臺灣博碩士論文加值系統

香蕉萎縮病毒(Banana bunchy top virus 簡稱BBTV)乃屬於Nanoviridae 科Babuvirus 屬之球型病毒。感染香蕉之典型的病徵為矮化、頂端新葉萎縮而叢生呈掃帚狀，假莖變細，葉片狹長直立，葉柄縮短。葉片、中肋、葉柄及假莖上常見暗綠色條斑。幼葉從葉緣逐漸萎黃且葉緣常呈焦枯，嚴重影響香蕉植株之生育。除蚜蟲傳播外，本病也可藉由香蕉吸芽苗傳播，近年來香蕉產業栽培以組織培養苗為主，故為防止此病毒經由組培苗傳播，香蕉親本於組培前必須經病毒檢測，確定無病毒感染後才能大量繁殖。BBTV由於病毒顆粒微小且僅存於感病植株之篩管細胞中，故濃度偏低，不易經由傳統方式純化病毒顆粒，以致影響檢定用病毒抗血清之製作。過去本研究室曾嘗試應用細菌表達載體之策略製備BBTV之CP 抗體，但其表達效果不佳，而影響抗體之製備與應用性。後續陳姿蓉與林益德兩位學長成功應用細菌表現載體技術，表達齒舌蘭輪斑病毒 (ORSV)及蕙蘭嵌紋病毒 (CymMV) 兩種蘭花病毒鞘蛋白成為一個具有兩種病毒抗原性的重組蛋白。且其中一種以CymMV 鞘蛋白N端序列接續ORSV 鞘蛋白C端序列之載體架構 (CyOrN)，其表現量高且穩定。因此本研究嘗試藉由基因重組技術將BBTV之鞘蛋白基因CP序列嵌入前述之CyOrN表現載體中，能以CymMV 之N端序列為前驅序列，帶動BBTV CP基因之合成，促進重組蛋白之表現與累積，以便能順利將其純化供檢定用抗體之
製備。實驗過程先根據GenBank上所登錄之BBTV CP基因序列，設計可增幅其全長、N端及C端序列且可於 5’端及3’端分別插入Pst1及Xho1限制酵素切位之三組引子對。利用此等引子對進行聚合酶連鎖反應 (polymerase chain reaction, PCR) 反應，從南投草屯採集之香蕉萎縮病樣品中確實可分別增幅出符合預期之541 bp、304 bp、198 bp之產物。經選殖於 pGEM-T Easy Vector並加以解序分析後，證實此三種產物乃分別為BBTV 鞘蛋白基因全長、N端及C端之序列。由於CyOrN表現載體之構築中CymMV先驅序列之後乃以Pst1切位與ORSV之序列連結，而ORSV序列之後則以Xho1切位連結於pET-28b(+) 載體。故上述三種不等長的BBTV CP基因增幅產物可經由此二限制酶之剪切順向構築於CyOrN載體中，而分別獲得CyBTFCP、CyBTNCP、CyBTCCP等三個不同表現載體之構築。後續經分別轉型於E.coli strain Rosetta寄主細菌並進行蛋白誘導表現，證實可分別獲得26 kDa (CyBTFCP)、17 kDa (CyBTNCP)、13 kDa (CyBTCCP) 之表現蛋白，其大小符合構築前之預期。並且發現此等利用CymMV N端59個胺基酸作為先驅序列之三種表達載體構築確實均較單獨表達BBTV CP之載體構築能夠明顯提升其蛋白表達量。另外經由Western blotting分析確認該三種重組蛋白均可與CymMV之專一性抗體產生反應，證實三者均穩定呈現CymMV鞘蛋白之抗原性。因此理論上此三種重組蛋白也應同時表現有BBTV之鞘蛋白序列及抗原性。我們將含全長BBTV序列及N端序列之二個表現載體構築培養於細菌寄主中大量誘導蛋白之合成，純化後進行兔子免疫注射，已分別獲得anti-CyBTFCP及anti-CyBTNCP二種對應之抗體。測試此兩種抗體之反應性時發現，二者均可於SDS-immunodiffusion及Western blotting tests中與同源之表現蛋白及CymMV感病組織產生明顯專一性反應，證明此二重組蛋白的確已順利誘導出對應CymMV之抗體。故理論上此等重組表現蛋白之BBTV CP序列也應可誘導出對應BBTV CP之抗體。此一推論在後續應用此二抗體於Western blotting test中能與BBTV CP之細菌表現蛋白產生專一性反應而獲得證實。後續在應用此二抗體於indirect ELISA中測試其與CymMV感病蘭花及BBTV感病香蕉樣品之反應性時發現，對應全長BBTV CP序列之重組蛋白抗體 (anti-CyBTFCP) 只與感染CymMV之蘭花樣品產生強烈之專一性反應，但不與感染BBTV之香蕉組織產生反應。然而對應BBTV CP N端序列之重組蛋白抗體 (anti-CyBTNCP) 除可與CymMV感病蘭花產生強烈反應外，也可與感染BBTV香蕉組織產生正反應。本研究證明應用細菌表現載體策略，可順利表達出同時含有CymMV與BBTV兩種病毒抗原性的重組蛋白，並獲得具雙專一性之抗體。由於此二種抗體均與BBTV之表現蛋白產生良好反應，證明本研究利用CymMV為先驅序列表達另一種不易自行大量表達之病毒序列之策略的確
可行。
 

