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研究生:陳姿蓉
研究生(外文):Tzu-Jung Chen
論文名稱:可同時檢測齒舌蘭輪斑及蕙蘭嵌紋病毒之雙特異性抗體之製備與應用
論文名稱(外文):Preparation and Application of Polyclonal Antiserum with Double Specificity that Can Detect Odontoglossum ringspot and Cymbidium mosaic virus Simultaneously
指導教授:張清安張清安引用關係
指導教授(外文):Chin-An Chang
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:91
中文關鍵詞:免疫捕捉反轉錄聚合酶連鎖反應酵素連結免疫吸附法蕙蘭嵌紋病毒齒舌蘭輪斑病毒
外文關鍵詞:Immunocapture-RT-PCR(IC-RT-PCR)Enzyme-linked immunosorbent assay( ELISA)Odontoglossum ringspot virus(ORSV)Cymbidium mosaic virus(CymMV)
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我國蘭花產業近年來輸出的品目逐漸以種苗為重,為延續企業之商機,確保蘭花種苗的品質與健康為生產管理上之關鍵目標。針對目前能夠感染蘭花的近 30 種病毒中,以齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV) 及蕙蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV) 之發生最普遍影響也最嚴重。傳統檢測此二種病毒應用最普遍之技術為酵素連結抗體免疫吸附法 (Enzyme-linked immunosorbent assay, ELISA),必須分別使用對應ORSV及CymMV之專一性抗體進行檢測,但若能研發可同時偵測 ORSV 及 CymMV 二種病毒之抗血清,將可節省一半的時間、材料及人工,提升檢測效率。過去本研究室已成功應用重組蛋白技術將ORSV及CymMV之鞘蛋白基因之部分片段構築成為四種不同組合(CyOrN, OrCyN, CyOrH, 及OrCyH)之單一重組蛋白進行多元抗體之製備,並證實可同時偵測ORSV及CymMV二種病毒。前二者乃分別利用二病毒之CP基因之N端與C端重組而成,後二者則單以二病毒CP基因之N端進行重組。本研究則分別以二病毒CP基因之C端序列進行重組蛋白之構築,其中CyOrC代表CymMV之CP基因C端在前而ORSV之CP基因之C端接續在後所構成。而OrCyC則為前後相反之構築。此二重組蛋白表現載體經 IPTG 誘導後均可產生預期大小之重組蛋白,且經由 Western blotting 確認該二種重組蛋白均可與 ORSV 及 CymMV 之專一性抗體產生明顯反應,證實二者均含有 ORSV 及 CymMV 鞘蛋白之抗原性。將此二重組蛋白大量純化並進行兔免疫注射,分別獲得 anti-CyOrC及anti-OrCyC二種對應表現蛋白之抗體。測試其反應性發現二者均可應用於 indirect ELISA、SDS-immunodiffusion 及 Western blotting 中與同源重組蛋白、ORSV 及 CymMV 病毒抗原產生專一性反應。將此二抗體連同過去已經完成之另四種重組蛋白抗體同時進行160個蘭花樣品之臨床ELISA測試,並以反轉錄聚合酶連鎖反應(RT-PCR)對檢測結果具爭議性之樣品進行再確認。結果顯示六種雙特異性重組蛋白抗體中,以具有CymMV N端及ORSV C端所構築產生之Anti-CyOrN在本研究之三次重覆試驗中表現效果最好,其中代表正確偵測出感病樣品的靈敏度(Sensitivity)達99%﹔而代表正確確定非感染樣品的專一性(Specificity)則高達100%。其他四種利用二個N端或二個C端所構築之雙特異性重組蛋白抗體之檢測結果在專一性上均有不錯之表現,在靈敏度上則與傳統單專一性抗體僅1~2 %的差異。此結果證實利用CymMV及ORSV鞘蛋白基因部分序列構築重組蛋白可製備出具有雙特異性之抗體,此等抗體在indirect ELISA、SDS-immunodiffusion 及 Western blotting等免疫偵測技術上已具有實際應用之價值。
本研究進一步探討應用此類具有雙特異性之重組蛋白抗體於免疫捕捉反轉錄聚合酶連鎖反應(Immunocapture-RT-PCR,IC-RT-PCR)上以同時偵測ORSV及CymMV之可行性。結果發現重組蛋白抗體在IC-RT-PCR之應用不如混和兩種單特異性抗體之效果,其原因可能在於此兩類型抗體與完整病毒顆粒之親和性有所差異。研究過程我們發現在IC-RT-PCR程序中,當病毒顆粒被抗體捕捉後可利用65℃乾熱處理10分鐘促使其病毒核酸釋出,此方法較部分前人研究使用增溫之0.5 % Triton X-100處理之結果更為穩定。另外發現在RT-PCR反應溶液中加入RNase inhibitor,可避免病毒核酸的被分解而提升增幅結果的穩定。其次我們也發現可將抗體預先吸附於試管並存放於室溫或4℃下至少半年,仍可穩定獲得IC-RT-PCR之檢測結果。總之,我們已發展出一個簡易穩定且可同時檢測ORSV及CymMV等兩種蘭花病毒之IC-RT-PCR流程。除了ORSV及CymMV外,此一流程也可適用於胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)、茉莉花病毒T (Jasmine virus T)及孤挺花嵌紋病毒(Hippeastrum mosaic virus, HiMV)之檢測。不過試驗過程中我們也發現所開發之IC-RT-PCR流程在面對部分低病毒濃度蘭花樣品時,其檢測效果猶不及商用RNA萃取試劑套組,推測該等蘭花樣品可能處於感染初期,組織中所累積之病毒濃度低於抗體所能捕捉之靈敏度,或者該等樣品組織中只存在病毒核酸,故未能被抗體所捕捉,因而影響RT-PCR之增幅結果。
總結,本研究所發展之六種雙特異性重組蛋白抗體確實可應用於ELISA同時檢測ORSV及CymMV,其檢測靈敏度與專一性均不亞於單特異性之傳統抗體,而具有實際應用之價值。但在IC-RT-PCR上,我們認為還是選用混合兩種單特異性抗體較為可靠。此一技術適合應用於大量樣品之初級篩檢,其靈敏度雖稍亞於利用商用RNA萃取試劑套組之效果,但每一樣品可節省萃取套組之成本約52~258元、作業時間縮短40~70分鐘及精簡人力之需求,具有明顯之經濟效益,值得產業界之參考應用。
