臺灣博碩士論文加值系統

香蕉萎縮病毒(Banana bunchy top virus, 簡稱BBTV)為危害香蕉之重要病原。幾乎全球各香蕉產地均受到此病毒之威脅。BBTV為單鍊型DNA病毒，可藉由蚜蟲以永續型方式傳播。病毒感染香蕉後可造成葉片直立叢生、葉幅縮小、葉緣黃化甚至焦枯，植株生育緩慢矮化，進而影響果實產量與品質。早年香蕉之栽培以無性繁殖之吸芽苗為主要繁殖體，因此BBTV常可藉由無病徵之帶病毒吸芽苗傳播。近年蕉苗之繁殖改由組織培養為之，故必須透過病毒檢定，篩選無病毒感染之親本母株作為繁殖材料，方能確保所生產之蕉苗免於BBTV之感染。BBTV之病毒顆粒微小，且僅存於感病植株之篩管細胞中，故病毒濃度偏低不易利用傳統純化方式獲得免疫用抗原以生產優質之病毒檢定用抗體。本研究採取藉由細菌表現載體(expression plasmid)於培養過程中大量表達病毒鞘蛋白之策略，將BBTV之鞘蛋白基因(coat protein, CP) 加以選殖並構築於pET-28b(+) (Novagen, Inc., Madison, WI, USA)上，經誘導使病毒鞘蛋白大量表現，再加以純化作為免疫用抗原以生產病毒檢定用抗體。本研究先根據文獻資料設計BBTV CP專一性引子對，以聚合酵素連鎖反應(polymerase chain reaction, PCR)由霧峰地區所採集之香蕉萎縮病樣品上增幅出一個符合預期之1000 bp產物。此產物經選殖於pCRII TOPO載體 (Invitrogen Corp., Carlsbad, CA, USA)再加以解序分析後，獲得一全長1023 bp之序列。此序列經比對後證實含有BBTV之CP序列。利用此序列設計三個構築(construct FL-2、construct FL、construct CR)分別包含NcoI 、EcoRI及XhoI限制酵素切位之專一性引子對(BBTV-up1及BBTV-dw1、BBTV-up1及BBTV-dw2、BBTV-up2及BBTV-dw3)，將BBTV CP基因之轉譯架構加以增幅並構築於表現載體pET-28b(+)上，其後將其轉型於E. coli strain Rosetta(DE3) 寄主內進行蛋白表現；經IPTG誘導結果分別得到21 kDa(construct FL-2)、19 kDa (construct FL)、14 kDa (construct CR)之表現蛋白，此蛋白經西方轉漬法(western blotting)證實可與購自Agdia Inc. (Elkhart, IN, USA)之BBTV抗體反應或His-tag (Promega Inc., Madison, WI, USA)反應，確定為細菌表現之BBTV鞘蛋白。將此蛋白大量純化後利用紐西蘭白兔進行免疫注射，獲得三株抗血清 (#115、#123、#121)。經測試後發現#115抗血清可應用於Indirect ELISA成功偵測出感染BBTV之香蕉植株。試驗中將香蕉植株依病徵之嚴重程度分成嚴重病徵型與輕微病徵型二類，另以無病徵植株作為對照。進行偵測時均以葉片之中肋部位做為採樣點，經ELISA測試結果發現嚴重病徵型樣品之病毒檢出率高達87% (20/23)，輕微病徵型樣品之病毒檢出率為70% (16/23)，顯示本研究所製備之抗體除可成功應用於田間已顯現BBTV病徵植株之感病確認外，亦可成功檢測出已經感染BBTV之輕微病徵香蕉。至於抗體#123與#121雖證實可應用於western blotting test中與BBTV CP反應，但無法應用於ELISA中與田間感病植株反應。可能由於製備完成於盛夏之際，病毒濃度大量減少，以致於無適當材料進行分析，顯示出氣溫的變化對於田間病毒濃度可能有很大的影響。

