞:BaMVCoat protein
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壹、中文摘要

竹嵌紋病毒(Bamboo mosaic virus, BaMV)是一單股正極性的 RNA 病毒,基因體具有五個轉譯區(open reading frames, ORFs),其中 ORF 5 可由次基因體核醣核酸轉譯出 25 kDa 的外鞘蛋白。竹嵌紋病毒的外鞘蛋白是個多功能的蛋白,參與病毒的包被和移動,在二維電泳顯示竹嵌紋病毒外鞘蛋白有多形現象。同屬於馬鈴薯病毒群的 Potato virus X ( PVX ) 其外鞘蛋白經過醣基化修飾可以穩定病毒結構,且具有 NTPase 活性可以參與病毒的細胞間移動。目前對於 BaMV 的外鞘蛋白是否轉譯後經醣基化修飾、是否具 NTPase 活性及其蛋白結構仍未知。本研究首先經過薄層層析法、酵素連結法和紫外線交聯法實驗證實 BaMV 外鞘蛋白不具有 ATPase 活性。為了分析在外鞘蛋白上是否有醣基化參與穩定蛋白結構,由醣染色及醣親和性管柱分析結果推測在竹嵌紋病毒上沒有醣蛋白的存在。另一部份,根據之前的研究,竹嵌紋病毒外鞘蛋白刪除 N 端 35 個胺基酸(Nd35)不會對病毒之感染造成影響,而外鞘蛋白 N 端刪除至 46 個胺基酸(Nd46)病毒無法在植物中累積。在純化 E. coli 表現的 BaMV CP 過程中,利用膠體過濾層析法發現 BaMV 全長和 Nd35 的病毒外鞘蛋白皆可形成多聚體。根據最近的文獻,推測 BaMV 外鞘蛋白上第四十五個胺基酸(苯丙胺酸)可能會參與外鞘蛋白之間的交互作用,與病毒的包被相關,所以本研究利用突變外鞘蛋白分析此位置胺基酸對病毒功能的影響。接種病毒於菸草結果得知刪除 N 端 44-46 個胺基酸,病毒就無法在植物中存活;突變 F45 成不同特性胺基酸會影響病毒形成。Nd44 和 Nd45 刪減株外鞘蛋白分別以三聚體和二聚體被純化出來,推測竹嵌紋病毒外鞘蛋白上的第 45 個胺基酸扮演著疏水性結合的角色,影響外鞘蛋白交互作用及包被核酸。
貳、英文摘要

Bamboo mosaic virus ( BaMV ) is a monopartite, positive-strand RNA virus that encodes five open reading frames ( ORFs ). ORF-5 encodes viral coat protein (CP) which is translated from a subgenomic RNA. The CP has multi-function including encapsidation of the viral RNA genomes and movement. Two dimentional gel electrophoresis revealed the polymorphism of BaMV CP. It has been known that the CP of Potato virus X ( PVX ) is glycosylated at N-terminus which stabilizes the PVX structure. PVX CP C-terminus has also been known to possess NTPase activity that is involved in cell-to-cell movement. Glycosylation, NTPase activity and the structure of BaMV CP are still unknown. Analyzing by thin layer chromatography, the coupled enzyme ATPase assay and UV cross-linking assay, we have confirmed that BaMV CP has no ATPase activity. Glycoprotein staining and glycoprotein affinity chromatography assays also confirmed that there is no glycoprotein exitence on BaMV CP. According to the previous studies, deletion of N-terminal 35 amino acids ( Nd35 ) in BaMV CP caused no obvious effect on the virus infectivity, but the virus couldn’t survive in N. benthamiana after deletion of N-terminal 46 amino acids in CP. It has been reported that amino acid F at the N-terminus of Potexvirus CP may play an important role in CP subunits interaction. Our recent gel filtration analysis demonstrated that wt CPs and Nd35 CPs over-expressed from Escherichia coli could form oligomer, however, Nd44 CPs and Nd45 CPs could only form trimer and dimer. In order to elucidate the subunits interaction of the residue F45 of BaMV CP associated with encapsidation, we mutate the CP at F45 to analyze the CP function. The results had revealed that the F45 plays a hydrophobic interaction among the subunits of BaMV CP and may affect encapsidation.
目 錄

壹、中文摘要 -----------------------------------------------------------i
貳、英文摘要 ----------------------------------------------------------ii
參、 前言
ㄧ、竹嵌紋病毒簡介 ------------------------------------------------1
二、植物病毒的外鞘蛋白 ---------------------------------------1
三、Potexvirus 外鞘蛋白的特性 -----------------------------3
(一)外鞘蛋白具有 ATPase 活性 -----------------------------------3
(二)外鞘蛋白的醣基化修飾 ---------------------------------------3
(三)外鞘蛋白的磷酸化修飾 ---------------------------------------4
(四)外鞘蛋白之間相互作用 -------------------------------------4
四、實驗目的 -------------------------------------------------------5

肆、 材料與方法
ㄧ、竹嵌紋病毒及外鞘蛋白之製備 ------------------------------------6
(一)BaMV 病毒純化 ----------------------------------------------6
(二)病毒顆粒解離出外鞘蛋白 -------------------------------------6
(三)構築外鞘蛋白 N 端突變之 BaMV 株 ----------------------------7
(四)構築 pET29a-N 端突變之外鞘蛋白 ---------------------------8
(五)外鞘蛋白突變株之選殖 ---------------------------------------9
(六)大腸桿菌表現外鞘蛋白之純化 --------------------------------10
二、ATPase 活性測試 ----------------------------------------------12
(一)酵素連結法 ------------------------------------------------12
(二)薄層層析法 ------------------------------------------------12
(三)紫外光交聯法 ----------------------------------------------12
三、外鞘蛋白醣基化分析 --------------------------------------------13
(一)電腦分析預測外鞘蛋白醣基化位置 ----------------------------13
(二)Cellufine 管柱分離醣基化的外鞘蛋白 ------------------------13
(三)醣染色 ----------------------------------------------------13
(四)醣蛋白偵測系統 --------------------------------------------14
四、外鞘蛋白 N 端突變株之接種測試 ----------------------------14
(一)菸草接種 --------------------------------------------------14
(二)原生質體接種 ----------------------------------------------15
(三)西方漬染免疫偵測 ------------------------------------------16
(四)北方墨點轉漬分析 ----------------------------------------17

伍、 結果
ㄧ、竹嵌紋病毒外鞘蛋白上可能不具有 ATPase 活性 ------------19
二、竹嵌紋病毒外鞘蛋白上可能不具有醣基化修飾 ----------------21
三、外鞘蛋白 N 端影響蛋白間之交互作用 ------------------------22

壹、 討論 -------------------------------------------------------------25
貳、 參考文獻 ---------------------------------------------------------30
參、 圖表 ------------------------------------------------------------38
肆、 附錄 -------------------------------------------------------------58
柒、參考文獻

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