Tissue culture plantlets have become the major exportation item for Taiwan’s Phalaenopsis industry in recent years. In order to maintain the competitiveness of this emerging business continuously, it is very important to ensure the quality and healthiness of the Phalaenopsis plantlets produced in Taiwan. At present, Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV) are recognized as the most widely spread and economically important viral agents to orchid industry among the 30 known viruses infecting orchids. Nowadays, enzyme-linked immunosorbent assay (ELISA) is the most commonly used virus detection technique in orchid industry worldwide. Traditionally, at least two antisera each specific to ORSV and CymMV are required to perform routine virus detection in ELISA. Traditionally, if there was an antiserum that can detect ORSV and CymMV simultaneously, the cost of time and materials will be reduced into at least half to conduct a regular ELISA test. In a previous study, our laboratory has successfully constructed four artificial recombinant proteins, namely CyOrN, OrCyN, CyOrH, and OrCyH, each all containing partial coat protein sequences of both ORSV and CymMV. Recombinant proteins CyOrN and OrCyN were constructed by linking N-termianl sequences of CP of either CymMV or ORSV with C-terminal sequences of CP of the other virus. On the other hand, proteins of CyOrH and OrCyH were constructed by fusing N-terminal sequences of CP of either CymMV or ORSV with the N-terminal sequences of other virus. These four recombinant proteins were shown to possess both antigenicity of coat proteins of ORSV and CymMV, and consequently the homologous, antisera of these four recombinant proteins could react with both ORSV and CymMV in ELISA, SDS-immunodiffusion and Western blotting tests. In this study, we attempted to prepare the other two possibilities of recombinant proteins (CyOrC and OrCyC) with combination of C-terminal sequences of ORSV and CymMV. Our result comfirmed that the bacterial expressed recombinant proteins of CyOrC and OrCyC also possessed both antigenicities of ORSV and CymMV, and the antisera prepared against these two new recombinant proteins could also detect both viruses in ELISA, SDS-immunodiffusion and Western blotting tests. To test the sensitivity and specificity of the antisera against six different recombinant proteins and compare with two other mono-specific antisera against ORSV and CymMV, we conducted a series indirect ELISA tests on a total of 160 orchid plants in our collection. For those non-detected samples by ELISA, we performed RT-PCR tests to confirm their infection status. The experiment was repeated three times at March 31, April 3, 2007, and January 8, 2008, respectively. Final results showed antiserum against CyOrN recombinant protein had highest sensitivity of 99% and specificity of 100 %. The other five antisera against five other recombinant protein constructions also had fairly high sensitivities (97-98%), except the lowest (94%) one of CyOrH. Furthermore, in these three experiments all six antisera consistently showed higher specificities (98-100%) than the traditionally made antisera against purified virus particles (97% in average). Putting together all these results, the antisera prepared against recombinant proteins containing partial sequences from coat protein gene of ORSV and CymMV could be practically applied in indirect ELISA for simultaneous detection of ORSV and CymMV. The results also implicated that recombinant proteins containing N-terminal sequence of CymMV down streamed with C-terminal sequence of ORSV had highest sensitivity and specificity which is comparable to those of mono-specific traditionally made antisera.
