臺灣博碩士論文加值系統

菜豆為台灣重要之豆類蔬菜，適應性廣，從南至北各縣市均有栽培，歷年栽培面積均可達2,000公頃，年產量約12噸以上。豆類之栽培可能受到多種病毒之危害，造成品質與產量降低，且豆類病毒均有種子帶毒之現象，導致防治上之困難。過去本省菜豆只有遭受胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)感染之記錄，2008年本實驗室於南投縣草屯鎮發現明顯嵌紋、葉部畸形、植株矮化等病徵之蔓性菜豆植株，經分子檢測證實為菜豆微斑駁病毒(Cowpea mild mottle virus, CpMMV)之分離株，分類上乃Carlavirus屬之病毒。文獻記載首次發現CpMMV是於東印度與迦納地區之豇豆，可造成葉片產生嵌紋、褪綠斑點以及葉部畸形等病癥，接著此病毒蔓延至非洲、中東、亞洲、南美洲、大洋洲地區也都發生之案例。CpMMV有別於一般Carlavirus屬病毒之蚜蟲媒介特性，而由粉蝨以非永續型方式傳播，因此對其蔓延非常快速。與其他Carlavirs屬病毒一樣，CpMMV之基因體包含一段稱為三重疊基因區(triple gene block, TGB)，所轉譯的三個蛋白(TGBp1、TGBp2與TGBp3)，其功能已在其他具類似構造之Potexvirus屬病毒中被證實其功能可能與感染初期病毒核酸之移動有關。由於CpMMV之傳播媒介與一般Carlavirus不同，為探討其功能與建立病毒之檢測方法，故本研究嘗試將此三種非結構性蛋白以細菌表達外源蛋白之技術加以純化，再製作此等蛋白質相對應之多元抗體，作為進行功能探討與病毒檢測之工具。本實驗根據NCBI databank所登錄之CpMMV全長基因體序列(HQ184471)設計對應TGB區域之專一性引子 (TGB-up1/TGB-dw1)，成功增幅並獲得預期之1200 bp片段，經解序分析後確認此序列涵蓋3個ORF區域。ORF2含696 bp、ORF3含321 bp、ORF4含207 bp。後續分別針對此三個TGB基因設計上下游各含酵素切位之三組專一性引子(ORF2-up、ORF2-dw；ORF3-up、ORF3-dw；及ORF4-up、ORF4 –dw)，分別成功增幅出符合預期大小之片段，再分別將其定向構築於pET-28b(+)外源基因表達載體上以進行蛋白質表現誘導，然後分別以製備型電泳(Preparative electrophoresis)與金屬離子親和性層析法 (Immobilized Metal Affinity Chromatography, IMAC)進行蛋白質回收純化並進行免疫注射，分別獲得TGB1-3等三種功能蛋白之抗體。本研究因ORF3與ORF4蛋白質分子量較小，以本實驗室慣用之製備型電泳(Preparative electrophoresis)無法有效率的回收，故更改以金屬離子親和性層析法 (Immobilized Metal Affinity Chromatography, IMAC)，已成功將CpMMV TGB之ORF3與ORF4之表現蛋白予以純化回收，並製備出所對應之多元抗體。利用所得之抗體針對接種CpMMV之菜豆葉片，於植株感染後之三、六、九天以ELISA與Western blot分別測試，結果發現ORF2蛋白由感染後三至九天於ELISA試驗下其讀值皆能達到健康對照葉片之兩倍值，同時在Western blot試驗下也能看見符合預期分子量大小約26 kDa之條帶，但至第十二天之後，不管ELISA或Western blot均無法測得蛋白累積之情況。ORF3蛋白之偵測方面，由接種後三至九天於ELISA試驗下其讀值皆無達到健康對照葉片之兩倍值，但接種第三天以Western blot測試，則能看見符合預期分子量大小約12 kDa之微量條帶累積，但第三天之後便無偵測到任何累積之情形。另外檢測ORF4蛋白之累積方面，只有於感染後第三天以ELISA與Western blot能夠測得健康葉片兩倍以上之讀值與符合預期分子量大小約10 kDa條帶，第六後則無法測得累積情形。此結果顯示，CpMMV之三種 TGB非結構性蛋白只於感染初期三至九天內可以在感染葉片組織內偵測到微量之累積情形，經過第九天後無法測出。此現象與相關文獻所提出之結果相符，其功能可能也與感染初期病毒核酸之移動有關。本研究已初步利用ORF2抗體針對感病葉片以穿透式電子顯微鏡(Transmission Electron Microscopy, TEM)進行免疫金顆粒標定試驗，雖然尚未取得足夠之證據證明其蛋白之功能，但推測此策略對探討TGB蛋白在感病組織內之分佈應屬可行之嘗試。

