據前人研究，外加大腸桿菌生產之口蹄疫病毒(Food-and Mouth disease virus, FMDV)外鞘重組蛋白，會促使BHK21細胞走向凋亡。為了研發利用植物做為分子農場(molecular farming)之用途，本研究表現口蹄疫病毒P1結構蛋白於轉基因馬鈴薯，並探討其葉部粗萃蛋白對BHK21細胞的毒殺能力。利用帶有口蹄疫病毒P1基因的農桿菌感染馬鈴薯（Solanum tuberosum L., var Cardinal）塊莖，經含有hygromycin的培養基初步篩選，獲得具有抗性的植株後，藉由聚合酶連鎖反應(PCR)及南方氏墨點法(Southern blot)分析，確認P1基因已成功地插入再生植株的基因體中。而反轉錄聚合酶反應(RT-PCR)分析可偵測到P1基因的mRNA表現，但西方墨點法(western blot)分析卻無法偵測到P1蛋白表現，推測應是蛋白質表現量過低而無法測得。細胞毒殺試驗中，取植株葉片進行蛋白質粗萃取，當在BHK21細胞加入10 g/ml的馬鈴薯粗萃蛋白時，可發現轉殖株(P1)較非轉殖株(WT)具有較強的細胞毒性，而以培養基無菌栽培的植株同樣具有細胞毒殺能力。BHK21細胞經由馬鈴薯粗萃蛋白處理後，有類似細胞凋亡的特徵，利用細胞核染劑Hoechst 33342進行染色，可看見細胞有明顯的染色質濃縮現象。進一步使用caspase活性偵測套組評估細胞內caspase的活性，結果顯示細胞內caspase-8, caspase-9以及caspase-3均有被活化的現象，表示馬鈴薯粗萃蛋白可能會經由活化caspase-8/-9/-3訊息傳導路徑引起BHK21細胞走向凋亡。此外，以pEGFP-VP1表現質體轉染至BHK21細胞的實驗發現，表現VP1重組蛋白時BHK21細胞型態會由梭形改變而呈現圓形，但此一外源VP1蛋白的產生是否確實會促使BHK21細胞走向凋亡，則還需要後續更多實驗加以證實。

It was reported that aqueous soluble recombinant DNA-derived VP1 (rVP1) protein of Food-and-Mouth disease virus (FMDV) could induce apoptosis of BHK21 cells. To study of using crop plants in molecular farming application, we introduce a FMDV structural protein gene, P1, into transgenic potato plants and to analyze the cytotoxic effect of potato crude extract on the growth of BHK21 cells. After introducing the gene expression cassettes containing P1 gene into potato tuber by Agrobacterium -mediated transformation, transgenic plants were selected and regenerated in media with hygromycin. PCR and Southern blot analysis confirmed that P1 gene had been stably and heritably inserted into the genome of the transgenic potatoes. The expression of P1 transcript has been assessed by RT-PCR. However, the P1 recombinant protein was currently undetectable from the western blot of transgenic plant. When BHK21 cells treated with 10 g/ml potato crude extract, it was found that the cytotoxic effect of crude extract from P1 transgenic plant was stronger than that of non-transgenic plant. In addition, those transgenic plants grown under tissue culture condition were also found to have cytotoxicity. The appearance of chromatin condensation in cells treated with potato crude extract after Hoechest 33342 staining may suggest that the potato crude extract could induce cell death through apoptosis. To further explore the possible pathway leads to cell apoptosis, we have analyzed the caspase activity of BHK21 cells treated with potato crude extract. The results have shown that the tested caspase-8, caspase-9 and the downstream caspase-3 of treated BHK21 cell were all activated. When transfected BHK21 cells with plasmid pEGFP-VP1, the cells were observed to become shrinkage under a fluorescence microscope. The correlation between expression of foreign VP1 protein and the morphological change of BHK21cell is waited to be confirmed.

