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研究生:王曉俐
研究生(外文):Hsiao-Li Wang
論文名稱:以轉基因菸草及馬鈴薯表現塵蟎過敏原DerpII、DerpV蛋白
論文名稱(外文):Development of transgenic tobacco and potato plants for expression of mite allergens Der p II and Der pV
指導教授:古新梅詹富智
指導教授(外文):Hsin-Mei KuFuh-Jyh Jan
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農藝學系
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:74
中文關鍵詞:內質網信號胜肽序列口服耐受性
外文關鍵詞:ER retention signal peptideoral tolerance
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於世界各地廣為盛行的過敏性疾病正困擾著數以萬計的兒童,而塵蟎為引起過敏性鼻炎、氣喘等最常見的主因,近年來新興的口服塵蟎過敏原療法可誘發口服耐受性而預防塵蟎過敏症發生,但口服過敏原疫苗純化困難且價格昂貴,所以未能普及,於是本論文於轉基因菸草及馬鈴薯中表現塵蟎過敏原蛋白Der p II及Der p V,希望建立高產低成本的植物口服疫苗模式系統。所構築的植物轉殖載體,是利用花椰菜嵌紋病毒35 S啟動子驅動昆蟲來源的塵蟎過敏原基因Der p II及Der p V使其能於植物中表現,並於塵蟎過敏原下游外加內質網信號胜肽序列 (ER retention signal peptide: SEKDEL),使外源蛋白停留在內質網而提高累積量。構築好的載體藉由農桿菌轉殖法產生轉基因菸草做為植物表現過敏原蛋白的模式系統,經聚合酶連鎖反應 (polymerase chain reaction, PCR)、反轉錄-聚合酶連鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR)及西方轉漬法 (western blotting) 之分析結果證明轉基因菸草確實可正確的表現塵蟎Der p II、Der p V基因與蛋白。為使植物表現的過敏原蛋白可直接口服食用,我們繼而進行馬鈴薯基因轉殖,除以PCR、RT-PCR證實Der p II、Der p V基因表現外,亦以南方墨點法 (Southern blotting) 進一步分析Der p II轉基因馬鈴薯染色體中外源基因嵌入數 (copy number),西方轉漬分析結果亦顯示轉基因馬鈴薯可正確的表現Der p II、Der p V蛋白。此外,外加SEKDEL信號胜肽序列之構築,所產生的轉基因菸草及馬鈴薯皆得到較高的Der p II、Der p V蛋白累積量。根據本論文研究結果,植物確實可表現塵蟎過敏原蛋白Der p II及Der p V,且蛋白於轉基因菸草及馬鈴薯中的表現量,皆可達總可溶性蛋白之0.01%,為發展抗塵蟎過敏症食用疫苗的研究,提供一植物表現的模式系統。
Allergic diseases have been increasing worldwide and causing problems for millions of children. Dust mite allergens are the most common cause of perennial allergic rhinitis and asthma . Edible allergen vaccine is a new form of treatment to reduce allergic response to allergens. The purified allergen vaccine is a very expensive treatment for allergic diseases. The focus of our research was to develop transgenic plants for expressing mite allergen as an edible and low cost vaccine for oral tolerance to allergy. We have generated plant expression and transformation vectors containing mite allergen genes Der p II and Der p V. The mite allergen genes were placed under control of 35S promoter of Cauliflower mosaic virus and nopaline synthase terminator of Agrobacterium tumefaciens. At the 3´-end of Der p II and Der p V, a nucleotide sequence coding for the hexapeptide Ser-Gln-Lys-Asn-Gln-Leu (SEKDEL) was added for retention of the protein in the endoplasmic reticulum (ER). The Der p II and Der p V genes were introduced into tobacco through the antigen gene-harboring A. tumefaciens-mediated plant transformation method as a model system. Expression of the introduced genes were confirmed by PCR, RT-PCR and Western blot analysis, which indicated that transformed plants expressed Der p II and Der p V and displayed structural and immunological characteristics identical to the Escherichia coli expressed protein. Our aim was to express mite antigens in edible plant, therefore, the same constructions was used to generate transgenic potato expressing Der p II and Der p V. Integration of the transgene in potato was also confirmed by PCR, RT-PCR and Southern blot analysis and expression of the Der p II and Der p V protein was confirmed by Western blot analysis. The transgeinc tobacco and potato plants expressing the transgene carrying the SEKDEL retention signal showed higher expression levels of Der p II and Der p V allergic proteins. The maximum level that could be detected was 0.01% of the total soluble proteins. This report first presented the expression of mite allergens in plant tissues with an immunogenic form and the transgenic plants generated by this method may provide a model system for development of low-cost edilbe oral vaccine.
Contents
Abstract in Chinese………………………………………………i
Abstract in English………………………………………………ii
Literature Review…………………………………………………1
Materials and Methods……………………………………………20 Construction of plant transformation vector containing Der p V gene…………………………………………………………………20
Construction of plant transformation vector containing Der p II gene…………………………………………………………………21
Tobacco transformation…………………………………………24
Potato transformation……………………………………………24
PCR detection of the Der p II/Der p V gene in transgenic plants………………………………………………………………27
Analysis of Der p II/Der p V gene transcription in transgenic plants by RT-PCR…………………………………………………28
Analysis of Der p II/Der p V protein expression in transgenic plants by Western blotting……………………………………28
Measurement of total soluble protein and quantification of Dp protein from plant extracts…………………………………29
Southern blot analysis………………………………………29
Results……………………………………………………………31
Transformation vectors for expression Der p II and Der p V in plants……………………………………………………………31
Tobacco plant transformation and PCR screening of the Der p II/Der p V transgenic tobacco plants……………………33
Western blotting analysis of the Der p II/Der p V protein in transgenic tobacco……………………………………………37
Potato plant transformation and PCR screening of the Der p II/Der p V transgenic potato plants………………………42
RT-PCR analysis of the Der p II/Der p V transcription in transgenic potato………………………………………………46
Western blotting analysis of the Der p II/Der p V protein in transgenic potato………………………………………………48
Southern blot analysis………………………………………52
Discussion………………………………………………………54
References………………………………………………………58
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