In the second part of this study, we tested the feasibility of using the six antisera specific to both ORSV and CymMV in immuno-captured RT-PCR (IC-RT-PCR) for capturing particles of both viruses. In the experiment, equal volume mixture of the two traditionally made antisera mono-specific to ORSV and CymMV were used for comparison. The result showed using the mixture of two mono-specific antisera in IC-RT-PCR, the sensitivity of 1 pg-100 fg/ml of virus detection could be reached. However, with the double specificity antisera to either of the six recombinant proteins, the sensitivity of detection was about 100 times lower than the mixture of two mono-specific antisera. This is probably due to the differences in the trueness of tertiary structure of epitopes residing in the artificially constructed recombinant proteins comparing with those in the intact virus particles. Nevertheless, an feasible and efficient IC-RT-PCR protocol for simultaneous detection of ORSV and CymMV was developed. In the developed protocol, we improved a procedure of 65 C dry heat treatment for 10 min that could release effectively the viral RNA from captured virions. We also found that by applying RNase inhibitor in the reaction mixture, consistent amplification result of RT-PCR could be improved. In addition, we found that the antibody coated eppendorf tubes could be preserved for at least 6 months without any detrimental effect on the immuno-capturing and later the RT-PCR amplification. Therefore, by the use of antibody pre-coated tubes, IC-RT-PCR could be performed immediately after sample arrival, saving the tedious RNA purification procedures. Furthermore, the developed IC-RT-PCR procedure could also be used for the detection of at least three other viruses, i.e. Cucumber mosaic virus, Jasmine virus T and Hippeastrum mosaic virus. Although the developed IC-RT-PCR procedure seemed to be feasible for simultaneous detection of ORSV and CymMV in orchid tissues, we did encounter some orchid samples that could not be detected by our IC-RT-PCR protocol. Apparently, these samples accumulated very low concentration of ORSV or CymMV virions in their tissues that beyond the sensitivity level of our IC-RT-PCR. Secondly, these samples might be at the begining stage of infection that contained only RNAs in their tissues so that they could not be detected by IC-RT-PCR. This findings suggested that at certain crucial stage such as virus indexing of orchid mother stocks before mericloning, conventional RT-PCR should be a better choice than IC-RT-PCR. Nevertheless, with the improvement accomplished in this study, the developed IC-RT-PCR is certainly a good alternative as virus indexing techniques especially suitable for large number of sampling during field survey.