中文摘要…………………………………………………………………… I
英文摘要…………………………………………………………………… II
縮寫表……………………………………………………………………… III
目錄………………………………………………………………………… IV-V
圖目錄……………………………………………………...……..………… VI
前言………………………………………………………………………… 1-8
材料與方法........................................... 9-22
大腸桿菌的培養及保存………………………………………………… 9
大腸桿菌勝任細胞製備………………………………………………… 9
熱休克轉型大腸桿菌…………………………………………………… 9
小量質體DNA抽取(Alkali lysis method)……………………………… 10
DNA電泳分析………………………………………………………… 10
DNA之定量…………………………………………………………… 11
瓊膠中DNA片段的回收 (Gel-MTM Gel Extraction System, Viogene).. 11
PCR產物純化 (QIAquick® PCR Purification Kit, QIAGEN)………… 12
含CaMV35S啟動子之植物表現載體，p1390/35S/P1………………… 12
構築大腸桿菌表現載體pCRII-VP1…………………………..………… 12
馬鈴薯組織培養………….……………………………………………… 13
植物基因組DNA(genomic DNA)之萃取……………………………… 13
以聚合酶連鎖反應(PCR)檢測馬鈴薯轉殖株………………..………… 14
南方氏墨點法(Southern blotting hybridization ………………………… 15
植物RNA之萃取 (Trizol, Invitrogen)…………..……………………… 16
反轉錄聚合酶反應(reverse transcription-polymerase chain reaction)…17
植物可溶性蛋白的粗萃取………………………………………………. 17
蛋白質定量- Braford method………………………….………………… 18
西方墨點法(western blot)……………………………………………… 18
細胞培養………………………………………………………………….19
細胞存活率分析（Cell viability assay）…………………………………20
細胞核染色(Hoechst 33342)…………..………………………………… 20
C aspase-3活性分析(Caspase-3 activity assay)……..……………..…… 21
Caspase-8及-9活性分析(Caspase-8 and -9 activity assay)………….…. 21
BHK21細胞轉染………………………………………………………… 21
統計方法………………………………………………………….……… 22
結果………………………………………………………………………… 23-32
一、馬鈴薯擬轉殖株之馴化與栽培…………………………………… 23
二、以聚合酶連鎖反應(PCR)及南方氏墨點法再次確認馬鈴薯轉殖株 24
三、轉基因馬鈴薯RT-PCR檢測結果 ………………………………… 25
四、大腸桿菌表現載體pCRII-VP1之構築與蛋白質表現…………… 25
五、轉基因馬鈴薯蛋白質檢測結果 …………..……………………… 26
六、馴化栽培後的轉基因馬鈴薯其粗萃蛋白對BHK21細胞之影響… 26
七、馴化栽培前的轉基因馬鈴薯粗萃蛋白對BHK21細胞之影響…… 28
八、馬鈴薯粗萃蛋白造成BHK21細胞走向細胞凋亡………………… 29
九、馬鈴薯粗萃蛋白引起BHK21細胞內caspase-3或caspase-3 like的活化…29
十、馬鈴薯粗萃蛋白引起BHK21細胞內caspase-8的活化………….. 30
十一、馬鈴薯粗萃蛋白引起BHK21細胞內caspase-9的活化……….. 31
十二、pEGFP-VP1重組蛋白表現對BHK21轉型細胞型態的影響…… 31
討論………………………………………………………………………… 33-40
一、馬鈴薯之馴化與栽培……………………………………………… 33
二、轉基因馬鈴薯之分子檢測………………………………………… 34
三、轉基因馬鈴薯P1蛋白質表現……………….……………………… 34
四、轉基因馬鈴薯粗萃蛋白對BHK21細胞生長之影響……………… 36
五、馬鈴薯粗萃蛋白促使BHK21細胞凋亡之訊息傳導路徑………… 38
六、利用pEGFP-VP1轉染之BHK21細胞型態的改變……………… 39
參考文獻…………………………………………………………………… 41-45
圖…………………………………………………………………………… 46-65
附錄………………………………………………………………………… 66

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