Common bean (Phaseolus vulgaris) is one of the important legume crops in Taiwan. Its wide adaptability makes it suitable for growing form south to north in this island. Its annual cultivated area and production are over 2,000 hectares and about 12 tons, respectively. Common bean is acrop prone to be infected by various viruses which have severely interfered its production and quality worldwide. However, the Cucumber mosaic virus(CMV) is the only virus being reported on common bean so far in Taiwan.
Common bean plants with symptoms of mosaic, leaf shrinkage and dwarf had been collected form nantou County in 2008. Cowpea mild mottle virus(CpMMV), a Carlavirus, has been identified as the causal agent. CpMMV was first found on cowpea in east India and Ghana, and spreaded to Africa, the Middle East and Asia later on. It causes symptoms of mosaic, chlorotic spots, and leaf distortion on cowpea, and can be transmitted by whitefly (Bermisia tabaci) in a non-persistent manner. As a Carlavirus, the genome of CpMMV contains an overlapping gene region called the triple gene block, (TGB), which encodes three functional proteins mediated the virus movement in the early stage of infection in planta.
The objectives of this thesis are to produce antisera against individual functional protein of CpMMV TGB and to establish possible detection methods for detecting CpMMV by using antisera produced in serological tests such as enzyme-linked immune-sorbent assay (EELISA) and western blotting test.
For the cloning of full-length TGB, corresponding primers (TGB-up1/TGB-dw1) were designed based on the full-length sequence of CpMMV (HQ184471) available on NCBI GenBank database. A cDNA fragment of about 1200 bp was obtained after reverse-transcription polymerase chain reaction (RT-PCR) and was cloned into pGEM-T easy vector. Sequence analysis has confirmed the cloned cDNA harbored all three ORFs, i.e. ORFs 2, 3, and 4, of TGB. The ORF2 consists of 696 bp whereas ORFs 3 and 4 contain 321 bp and 207 bp respectively. Individual ORFs of TGB were further cloned into bacterial expression vector pET-28 (b+) separately after PCR amplification with primer sets of (ORF2-up/ORF2-dw; , ORF3-up /ORF3-dw; , and ORF4-up /ORF4-dw), accordingly. Each expression constructs were then transformed individually into E. scherichia coli (strain BL21) for the over-expression of each recombinant proteins. The over-expressed proteins of 26 kDa, 12 kDa and 8 kDa, corresponding to the products of ORFs 2, 3 and4, respectively. , can be visualized on SDS-PAGE. Bacterial expressed ORF2 protein was purified by using preparative gel electrophoresis. As the protein products of ORF3 and ORF4 were too small to be purified as ORF2 protein, the metal ion affinity chromatography (or immobilized metal affinity chromatography, IMAC) was used as an alternative. The purified proteins were then used as antigen for the production of specific polyclonal antibodies in rabbit. The obtain antibodies were used to detect the CpMMV infection. CpMMV-infected leaves of common bean were collected three, six and nine days post inoculation (dpi). The presence of the protein products of CpMMV ORFs 2, 3, and 4 in inoculated and systemic leaves were tested by ELISA and Western blot analyses.
The ORF2 protein of 26 kDa can be detected in the inoculated and systemic cowpea leaves of 3-9 dpi was not detectable in infected leaves of 12 dpi, in ELISA and Western blot tests. However, the protein of CpMMV ORF3 only can be detected in the leaves of 3 dpi in immunoblots while the ORF4 protein was detectable in the leaves of 3 dpi with both ELISA tests and iimmunoblots.
These result indicated that the CpMMV TGB proteins accumulated only in the early stage of infection, which is in accord with those data showed in earlier documentations.
In brief, the results of this thesis showed, the prepared antisera ahainst CpMMV TGB are suitable for applying to detect target proteins in vitro and in vivo.

書名頁
碩士論文授權書
論文口試委員審定書(中文版)
論文口試委員審定書(英文版)
中文摘要……………………………………………………………..……….. I
英文摘要……………………………………………………………..…….... III
誌謝………………………………………………………….………..……... VI
表目錄………………………………………………………….………..….. VII
圖目錄………………………………………………………….………..……IX
目錄……………………………………………………….………..……….. XII
壹、 緒論…………………………………………………………….………. 1
貳、 前人研究……………………………………………………….………. 5
參、 材料與方法……………………………………………….……………. 9
(一) 感病菜豆樣品來源、採集與保存…………………….…….….. 9
(二) CpMMV三重複基因區塊TGB基因之選殖………….…….… 10
(三) TGBs蛋白基因之細菌表現載體構築……………….…….….. 14
(四) 表現蛋白之分析…………………………………….…………. 15
(五) 血清學試驗(serological test) ……………………….……........ 19
肆、 結果…………………………………………………………….……… 23
一、 CpMMV三重疊基因區(Triple gene block, TGB)基因選殖…... 23
二、 酵素結合免疫吸附法(Enzyme-linked immunosorbent assay, ELISA) …………………………………………………………... 23
三、 ORFs之蛋白表現與系統篩選…………………………………... 24
四、 蛋白質大量表現與純化…………………………………………. 25
五、 血清學試驗(serological test)……………………………………. 26
伍、 討論……………………………………………………………………. 29
陸、 參考文獻…………………………………………………………….... 33
表、…………………………………………………………………………… 39
圖、……………………………………………………………………………. 41