Banana bunchy top virus (BBTV) is one of the most devastating banana diseases (Musa spp.) around the world. Its genome consists of single-stranded DNA and it is transmitted in a persistent manner by vector aphid (Pentalonia nigronervosa). The symptoms induced by BBTV include chlorotic margin, narrow erect and witches’ broom-like leaves. The infected plants are usually evidently stunted and their yield and quality of banana fruits are significantly affected. Unlike the conventional means of propagation by vegetative suckers, bananas are presently propagated mainly by tissue culture plantlets. Screening for virus-free banana mother plants for mass production of tissue culture plantlets is a crucial and necessary step in modern banana production system. Therefore, to development a sensitive and efficient virus detection technique is always an attractive research topic. In the past decades, serological methods are widely and routinely used for the detection of BBTV. Monoclonal antibodies are normally used as detection kits for BBTV because the preparation of polyclonal antibodies by conventional means is very difficult comparing with other viruses diseases. This is because purification of BBTV virus particles from infected banana tissues is highly difficult. In this research, we took the approach of cloning and expressing the BBTV CP gene in bacteria and using the bacteria expressed CP as immunogen for antiserum preparation. We designed a set of primer (BBTV-up/ BBTV-dw) according to BBTV’s coat protein (CP) gene sequences documented in the GenBank and successfully amplified a 1023 bp DNA product by polymerase chain reaction (PCR) from banana specimens collected from Taichung County. The PCR product was later cloned and sequenced and found that it contained the coat protein gene of BBTV. In order to clone the CP gene into the bacterial expressing vector plasmid, pET-28b(+), we designed three types of construction of BBTV CP expression cassette. Using three sets of primers (BBTV-up1/ BBTV-dw1, BBTV-up1/ BBTV-dw2 and BBTV-up2/ BBTV-dw3) designed in this study, the complete or part of BBTV CP gene were successfully cloned and directionally constructed into pET-28b(+), allowing the bacteria (E. coli strain Rosetta) to express three different types of BBTV CP related fusion proteins. The three different constructs, namely FL-2, FL and CR, can synthesize fusion proteins with molecular weights of 21 kDa, 19 kDa and 14 kDa, respectively. These three proteins were shown serologically reacted to an antiserum to BBTV purchased from Agdia Inc., USA, indicating that they were originated from BBTV CP gene. Three antisera with the code numbers of #115, #123, and #121 were subsequently produced by immunizing three rabbits with the aforementioned fusion proteins synthesized by constructs FL-2, FL and CR, respectively. In SDS-immunodiffusion tests, these three antisera were found not feasible to detect infected banana plant antigens. However, they all can be used for the detection of BBTV CP in Western blotting tests. In ELISA, only the antiserum #115 was confirmed to be useful in the detection of BBTV infected plants in the field. The other two antisera need further experiments to confirm their feasibility to be applied in ELISA tests. Using antiserum #115, higher percentage of BBTV detection was always obtained by taking midribs as sample for ELISA indexing than taking the interveinal tissues. Of 23 midrib sampling from different banana plants with severe marginal necrosis symptoms, 20 of them were positively detected in ELISA. For those three samples non-detected were subsequently confirmed as BBTV-infected ones by PCR, indicating some severely BBTV-infected plants might accumulate too low of virus concentration in the midribs to be detected by ELISA. On the contrary, when taking the midribs from banana leaves with mild or no symptoms for ELISA indexing, 100% of detection was obtained. This result indicates that the antiserum #115 against bacteria expressed BBTV CP is feasible to be applied in ELISA for the detection of BBTV in banana fields.

書名頁
國科會授權書
論文口試委員會審定書
中文摘要………………………………………………………… I
英文摘要………………………………………………………… IV
目錄……………………………………………………………… VI
誌謝……………………………………………………………… VIII
表目錄…………………………………………………………… X
圖目錄…………………………………………………………… XI
壹、 緒論……………………………………………………….. 1
貳、 前人研究………………………………………………….. 4
一、香蕉萎縮病之分佈與發現過程…………………… 4
二、香蕉萎縮病對台灣香蕉產業之影響……………… 5
三、香蕉萎縮病毒之生物、分子學特性及寄主範圍… 6
四、香蕉萎縮病毒之偵測技術發展…………………. 9
參、材料與方法………………………………………………. 11
一、罹病香蕉樣品之來源、採集與保存……………. 11
二、香蕉萎縮病毒鞘蛋白基因之選殖………………. 11
三、BBTV鞘蛋白基因之細菌表現載體之構築……… 15
四、已構築之表現載體之轉型………………………… 16
五、表現蛋白的分析..………………………………….. 17
六、表現載體於寄主細菌中大量表現之誘導………… 18
七、表現蛋白之回收與純化…………………………… 18
八、抗血清之製備……………………………………… 19
九、血清反應……………………………………………. 20
肆、結 果……………………………………………………… 23
一、BBTV鞘蛋白基因之選殖與解序…………………. 23
二、表現載體構築設計………………………………… 23
三、三種表現載體構築之蛋白表現分析……………… 24
四、BBTV 鞘蛋白基因各構築選殖株之蛋白大量表現
與純化..……………………………………………. 25
五、BBTV抗血清之製備與應用……………………… 25
伍、討 論…………………………………………………….. 29
陸、參考文獻………………………………………………….. 35
表……………………………………………………………….. 39
圖……………………………………………………………….. 42
附錄……………………………………………………………… 66

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