書名頁
國科會授權書
碩士論文授權書
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 ------------------------------------------------- V
誌謝 ----------------------------------------------------- IX
目錄 ----------------------------------------------------- X
表目錄 --------------------------------------------------- XIII
圖目錄 --------------------------------------------------- XIV
壹、前言 ------------------------------------------------- 1
貳、前人研究 --------------------------------------------- 7
一、蘭花之簡介 --------------------------------------- 7
二、台灣蘭業之發展概況 -------------------------------- 7
三、蘭花病毒之鑑定 ----------------------------------- 8
四、蘭花病毒檢測方法之發展概況------------------------ 12
五、蘭花產業與病毒檢測 ------------------------------- 17
參、材料與方法 ------------------------------------------- 18
一、CyOrC及OrCyC重組蛋白表現載體之構築 ------------ 18
二、表現蛋白的分析 ----------------------------------- 24
三、重組蛋白抗體之製備 ------------------------------- 27
四、雙特異性多元抗體於Indirect ELISA之反應性比較 ------ 29
五、利用純化試劑套組檢測ORSV及CymMV ------------- 30
六、利用免疫捕捉反轉錄聚合酶連鎖反應
(Immunocapture-RT-PCR) 檢測ORSV及CymMV ------- 31

七、利用免疫捕捉反轉錄聚合酶連鎖反應
(Immunocapture-RT-PCR) 檢測其他植物病毒 ----------- 33
肆、結果 ------------------------------------------------- 34
一、可表現ORSV及CymMV C端序列之重組蛋白
之構築設計 --------------------------------------- 34
二、ORSV及CymMV C端序列之增幅與選殖 ------------ 34
三、C端序列重組蛋白表現載體之構築 ------------------- 35
四、表現載體之選殖與轉型 ----------------------------- 36
五、表現蛋白的分析 ----------------------------------- 37
六、C端重組蛋白之大量表現與純化 --------------------- 38
七、重組表現蛋白之抗原性確認 ------------------------- 38
八、重組表現蛋白之抗血清製備與反應性分析 ------------- 39

九、應用雙特異性抗體於免疫捕捉反轉錄聚合酶連鎖反應
(Immunocapture-RT-PCR,IC-RT-PCR)中測試同時檢測
ORSV及CymMV之效果---------------------------- 41
十、應用IC-RT-PCR檢測其他植物病毒之效果評估 -------- 45
伍、討論 ------------------------------------------------- 46
陸、參考文獻 --------------------------------------------- 54
表 ------------------------------------------------------ 60
圖 ------------------------------------------------------ 64
附錄 ----------------------------------------------------- 87

表 目 錄
表一、六種可同時表現齒舌蘭輪斑(Odontoglossum ringspot virus)
及蕙蘭嵌紋病毒(Cymbidium mosaic virus)重組鞘蛋白之表現載體之名稱與構築序列內容 -------------------------- 60
表二、本研究所設計以增幅蕙蘭嵌紋及齒舌蘭輪斑病毒鞘蛋白C端序列之引子對名稱、序列中所含限制酶切位及預期增幅之片
