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研究生:蘇瑞媛
研究生(外文):Ruei-Yuan Su
論文名稱:轉殖萵苣表現豬瘟病毒表面結構蛋白之研究
論文名稱(外文):The Study On Transgenic Lettuce (Lactuca Sativa L.) Expressing The Surface Structural Protein Of Classical Swine Fever Virus
指導教授:尤進欽
指導教授(外文):Jinn-Chin Yiu
口試委員:郭純德方怡丹劉程煒尤進欽
口試委員(外文):Chun-Teh KuoYi-Tan FangCheng-Wei LiuJinn-Chin Yiu
口試日期:2012-06-29
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:園藝學系碩士班
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:87
中文關鍵詞:基因轉殖萵苣
外文關鍵詞:Transgenic Lettuce
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  • 被引用被引用:3
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豬瘟(Swine fever, Classical swine fever),又稱豬霍亂(Hog cholera, HC)為豬瘟病毒所引起,是一種豬高度傳染性的病毒性疾病,以嚴重的全身性出血為主徵,死亡率極高。目前最廣泛使用的猪瘟疫苗為減毒疫苗,其可能經個體繼代感染後有毒力迴歸的危險性。以植物做為生物反應器的基因工程疫苗能克服減毒疫苗的缺點,因此本試驗將豬瘟病毒上具有抗原決定位的E2醣蛋白,利用轉殖萵苣發展次單位疫苗以抵抗豬瘟疾病。
本研究將豬瘟病毒表面結構蛋白E2基因構築於pBI121轉殖載體,再藉由農桿菌轉殖法將E2基因轉殖至萵苣中。實驗使用子葉做為培植體誘導芽鞘再生,並以kanamycin進行篩選,`福山´、`翠花´及`綠蘿蔓´萵苣植株的再生率分別為6.7%、2.5%及5.6%。藉由PCR分析鑑定擬轉殖株葉片的基因組DNA,證實E2基因插入萵苣基因組中。利用RT-PCR分析顯示,E2基因能正常的轉錄mRNA。西方墨點分析證明,轉殖株能正確轉譯合成目標蛋白。透過分子分析,T0代萵苣植株轉殖成功率分別為0.38%、0.16%及2.27%。T1代植株皆可檢驗到E2基因的表現及目標蛋白的累積,顯示目標基因能穩定遺傳至後代。子代植株進行ELISA分析,`福山´萵苣E2蛋白的表現量約占總可溶性蛋白含量的0.0031至0.0093%、`翠花´萵苣為0.0012至0.0031%及`綠蘿蔓´萵苣為0.0020至0.0047%。本試驗結果顯示,利用萵苣做為生物反應器表現E2蛋白是可行的,未來將進一步地進行動物試驗以證實其保護效力。

Swine fever, also called “classical swine fever” (CSF) or “hog cholera” (HC) is caused by classical swine fever virus (CSFV). It is a highly infectious viral disease in pigs that is typified by severe systemic hemorrhage and high mortality. Currently, the most widely used vaccines for controlling CSF are attenuated vaccines, which have the risk of virulence regression through individual subculture infections. Vaccines genetically engineered using plant bioreactors can overcome the disadvantages of attenuated vaccines. Therefore, we transferred the E2 glycoprotein containing epitopes on CSFV into lettuce to develop subunit vaccines for controlling CSF.
We constructed the E2 gene, which is a surface structural protein of CSFV, in the transformation vector pBI121, and used the agrobacterium-mediated transformation method to transfer the E2 gene into lettuce. We used cotyledon explants to induce coleoptile regeneration, and then employed kanamycin for selection. The regeneration rates for “Fukuyama,” “Cui-Hua,” and “Romaine” lettuce plants were 6.7%, 2.5%, and 5.6%. We used polymerase chain reactions (PCR) to analyze the DNA of the intended transformed leaf genome and verified that the E2 gene could be inserted into the lettuce genome. The results of the reverse transcription (RT) PCR analysis showed that the E2 gene can perform normal transcriptions of mRNA. Western blot analysis verified that the transformed plants can accurately translate and synthesize the target protein. The molecular analysis results showed that the success rate of the T0 generation-transformed lettuce plants was 0.38%, 0.16%, and 2.27%. The T1 generation plants all showed E2 gene expression and accumulation of the target proteins, indicating that the target genes were steadily inherited in later generations. Enzyme-linked immunosorbent assay (ELISA) analysis of the progeny plants showed that the E2 protein expression in the Fukuyama lettuce accounted for 0.0031% to 0.0093% of the total amount of soluble protein, whereas that in the Cui-Hua lettuce accounted for 0.0012% to 0.0031%, and that in the Romaine lettuce accounted for 0.0020% to 0.0047%. These results demonstrated that using lettuce as a bioreactor to express E2 protein is feasible. We plan to conduct animal experiments in the future to verify its protective efficacy.

圖目錄 III
表目錄 V
中文摘要 1
英文摘要 2
前言 3
前人研究 4
一、豬瘟病毒介紹 4
二、豬瘟病毒的複製過程 5
三、致病及傳染機制 5
四、感染症狀 6
五、臨床症狀 6
六、病理變化 7
七、豬瘟預防方式 7
八、接種 7
九、植物生產人類及動物疾病抗原蛋白 9
十、轉殖植物生產E2疫苗之研究 19
材料與方法 25
一、試驗材料 25
二、試驗方法 25
1.植物之轉型與再生 25
(1)農桿菌中基因之抽取 25
(2)農桿菌中基因之確認 25
2.培植體培養、農桿菌基因感染與植株再生 26
(1)培植體培養 26
(2)農桿菌培養 26
(3)共同培養 26
(4)轉殖基因植株再生 27
(5)馴化出瓶 27
3.再生植株之分子層次分析 28
(1)植物基因體DNA之萃取 28
(2)聚合酶鏈鎖反應(Polymerase chain reaction;PCR) 28
(3)植物總RNA之萃取 28
(4)反轉錄聚合酶鏈鎖反應(Reverse transcriptase polymerase chain reaction;RT-PCR) 29
(5)葉片總可溶性蛋白之萃取 29
(6)植物葉片總可溶性蛋白含量之測定 29
(7)西方墨點 30
(8)轉殖株子代之種植與鑑定 30
(9)轉殖株子代之種子對kanamycin的抗藥性測試 31
(10)轉殖株子代之種子E2蛋白之定量分析 31
結果 32
一、農桿菌質體之確認 32
二、基因轉殖植株之再生情形 32
(1)再生流程 32
(2)萵苣再生情形綜合分析 32
三、轉殖再生萵苣之分子分析 33
(1) PCR分析 33
(2) RT-PCR分析 33
(3)再生植株的採種情形 33
四、轉殖株之子代分析 34
(1)`福山´品種之萵苣 34
(2)`翠花´品種之萵苣 34
(3)`綠蘿蔓´品種之萵苣 35
(4)蛋白質含量測定 36
討論 69
1.植株之再生及採種情形 69
2.基因轉殖萵苣的分子分析 71
3.基因轉殖萵苣的植株子代種植與鑑定 72
參考文獻 75
附件 85
附件一、E2基因核酸序列 85
附件二、PBI121-D之質體圖 86
附件三、NPTII基因核酸序列 87

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