Banana bunchy top virus (BBTV) is taxonomically belonging to the genera of Babuvirus of Nanoviridae family. It infects banana systemically and induces symptoms of stunting, shortening of internodes, small and upward leaves and witches’ broom like of apical stem. Dark greenish lines can be observed on the diseased leaves, midribs, and petioles and the leaf margins turn frequently from yellowing into necrotic that weaken significantly the vigor of the infected plants. Besides aphid transmission, the virus can also be easily transmitted by vegetatively propagated suckers. Nowadays, since industrialized production of banana depends mainly on tissue cultured plantlets as growing materials, the virus indexing of mother plants before tissue culture cloning becomes highly important. Traditionally, detection of BBTV can be approached by both serological and molecular based techniques. However, production of antibody against BBTV is always confined by the difficulty of purifying virions from infected banana tissues. Our laboratory once tested the possibility of producing recombinant coat protein (CP) of BBTV by bacterial expressing technology, but the result was not satisfactory due to low expression of its CP in pET system. In a separate study, we found that a construction of pET-28b(+) expression cassette, namely CyOrN, using a 177 bp sequence encoding 59 amino acid residues from the N-terminal region of coat protein (CP) gene of Cymbidium mosaic virus (CymMV) as upstream leader sequence followed by partial sequences of Odontoglossum ringspot virus (ORSV), could generate significant quantity of expressed recombinant viral proteins. In this study, we took the advantage of this unique expression cassette, CyOrN, by maintaining the sequence of CymMV but replacing the following ORSV sequences with the complete or partial CP gene sequences of BBTV and tested the differential accumulation of different recombinant BBTV coat proteins. The CP gene of BBTV was obtaine by amplification and cloning in pGEM-T Easy vector using primer pairs designed in this study. To facilitate later directional cloning into expressing cassette of CyOrN for replacement of ORSV sequences, we designed the primers by creating a Pst1 site at the 5’-terminus and Xho1 site at the 3’-terminus of the amplified BBTV CP genes. After amplification using the designed primers, the BBTV amplicons were then digested with Pst 1 and Xho 1 restriction enzymes and directionally cloned into CyOrN expression plasmid separately. Three constructions of pET-28b expression plasmids, namely CyBTFCP, CyBTNCP and CyBTCCP, expressing full length (541 bp), N-terminal (304 bp), and C-terminal (198 bp) of BBTV CP gene sequences were made and tested, respectively. These plasmids were then separately transformed into host bacteria, E. coli strain Rosetta, and analyzed its protein expression by induction the bacteria cultures with IPTG. In agarose gel electrophoresis, recombinant proteins with expected sizes of 26 kDa, 17 kDa and 13 kDa were located in the bacterial lysates containing expression plasmids of CyBTFCP, CyBTNCP and CyBTCCP constructs, respectively. These proteins were confirmed by Western blotting tests all reacting positively with antisera against CymMV. The expression levels of these three recombinants were all significantly higher than the control clone of pET-28b expressing only BBTV CP sequence. Two of the expression constructs (CyBTFCP and CyBTNCP) were selected for mass production of their recombinant proteins in IPTG induced bacteria cultures. After purification the recombinant proteins by preparative PAGE, they were used as immunogens to produce their respective antisera (anti-CyBTFCP and anti-CyBTNCP) in rabbits. Both antisera were shown in SDS-immunodiffusion and Western blotting tests to react strongly and specifically with their homologous recombinant proteins and to CymMV CP, indicating the recombinant proteins had indeed raised antibodies against epitopes from CymMV CP sequences. Therefore, these recombinant proteins should theoretically be able to induce antibodies against BBTV CP due to the co-expression of BBTV and CymMV CP sequences in the same protein. This was later confirmed by the revelation of reactivity of both antisera with bacteria expressed BBTV CP in Western blotting test. In the later experiments, we tested the reactivities of both antisera in indirect ELISA with BBTV-infected bananas and CymMV-infected orchids and found that the antiserum against full length BBTV CP (anti-CyBTFCP) could only strongly react with CymMV-infected orchids but not with BBTV-infected bananas. However, antiserum against N-terminal sequences of BBTV CP (anti-CyBTNCP) could give positive reactions against both CymMV-infected and BBTV-infected banana tissues, although the reactivities with BBTV were not as high as those against CymMV. Altogether, we have shown in this study that by the use of CymMV leader sequence in the same pET-28b expression vector, different size of BBTV CP can be expressed and the expression level was significantly higher than the control pET-28b plasmid expressing only BBTV CP sequence. We also showed that the expressed recombinant proteins of CymMV and BBTV can induce both antibodies against CymMV and BBTV. Furthermore, at least one of the antiserum against N-terminal sequence of BBTV CP can be used in indirect ELISA and positively detects BBTV infection in bananas.

