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研究生：	呂詩偉 Lu, Shih-Wei
論文名稱：	發展高表現量的家禽里奧病毒之植物性疫苗 Develop plant-based vaccine for poultry to against avian reovirus
指導教授：	張清俊 Chang, Ching-Chun
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology
論文出版年：	2008
畢業學年度：	96
語文別：	中文
論文頁數：	111
中文關鍵詞：	植物疫苗、菸草基因轉殖、σC蛋白質、家禽里奧病毒
外文關鍵詞：	transgenic tobacco, plant-based vaccine, avian reovirus (ARV), sigma C protein
相關次數：	點閱：86 下載：1
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植物基因轉殖技術近年來已廣泛被應用在各種基礎研究領域與開發實用性生物技術產品，其中利用農桿菌法遞送外源基因至植物細胞核更是發展多年且成熟的技術。隨著植物生物技術的發展，以植物生產動物疫苗能降低成本、較為安全、可發展為口服疫苗、可表現於貯存組織，運送方便等優點，許多以植物生產疫苗的研究，在動物試驗中證實是可引發動物的免疫反應，並可以對抗病原菌的感染。經由前人的研究，家禽里奧病毒之外鞘膜蛋白(outer capsid protein) σC可誘發中和抗體，因此，我們利用轉殖菸草的表現系統製做家禽里奧病毒(avian reovirus；ARV)之植物性疫苗。在本實驗室學長曾將S1基因轉殖到苜蓿中，但蛋白質(σC)表現量有過低的問題。因此，此實驗之目的，在不改變胺基酸序列為前提下修改病毒S1基因上的DNA序列，去掉potential intron、mRNA instability element、並且將部分codon usage更替成菸草使用頻率較高者，來增加蛋白質的表現量。藉由這種方式希望可以提升病毒基因在植物表現量過低的問題，以此做為雞隻的植物性疫苗，希望能更有效率誘發雞隻產生中和抗體，以達抵抗家禽里奧病毒感染之目的。

Many feeding experiments have demonstrated that transgenic plants with overexpression the surface antigen of pathogen can confer animals to induce immunity to against pathogen attack. Previously, in our lab, we have expressed the σC protein of avian reovirus (ARV) in alfalfa. However, the level of σC protein expression is low in transgenic alfalfa cells, which might limit its practical application. To increase σC protein expression, we modified the S1 gene by eliminating potential introns, mRNA instability elements and optimizing the codon to more frequently use in plants. We constructed four plant nuclear expression vectors (p35S1, pActS1, p35UmS1 and p35TmS1) which carry S1 or modified S1 gene (mS1) with different promoters and target to different subcellular compartments. Transformations of σC expression constructs into tobacco nucleus were carried out by Agrobacteria-mediated method. The transformants were selected with media containing kanamycin (100 μg/ml). PCR analysis confirmed that the integration of S1 or mS1 gene into the tobacco chromosome. Western blot analysis was carried out to determine the expression level of σC protein in transgenic tobacco. The highest expression levels of σC protein in selected p35S1, pActS1, p35UmS1 and p35TmS1 transgenic lines were 0.0125%, 0.0210%, 0.0013% and 0.0002% of the total soluble proteins, respectively. Animal experiments to test the immunity of mice by injecting or feeding the plant–expressed σC proteins will be performed.

目錄
第一章、前言..............................................1
第二章、文獻探討..........................................2
     1. 疫苗的重要性及其優缺點............................2
     2. 以植物生產動物疫苗的優點..........................4
     3. 如何利用植物來生產動物疫苗........................5
        3.1 細胞核基因轉殖................................5
        3.2 葉綠體基因轉殖................................6
        3.3 病毒表現系統..................................7
     4. 利用植物來做為動物疫苗的發展現況..................9
        4.1 以植物開發人體疫苗............................9
        4.2 以植物開發畜產體疫苗.........................12
     5. 以植物生產疫苗有待解的問題.......................15
        5.1 修改codon usage 以增加外源蛋白的表現量.......15
     6. 研究目標-家禽里奧病毒............................16
第三章、材料與方法 ......................................18
     1. 實驗材料.........................................18
     2. 基因合成.........................................18
        2.1 設計用於合成S1基因的引子.....................18
        2.2 合成mS1基因..................................19
        2.3 合成transit peptide..........................20
     3. 植物基因轉殖載體的構築...........................20
        3.1 p35UmS1和p35TmS1表現載體的構築...............20
        3.2 構築表現載體所使用的方法.....................21
     4. 菸草基因轉殖.....................................29
     5. 基因轉殖植株之篩選...............................33
     6. 獲得同質純系之轉殖植株( homozygous lines)........35
     7. 偵測蛋白質表現量.................................36
第四章、結果.............................................41
     1. 合成mS1基因......................................41
     2. 獲得持續性表現家禽里奧病毒mS1基因之菸草轉殖品系..41
     3. 轉殖植株之孟德爾分離率分析.......................42
     4. 西方墨點法分析轉殖植株之結果.....................43
第五章、討論.............................................44
     1. 修改S1基因所遭遇到的問題與解決方式...............44
     2. 菸草農桿菌轉殖效率...............................44
     3. 轉殖植株不符合孟德爾遺傳定律.....................45
     4. 基因修改之轉殖植株未如預期增加外源蛋白質表現量...46
第六章、參考文獻.........................................47
附錄一、水稻葉綠體基因轉殖系統之建立.....................75
附錄二、以細胞核轉殖在菸草中表現轉化酵素cellulase........99
附錄三、藥品與溶液配方..................................107

圖目錄
圖一、以PCR分別將mS1的13個片段合成出來...................54
圖二、以primer extension方式將mS1的小片段串接合成出來....54
圖三、原始S1基因與修改過的mS1基因之序列比對..............55
圖四、農桿菌雙偶型載體p35S1、pActS1、p35UmS1與p35TmS1....56
圖五、以PCR檢測p35S1、pActS1菸草轉殖植株的基因體中
      是否含有kanamycin resistance基因...................57
圖六、以PCR檢測p35UmS1菸草轉殖植株的基因體中
      是否含有kanamycin resistance基因...................58
圖七、以PCR檢測p35UmS1菸草轉殖植株的基因體中
      是否含有kanamycin resistance基因...................59
圖八、以PCR檢測p35TmS1菸草轉殖植株的基因體中
      是否含有kanamycin resistance基因...................60
圖九、以PCR檢測p35TmS1菸草轉殖植株的基因體中
      是否含有kanamycin resistance基因...................61
圖十、以西方墨點法分析p35S1、pActS1 菸草轉殖品系的
      σC表現量...........................................62
圖十一、以西方墨點法分析p35UmS1、p35TmS1 菸草轉殖
      品系的σC表現量.....................................63

表目錄
表一、比較利用各種表現系統來生產疫苗的優缺點.............64
表二、以植物生產疫苗的優點...............................64
表三、各種植物表現系統的比較.............................65
表四、以植物做為動物疫苗的發展狀況.......................66
表五、本實驗所使用之引子.................................70
表六、mS1基因所更改之codon...............................71
表七、 p35S1菸草轉殖植株之孟德爾分離率分析...............72
表八、 pActS1菸草轉殖植株之孟德爾分離率分析..............72
表九、 p35UmS1菸草轉殖植株之孟德爾分離率分析.............73
表十、 p35TmS1菸草轉殖植株之孟德爾分離率分析.............74
                                    

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校內：2010-08-22公開
校外：2010-08-22公開

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