臺灣博碩士論文加值系統

竹嵌紋病毒 (Bamboo mosaic virus) 為單一正股 RNA 病毒，具有五個主要轉譯架構區，其中第一個轉譯架構區可轉譯出大小約155 kDa 的複製酵素，從N端到C端分別為戴帽酵素活化區、類解旋酵素活化區，以及核糖核酸聚合酵素活性區；第五個轉譯架構區由約1 kb 的次基因體核糖核酸轉譯，產生病毒包被所需的外鞘蛋白質。經前人的研究利用酵母菌雙雜交篩選得知，竹嵌紋病毒之類解旋酵素與外鞘蛋白質有交互作用，因此本實驗在探討兩個蛋白質交互作用在病毒的生命週期中所扮演的角色。首先推測兩者交互作用可能與病毒的複製相關，藉由菸草原生質體胞內試驗顯示，大量表達類解旋酵素會降低病毒外鞘蛋白質的累積量，為進一步確定會影響病毒複製，藉由北方墨點法測定 RNA 的累積量，結果並沒有偵測到任何 RNA 的存在，因此未來將進行 real-time PCR 測定。此外我們利用細菌雙雜交篩選系統，藉由 error-prone PCR 方法隨意突變外鞘蛋白質核苷酸片段，尋找兩個蛋白質之間交互作用的胺基酸殘基位置，其結果顯示，外鞘蛋白質上分別有4個胺基酸與1個胺基酸殘基位置突變降低與類解旋酵素之間交互作用；分別為 I130M、D170N、A209G、N218K 以及 N210S，另外，藉由 pull down assay ，結果顯示這些胺基酸殘基位置突變減少與類解旋酵素之間交互作用。為了找到更多兩個蛋白質之間交互作用的重要胺基酸殘基位置，須再作更多的篩選，未來將針對這些突變點,藉由菸草作為分析的載體，以便更深入了解兩個蛋白質之間交互作用的生理意義。

Bamboo mosaic virus (BaMV) has a single-strand RNA genome which consists of five open reading frames (ORFs). ORF 1 encodes a ~155 kDa replicase that contains a capping enzyme domain, a helicase-like domain, and a RNA dependent RNA polymerase. ORF5 encodes the viral coat protein. Previous studies showed a interaction between the helicase-like domain and the coat protein of BaMV by yeast-two-hybrid screening. Hence, I focused on investigating the biological significance of the interaction between the helicase-like domain and the coat protein of this virus in this study.
First, I investigated the effect of the helicas-like domain on coat protein accumulation by overexpressing extra helicase-like domain in protoplast assay. Preliminary results showed that the coat protein accumulation was reduced when the helicase-like domain was additionally expressed. To verify the reduction of BaMV replication, I will perform real-time PCR to assay the RNA accumulation of BaMV. I also randomly mutated amino acids on the coat protein by the error-prone PCR to find out the interaction sites with the helicase-like domain in a bacteria-two-hybrid screening. The screening identified two mutant coat proteins which one with 4 amino acid substitutions (I130M, D170N, A209G and N218K) and another with 1 amino acid substitution (N210S) that has weaken interaction with the helicase-like domain . Furthermore, I confirmed that two mutant coat proteins reduce the interaction ability with the helicase-like domain by pull down assay. In the future, I will continue to search for more interaction sites between the helicase-like domain and the coat protein. In order to elucidate more biological significance of the interaction, we will mutate these amino acids respectively which I have found to apply in N. benthamiana.

目次
第一章、前言
竹嵌紋病毒................................................1
竹嵌紋病毒類解旋酵素......................................2
外鞘蛋白質的研究..........................................3
本實驗目的................................................5

第二章、材料與方法
第一節、檢測 BaMV 之外鞘蛋白質 (Cp) 與類解旋酵素 (HLD) 之間交互作用關係
壹、蛋白質表達質體的構築..............................7
一、菌株、載體及培養基..............................7
二、質體的構築......................................9
三、大腸桿菌 (E. coli) 之轉型作用..................11
四、E. coli 質體 DNA 之抽取........................13
貳、 蛋白質與蛋白質之間交互作用的分析................14
一、判定兩個蛋白質之間交互作用方式.................14
二、尋找 Cp 中與 HLD 之間交互作用的重要胺基酸殘基位置.......................................................14
参、蛋白質電泳的分析.................................16
肆、西方墨點法.......................................17
ㄧ、蛋白質電泳轉移.................................17
二、免疫呈色反應...................................17
伍、體外分析蛋白質與蛋白質之間交互作用...............18
一、BaMV 之 HLD 與 Cp 的質體構築...................18
二、BaMV HLD 的備製及純化..........................18
三、BaMV Cp 的備製.................................23
四、Pull down assay................................24
第二節、菸草原生質體胞內試驗蛋白質與蛋白質之間交互作用的關係
壹、DNA 質體的構築...................................25
一、質體 pCBG、pCB、pCB△Cp......................25
二、質體 PBI221..................................25
三、質體 pBI221-HLD 與質體 pBI221-muHLD..........26
貳、 菸草原生質體之製備..............................26
一、緩衝液配置...................................26
二、原生質體之細胞壁水解.........................27
三、原生質體的純化...............................28
四、菸草原生質體細胞的計算.......................28
五、菸草原生質體之轉形作用及培養.................29
參、菸草原生質體蛋白的萃取及定量.....................29
一、萃取.........................................29
二、定量.........................................29
肆、影響 BaMV 複製的分析.............................30
一、西方墨點.....................................30
二、北方墨點.....................................30
三、即時定量聚合酶連續反應.......................34