段長度 --------------------------------------------- 60
表三、應用六種重組鞘蛋白所製備之雙特異性抗體於Indirect ELISA 檢測蕙蘭嵌紋病毒( CymMV )及齒舌蘭輪斑病毒(ORSV)之靈敏度比較 ------------------------------------------- 61
表四、應用六種重組鞘蛋白所製備之雙特異性抗體於Indirect ELISA 檢測蕙蘭嵌紋病毒( CymMV )及齒舌蘭輪斑病毒(ORSV)之專一性比較 ------------------------------------------- 61
表五、利用商業RNA純化試劑套組進行RT-PCR與免疫捕捉反轉錄聚合酶連鎖反應(IC-RT-PCR)之成本評估 ---------------- 63
表六、利用商業RNA純化試劑套組進行RT-PCR與免疫捕捉反轉錄聚合酶連鎖反應(IC-RT-PCR)消耗時間之評估 ------------ 63

圖 目 錄
圖一、構築表現載體 CyOrC/pET-28b(+)之流程 ---------------- 64
圖二、構築表現載體 OrCyC/pET-28b(+) 之流程 --------------- 65
圖三、CyOrC構築於表現載體 pET-28b(+) 上之上下游相關位置與序列 --------------------------------------------- 66
圖四、OrCyC構築於表現載體 pET-28b(+) 上之上下游相關位置與序列 --------------------------------------------- 67
圖五、成功轉型表現載體 CyOrC/pET-28b(+)、OrCyC/pET-28b(+)
之菌株篩選 ----------------------------------------- 68
圖六、應用電泳(SDS-PAGE)及西方轉漬(western blotting)分析表現載體CyOrC/pET-28b(+)經IPTG誘導後之表現蛋白-------- 69
圖七、應用電泳(SDS-PAGE)及西方轉漬(western blotting)分析表現載體 OrCyC/pET-28b(+)經IPTG誘導後之表現蛋白 ------- 70
圖八、六種不同構築所表現之重組鞘蛋白於純化前後之分子量與免疫特性確認 --------------------------------------- 71
圖九、六種不同構築方式所表現之重組蛋白之抗原性確認 ------- 72
圖十、利用 SDS 免疫擴散法分析二種由齒舌蘭輪斑病毒及蕙蘭嵌紋病毒C 端序列所構築之重組細菌表現鞘蛋白之抗血清之免疫反應特性 ------------------------------------ 73
圖十ㄧ、利用西方轉漬法分析二種由齒舌蘭輪斑病毒及蕙蘭嵌紋病毒C端序列所構築之重組細菌表現鞘蛋白之抗血清免疫反應特性 --------------------------------------- 74
圖十二、應用混合傳統單專一性ORSV及CymMV抗體於免疫
捕捉反轉錄聚合酶連鎖反應(IC-RT-PCR)以同時檢測蘭
花病毒ORSV及CymMV之最佳化抗體稀釋倍數測試---- 75
圖十三、應用混合傳統單專一性ORSV及CymMV抗體於免疫
捕捉反轉錄聚合酶連鎖反應(IC-RT-PCR)以同時檢測蘭
花病毒ORSV及化蘭花樣品汁液稀釋倍數測試 --------- 77
圖十四、比較免疫捕捉反轉錄聚合酶連鎖反應(IC-RT-PCR)程序中以預熱65℃之Triton X-100處理或直接以65℃處理
10分鐘對檢測結果之影響 --------------------------- 79
圖十五、比較免疫捕捉反轉錄聚合酶連鎖反應(IC-RT-PCR)程序中添加RNase inhibitor對檢測ORSV及CymMV病毒
之影響-------------------------------------------- 80
圖十六、本研究所發展之免疫捕捉反轉錄聚合酶連鎖反應
(IC-RT-PCR)流程於檢測CymMV與ORSV之靈敏度 ---- 81
圖十七、六種不同構築方式之雙特異性抗體於免疫捕捉反轉錄聚合酶連鎖反應(IC-RT-PCR)之應用效果 -------------- 83
圖十八、免疫捕捉反轉錄聚合酶連鎖反應所使用之預先吸附抗體離心管保存期限測試 --------------------------- 84
圖十九、比較本研究所發展之免疫捕捉反轉錄聚合酶連鎖反應
(IC-RT-PCR)與應用試劑套組純化病毒RNA之傳統
RT-PCR對檢測ORSV及CymMV之效果 ------------- 85
圖二十、應用免疫捕捉反轉錄聚合酶連鎖反應(IC-RT-PCR)檢測三種植物病毒之結果 ------------------------------ 86
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