表目錄

表一、本研究所設計與使用的寡核苷酸之引子…………………………… 39
表二、本研究解序獲得之Cowpea mild mottle virus (CPMMV)三基因體
(TGB)區域之序列與其重疊之轉譯架構…………………………. 40

圖目錄

圖一、Carlavius屬基因體結構包含6個開放閱讀區………..…………… 41
圖二、對應豇豆微斑駁病毒(Cowpea mild mottle virus, CPMMV)TGB
之專一性引子對進行基因增幅分析結果…………………….......... 42
圖三、豇豆微斑駁病毒(Cowpea mild mottle virus，CPMMV) TGBs蛋
白基因於pET-28b(+)載體構築示意圖………………………......... 43
圖四、豇豆微斑駁病毒(Cowpea mild mottle virus, CPMMV)含酵素切
位引子對增幅ORF2基因分析結果…………………………........... 44
圖五、豇豆微斑駁病毒(Cowpea mild mottle virus, CPMMV)含酵素切
位引子對增幅ORF3基因分析結果…………………………........... 45

圖六、豇豆微斑駁病毒(Cowpea mild mottle virus, CPMMV)含酵素切
位引子對增幅ORF4基因分析結果………………………............... 46
圖七、外源基因表達載體pET-28b(+)之ORF2基因之酵素剪切結果…. 47
圖八、外源基因表達載體pET-28b(+)之ORF3基因之酵素剪切結果…. 48
圖九、外源基因表達載體pET-28b(+)之ORF4基因之酵素剪切結果…. 49
圖十、比較兩種細菌表達系統對構築於pET-28b(+)之ORF2進行蛋白
表達之分析………………………………………………………...... 50
圖十一、比較兩種細菌表達系統對構築於pET-28b(+)之ORF3進行蛋
白表達之分析……………………………………………………. 51
圖十二、比較兩種細菌表達系統對構築於pET-28b(+)之ORF4進行蛋
白表達之分析…………………………………………………….. 52
圖十三、ORFs表現蛋白回收產物結果分析……………………………… 53
圖十四、SDS-免疫擴散反應(SDS-immuno-diffussion test)分析ORFs蛋
白抗體之反應性分析…………………………………………...... 54
圖十五、ORF2蛋白抗體針對接種CpMMV三天之葉片組織ELISA分
析結果…………………………………………..…………………. 55
圖十六、ORF2蛋白抗體針對接種CpMMV六天之葉片組織ELISA分
析結果…………………………………………..…………………. 56
圖十七、ORF2蛋白抗體針對接種CpMMV九天之葉片組織ELISA分
析結果…………………………………………..…………………. 57
圖十八、ORF2蛋白抗體針對接種CpMMV十二天之葉片組織ELISA
分析結果……………………………………..…………………… 58
圖十九、ORF3蛋白抗體針對接種CpMMV三天之葉片組織ELISA分
析結果……………………………………..……………………… 59
圖二十、ORF3蛋白抗體針對接種CpMMV六天之葉片組織ELISA分
析結果………………………………………..…………………… 60
圖二十一、ORF3蛋白抗體針對接種CpMMV九天之葉片組織ELISA
分析結果……………………………………………………..… 61
圖二十二、ORF4蛋白抗體針對接種CpMMV三天之葉片組織ELISA
分析結果……………………………………………………..… 62
圖二十三、ORF4蛋白抗體針對接種CpMMV六天之葉片組織ELISA
分析結果……………………………………………………..… 63
圖二十四、ORF4蛋白抗體針對接種CpMMV九天之葉片組織ELISA
分析結果……………………………………………………..… 64
圖二十五、ORF2蛋白抗體針對接種CpMMV後第三天至第十二天之
葉片組織西方墨點法分析結果……………………..………… 65
圖二十六、ORF3蛋白抗體針對接種CpMMV後第三天至第九天之葉
片組織西方墨點法分析結果……………………..…………… 66
圖二十七、ORF4蛋白抗體針對接種CpMMV後第三天至第九天之葉
片組織西方墨點法分析結果……………………..…………… 67

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