第三章、 結果
第一節、檢測細菌雙雜交系統中，HLD 與 Cp 之間交互作用...36
一、pKT25-HLD、pUT18-Cp、pKT25-Cp 及 pUT18-HLD 質體的構築.......................................................37
二、檢測 T25-HLD 與 Cp-T18 於 E. coli 中之表現.......38
三、在細菌雙雜交系統，檢測 T25-HLD 與 Cp-T18 之專一性活性反應...................................................38
第二節、細菌雙雜交系統尋找 Cp 與 HLD 之間交互作用的重要胺基酸殘基位置.............................................40
一、藉由error-prone PCR 方法製造大量突變質體 pUT18-muCp基因庫，並計算error-prone PCR之突變效率..................40
二、利用細菌雙雜交系統之選擇性培養基，挑選會影響與HLD之間交互作用的突變Cp.......................................41
三、Pull down assay 在細菌雙雜交系統中挑選 muCp 胺基酸殘基突變位置影響與 HLD 之間交互作用的程度......................43
第三節、測定 HLD 與 Cp 之間交互作用對於BaMV的複製之影響...44
一、原生質體胞內測定大量表達 HLD 對 BaMV Cp 累積量之影響..45
二、原生質體胞內測定 NTPase 突變之 HLD 對 BaMV Cp 累積量之影響......................................................45
三、 原生質體胞內測定大量表達 HLD 對 BaMV Cp 累積量在不同時間點之影響..............................................46
四、原生質體胞內測定大量表達 HLD 對缺乏 Cp 的 BaMV 複製之影響......................................................46

第四章、 討論...........................................48

參考文獻
一、 中文參考文獻.......................................54
二、 英文參考文獻.......................................54

表目次
表1、PCR 所用引子之序列..................................58
表2、細菌雙雜交系統之選擇性培養基，篩選突變 Cp 之質體
pUT18-muCp 及檢測分析之統計..............................59
表3、定序結果估算 error-prone PCR 突變效率...............60

圖目次
圖一、竹嵌紋病毒基因體架構...............................61
圖二、質體 pKT25-HLD 之圖譜與特徵........................62
圖三、質體 pUT18-Cp 之圖譜與特徵.........................63
圖四、質體 pUT18-HLD 之圖譜與特徵........................64
圖五、質體 pKT25-Cp 之圖譜與特徵.........................65
圖六、質體 pBI221-HLD 之圖譜與特徵.......................66
圖七、質體 pBI221-△HLD 之圖譜與特徵.....................67
圖八、檢測 T25-HLD 與 Cp-T18 於 E. coli 中之表現.........68
圖九、在細菌雙雜交系統篩選用培養基，檢測 T25-HLD 與 Cp-T18
之專一性活性反應.........................................70
圖十、在細菌雙雜交系統選擇性培養基，檢測 T25-HLD 與 Cp-T18
之專一性活性反應.........................................71
圖十一、Error-prone PCR 突變 Cp 與細菌雙雜交系統之選擇性培養
基，尋找 Cp 胺基酸殘基突變位置影響與 HLD 之間交互作用之流
程圖.....................................................72
圖十二、由 M63 固態選擇性基礎培養基挑選質體 pUT18-muCp 及
檢測 muCp-T18 之表現.....................................73
圖十三、細菌雙雜交系統之 M63 固態選擇性基礎培養基，尋找
muCp-T18 影響與 HLD 交互作用.............................75
圖十四、突變 Cp 與野生菌株 Cp 序列比對圖.................77
圖十五、Pull down assay 測定 muCp-T18 與 HLD 之間交互作用之
影響.....................................................78
圖十六、原生質體胞內測定大量表達 HLD 對 BaMV Cp 累積量之影響.......................................................80
圖十七、胞內測定 NTPase 突變之 HLD 對 BaMV Cp 累積量之影
響.......................................................81
圖十八、大量表達 HLD 對 BaMV Cp 累積量在不同時間點之影
響.......................................................82
圖十九、比對同屬於 potexvirus 之病毒 TVX、PapMV、PVX 及 LVX
之 Cp 片段胺基酸序列.....................................83
圖二十、PVX Cp 與 BaMV Cp 序列以 local alignment 之相同性比
對圖.....................................................84
圖二十一、探討 BaMV 之 Cp 與 HLD 之間交互作用對於病毒的生
理意義之策略圖...........................................85

附表目次
附表1、GeneMorph® II Random Mutagenesis Kit 說明 error-prone PCR 之調控突變的條件...............................86

附圖目次
附圖一、細菌雙雜交系統之模式圖............................87
附圖二、細菌雙雜交系統之篩選方式模式圖....................88
附圖三、細菌雙雜交系統，質體 pKT25 及 pUT18 之圖譜與特徵..89
附圖四、質體 pCBG、pCB、pCB△Cp 之圖譜與特徵..............90
附圖五、pET-Dute-HLD 之圖譜與特徵.........................91
附圖六、pBI221 之圖譜與特徵...............................92
附圖七、PVX 藉二級結構預測，模擬空間上之三級結構模型圖....93

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