目錄
書頁名
碩士班論文授權書
論文口試委員會審定書 (中文版)
論文口試委員會審定書 (英文版)

中文摘要 I
英文摘要 IV
致謝 VII
目錄 VIII
表目錄 X
圖目錄 XI
壹、前言 1
貳、前人研究 4
一、香蕉萎縮病歷史簡介與區域分布 4
二、香蕉萎縮病之病徵與病因研究 6
三、香蕉萎縮病之媒介 7
四、香蕉萎縮病毒基因體研究及分子學特性 10
五、偵測技術發展及其防治方法 12
六、蘭花簡介及CyOrN重組蛋白之構築 13
參、材料與方法 16
一、罹病香蕉樣品之來源與保存 16
二、香蕉萎縮病病毒(Banana Bunchy top virus，BBTV)鞘蛋白基因之選殖 16
三、BBTV三種組合表現載體構築之引子設計 22
四、CyOrN重組蛋白之細菌表現載體構築 24
五、表現蛋白之分析 27
六、表現載體於寄主細菌 (E.coli strain Rosetta) 之大量表現 28
七、表現蛋白之回收與純化 28
八、重組蛋白抗體之製備 30
九、抗血清之反應 30
肆、結果 33
一、利用CymMV N端177 bp為前驅序列引領表達BBTV全長、N端及C端鞘蛋白之載體構築 33
二、各引子對所增幅之片段選殖 33
三、三種重組蛋白表現載體之構築 34
四.表現載體之轉型 36
五.表現蛋白之誘導與分析 36
六、重組表現蛋白之抗血清製備與反應分析 38
七、二種重組蛋白抗體與BBTV 抗原反應性之確認 39
伍、討論 41
陸、參考文獻 47
表………………………………………………………………………………53
圖………………………………………………………………………………54
附錄……………………………………………………………………………77

