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研究生:郭庭豪
研究生(外文):Ting-Hao Guo
論文名稱:蟑螂過敏原Per a 3蛋白在轉基因煙草之表現分析
論文名稱(外文):Analysis of the Expression of Cockroach Allergic Protein Per a 3 in Transgenic Tobacco
指導教授:江主惠
指導教授(外文):Chu-Hui Chiang
口試委員:江主惠鄭櫻慧李泰林
口試委員(外文):Chu-Hui ChiangYing-Huey ChengTai-Lin Lee
口試日期:2014-07-31
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
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:2014
畢業學年度:102
語文別:中文
論文頁數:51
中文關鍵詞:蟑螂轉基因訊息胜肽過敏原食用疫苗
外文關鍵詞:cockroachtransgenesignal peptideallergenedible vaccine
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蟑螂為重要室內過敏原,會引發呼吸道過敏疾病,在台灣盛行率為第二位,僅次於塵蟎。研究發現,使用單一種過敏原進行免疫治療比使用蟑螂蛋白粗萃物進行治療的效果更佳,因此,本研究目的為分析蟑螂過敏原Per a 3蛋白在轉基因煙草中的表現情形,以評估此植物表現系統作為蟑螂過敏症的預防或治療之可行性。之前本實驗室已構築完成含Per a 3的轉基因煙草,共有7種構築方式,得到71個擬轉殖株品系,包含有不帶任何訊息胜肽的Per a 3構築,或在其5’端連接不同的訊息胜肽,分別來自α-澱粉酶 (α-amylase)、過氧化氫酶,以及熱休克蛋白(HSC70)或3’端含內質網保留胜肽 (KDEL) 之構築。為了確認Per a 3 基因是否成功的插入植株染色體,本研究利用PCR技術成功的擴增出對應Per a 3的DNA片段。當以西方墨點法偵測Per a 3蛋白在轉基因煙草表現時,可發現含有訊息胜肽α-amylase構築的植株,偵測到Per a 3蛋白表現的比例最高,約42%,而不帶任何訊息胜肽的Per a 3構築則完全偵測不到Per a 3蛋白表現。當進一步將含有α-amylase訊息胜肽的煙草植株進行馴化並種植在溫室,一個月後發現原本在瓶苗階段可偵測到的Per a 3蛋白,卻完全測不到其表現。為了觀察Per a 3蛋白的消長,本研究進一步收集馴化後5、10、15、20、25及30天後的煙草上位葉片進行蛋白質偵測,發現到第20或25天以後,Per a 3蛋白表現已完全被抑制,推測可能是植物啟動基因靜默的機制所致。為解除植物基因靜默機制,我們將具有抑制基因靜默能力的植物病毒基因NSs、HC-Pro及P19,以農桿菌注射方式送入煙草葉片,結果發現當注射含有NSs或P19基因的葉片時, Per a 3蛋白可恢復但呈現部份分解的情形。由以上結果顯示,轉基因植物確實可用來生產Per a 3蛋白,但在構築時應包含有適當的訊息胜肽,此外,馴化後之植物應於適當的時間進行採收,以進行蛋白質的回收。
Cockroach is the second leading allergen in Taiwan, only next to house dust mite. Previous studies showed that more than half of the asthmatic patients in Taiwan are allergic to cockroach. There is increasing data showing that immunotherapy with specific recombinant allergens or epitopes rather than crude mixtures, may be a more effective regimen. We have developed a transgenic plant system for producing cockroach allergen Per a 3. The objective of this study is to characterize the expressing pattern of Per a 3 protein in the transformants. Seven Per a 3 constructs were obtained with or without an additional signal sequence, including α-amylase (α- amylase), peroxide catalase, and HSC70 (heat shock proteins) at its 5’ end and an endoplasmic reticulum (ER) retention signal KDEL at its 3’ end. Putative transgenic tobacoo plants were selected in kanamycin medium and confirmed by PCR amplification. Western blot detection indicated that transgenic plants with the signal peptide α-amylase have the highest proportion of Per a 3 protein expression of about 42%, while the transgenic lines with no signal peptide were not detectable of Per a 3 protein. Per a 3 expressed transgenic plants were acclimated and grew in greenhouse. After a month or more, it was found that no Per a 3 protein was detected in these plants. In order to track the changed of protein expression, western blot analyses were used to detect Per a 3 protein from leaves collected at 5, 10, 15, 20, 25 and 30 days after acclimation. It was found that at 20 or 25 days past acclimation, the expression of Per a 3 protein was completely inhibited, presumably due to gene silencing mechanism. In order to rescue the silenced transgenic plants, three gene silencing suppressers from plant viruses, including NSs, p19, and HC-Pro were introduced into Per a 3 silenced transgenic tobacco via Agrobacterium. It was found that transgenic plants injected with NSs gene and p19 gene could recover the expression of Per a 3 though the protein shown degradation. We conclude that transgenic plant system can be used to produce Per a 3 protein which constructed together with a suitable signal peptide and the transgenic plants need to be harvest at appropriate time.
封面內頁
簽名頁
中文摘要 iii
英文摘要 v
誌謝 viiviivii
目錄 viiiviiiviiiviii
圖目錄 xi
表目錄 xii
第一章 前言
1.1 台灣主要過敏原 1
1.2 蟑螂造成的過敏 3
1.3 美國蟑螂之過敏原 4
1.4 利用轉基因植物表現Per a 3蛋白 5
1.5 以植物病毒基因產物對抗植物基因靜默 5
第二章 材料與方法
2.1 實驗材料 8
2.1.1煙草品種 8
2.1.2轉殖的基因與轉基因煙草株系 8
2.1.3組織培養藥品 9
2.1.4 植物賀爾蒙 9
2.1.5 抗生素 10
2.1.6 含基因靜默suppressor (P19、NSs與HC-Pro)
之農桿菌 10
2.1.7 Per a 3的單株及多株抗體 10
2.2 轉基因植物之核酸分析 11
2.2.1 轉基因植物的繼代培養 11
2.2.2 轉基因植物總DNA的抽取 11
2.2.3 使用聚合酶鏈鎖反應偵測植物染色體中的
轉基因 11
2.3 轉基因植物之蛋白表現分析 12
2.3.1 轉基因植物總蛋白質的抽取 12
2.3.2 植物蛋白電泳分析 13
2.3.3 植物蛋白西方墨點法偵測 14
2.3.4 馴化煙草並觀察Per a 3在不同植物生長期或
葉片位置之表現量 15
2.3.5 將抑制基因靜默的病毒基因注射到轉基因
煙草 15
第三章 結果
3.1 轉基因煙草總DNA抽取與聚合酶鏈鎖反應 17
3.2 西方墨點法偵測轉基因煙草Per a 3蛋白之表現17
3.3 不同年齡之轉基因煙草的Per a 3蛋白表現 19
3.4 利用病毒蛋白NSs、P19及HC-Pro抑制基因
靜默 19
第四章 結論 21
參考文獻 37
附錄 41
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