表目錄

表一、構築三種構築不同重組表現蛋白所使用之引子對名稱、序列及增幅片段之預期長度及蛋白大小與預期分子量。 48

 
圖目錄
圖 一、表現載體CyBTFCP、CyBTFNP、CyBTCCP /pET-28b(+) 之構築流程 54
圖 二、CyBTFCP構築於表現載體pET-28b(+) 上之上下游相關位置與序列 55
圖 三、CyBTNCP構築於表現載體pET-28b(+) 上之上下游相關位置與序列 56
圖 四、CyBTCCP構築於表現載體pET-28b(+) 上之上下游相關位置與序列 57
圖 五、利用對應香蕉萎縮病毒（Banana bunch top virus，BBTV）全長鞘蛋白基因之引子對（BTFCP-up/dw）進行病毒PCR 增幅獲得541 bp產物之結果 58
圖 六、利用對應香蕉萎縮病毒（Banana bunch top virus，BBTV）全長鞘蛋白基因之引子對（BTFNP-up/dw）進行病毒PCR 增幅獲得304 bp產物之結果 59
圖 七、利用對應香蕉萎縮病毒（Banana bunch top virus，BBTV）全長鞘蛋白基因之引子對（BTCCP-up/dw）進行病毒PCR 增幅獲得198 bp產物之結果 60


圖 八、香蕉萎縮病毒(Banana bunchy top virus,BBTV) 全長鞘蛋白基因片段，選殖於篩選pGEM-T Easy Vector後轉型株之篩選 61
圖 九、香蕉萎縮病毒(Banana bunchy top virus,BBTV) 全長鞘蛋白N端基因片段，選殖於篩選pGEM-T Easy Vector後轉型株之篩選 62
圖 十、香蕉萎縮病毒(Banana bunchy top virus,BBTV) 全長鞘蛋白C端基因片段，選殖於篩選pGEM-T Easy Vector後轉型株之篩選 63
圖 十一、成功轉型表現載體 CyBTFCP/pET-28b(+) 之菌株篩選 64
圖 十二、成功轉型表現載體 CyBTNCP/pET-28b(+) 之菌株篩選 65
圖 十三、應用膠體電泳 SDS-PAGE 及西方轉漬 Western blotting 分析表現載體CyBTFCP/pET-28b(+) 經IPTG 誘導後之表現蛋白 66
圖 十四、應用膠體電泳 SDS-PAGE 及西方轉漬 Western blotting 分析表現載體CyBTNCP/pET-28b(+) 經 IPTG 誘導後之表現蛋白 67
圖 十五、應用膠體電泳 SDS-PAGE 及西方轉漬 Western blotting 分析表現載體CyBTCCP/pET-28b(+) 經IPTG誘導後之表現蛋白 68
圖 十六、兩種不同構築方式所表現之重組蛋白經純化後之抗原性確認 69
圖 十七、利用 SDS 免疫擴散法分析二種不同構築方式所表現重組蛋白抗血清之反應特性 70
圖 十八、利用 SDS 免疫擴散法分析兩種不同構築方式所表現之重組蛋白抗血清之反應特性 71
圖 十九、利用表現重組蛋白抗體 (anti- CyBTFCP) 於indirect ELISA檢測田間香蕉萎縮病中肋組織樣品之結果 72
圖 二十、利用表現重組蛋白抗體 (anti- CyBTNCP) 於indirect ELISA檢測田間香蕉萎縮病中肋組織樣品之結果 73
圖 二十一、利用西方轉漬法確認兩種不同構築方式所表現之重組蛋白抗血清與香蕉萎縮病毒 (Banana bunchy top virus, BBTV) 之細菌表現鞘蛋白之反應性 74
圖 二十二、田間感染香蕉萎縮病毒（Banana bunch top virus，BBTV）之香蕉植株外觀與葉片病徵圖 76

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