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學校名稱輔仁大學
系所名稱生命科學系
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學號492546197
研究生(中)廖智凱
研究生(英)Chih-Kai Liao
論文名稱(中)基因轉殖蕃茄表現單鏈人類介白素12之基因
論文名稱(英)Expression of Single-chain human interleukin-12 gene in transgenic tomato plants
其他題名
指導教授(中)蘇睿智
指導教授(英)Ruey-Chih Su
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國圖全文開放日期.2008.08.01
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學位類別碩士
畢業學年度94
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關鍵字(中)介白素12 連接子 單鏈人類介白素12 基因轉殖蕃茄 農桿菌轉型作用
關鍵字(英)interleukin-12, IL-12 linker single-chain human IL-12, schIL-12 transgenic tomato plants Agrobacterium-mediated transformation
摘要(中)相較其他的表現系統,利用植物生產醫療用蛋白具有價格低廉、安全與可長期貯存等品質與經濟上的利益,本研究目的是利用可食用的蕃茄植物生產人類介白素-12號蛋白質(human interleukin-12, hIL12),IL-12為p40與p35次單位藉由雙硫鍵摺疊形成的異型雙隅體蛋白質,是一種促發炎與多效性的細胞激素,在引發細胞免疫中佔有重要的地位,目前已應用於癌症治療、呼吸道發炎反應,以及病毒感染的治療與研究上,也常被利用來當作疫苗用的佐劑。為了避免在轉基因植物內形成p40同型雙隅體而抑制IL-12的生物活性,我們利用18個胺基酸序列的連接子(linker)將人類IL-12的p40與pP35次單位構築成單鏈的融合基因,送交定序確定無誤後,利用pET系統於大腸桿菌BL21(DE3)品系中表現,經IPTG誘導後,以人類的IL-12單株抗體可以偵測到約70 kDa的蛋白質。將IL-12的融合基因構築於CaMV 35S啟動子驅動的基因表現匣中,利用農桿菌(Agrobacterium)轉殖的方式把IL-12的融合基因表現匣送入蕃茄細胞中,在含有植物生長激素Zeatin與抗生素hygromycin的培養基中進行癒傷組織的篩選、分化與再生。經PCR檢測確認10株含有單鏈人類IL-12的基因轉殖蕃茄,其中4株以南方氏墨點法分析顯示僅含有一個轉殖基因的插入位置。而ELISA定量結果顯示在10株基因轉殖蕃茄中單鏈人類IL-12蛋白的表現量均很低。此外,本實驗中亦發現表現單鏈人類IL-12蛋白可能對植物體具有毒性。
摘要(英)The production of biopharmaceutical proteins in plants has many economic benefits compared to traditional expression system, including low cost, high safety, and long term storage. The aim of this study is to produce a disulfide-bonded heterodimeric cytokine, human Interleukin-12(hIL-12) in transgenic tomato plants . IL-12 is consisting of p35 and p40 subunits. It is an proinflammatory and pleiotropic cytokine that plays a crucial role in cell-mediated immunity. IL-12 has been extensively employed in clinical treatments for cancer immunotherapy, airway inflammation, infectious disease, and also used as a vaccine adjuvant. In order to avoid the antagonism of p40 homodimers, we have constructed a human single-chain IL-12(schIL-12) fusion gene in which p40 and p35 subunits were linked by a 18 amino acid linker. The hschIL-12 gene has been sequenuced and expressed in the pET E.coli system. Upon the IPTG induction, the expression of about 70 kDa human IL-12 protein can be detected by anti-human IL-12 monoclonal antibody. Subsequently, we constructed the pC02B-schIL12 recombinant plasmid which harboring schIL-12 gene that was driven by CaMV35S promoter, and introduced into tomato explants via Agrobacterium-mediated transformation. After transformation, transgenic tomato plants were selected and regenerated in culture medium containing Zeatin and hygromycin. The PCR results indicated that 13 transgenic plants containing the schIL12 fusion gene, and the Southern blot analysis showed that 4 transgenic plants contained only one copy transgene in genomic DNA. ELISA quantizative analysis of hIL-12 in transgenic tomato plants revealed that schIL-12 accumulates in very low levels both in leaves and fruits. It was suspected that the expression of schIL-12 in transgenic plants might be toxic to plant cells.
論文目次摘要.............................................................Ⅰ Abstract.........................................................Ⅱ 目錄..............................................................Ⅲ 表目錄............................................................Ⅶ 圖目錄............................................................Ⅷ 前言..............................................................1 介白素-12的發現與免疫功能相關研究.................................1 醫療用蛋白的發展................................................4 重組蛋白表現系統之介紹...........................................6 植物基因轉殖的方式...............................................8 農桿菌轉型法(Agrobacterium-mediated transformation)之原理.......9 植物表現醫療用蛋白之發展..........................................11 植物表現細胞激素之相關研究........................................12 本論文研究之目的.................................................13 材料與方法.........................................................14 1. 菌種、植物與引子..............................................14 2. 菌種的培養...................................................14 2.1 細菌的培養條件............................................14 2.2細菌的保存方式.............................................14 3. 少量抽取細菌質體DNA...........................................14 4. DNA之酵素處理................................................15 4.1 限制酵素之作用............................................15 4.2 DNA去磷酸化之製備.........................................15 4.3 DNA的接合作用............................................16 5. DNA洋菜膠體電泳分析...........................................16 6. 洋菜膠體DNA之分離與回收.......................................16 6.1 膠膜透析法................................................16 6.2 GFXTM PCR DNA與膠體DNA條帶純化套組.........................17 7. 細菌的轉型作用................................................17 7.1 大腸桿菌之轉型作用.........................................17 7.2 農桿菌之轉型作用...........................................18 8. 人類介白素-12基因重組質體之製備.................................18 8.1 單鏈人類介白素-12基因之構築.................................18 8.2 植物表現載體之製備.........................................19 8.3 植物表現單鏈人類介白素-12基因的重組質體之構築.................19 8.4 大腸桿菌表現單鏈人類介白素-12基因的重組質體之構築..............20 9. 大腸桿菌蛋白表現之分析..........................................20 9.1 誘導單鏈人類介白素-12基因的表現..............................20 9.2 SDS-聚丙烯胺膠體(SDS-polyacrylamide)之製備................20 9.3 大腸桿菌蛋白質之粗萃取......................................21 9.4 SDS-聚丙烯胺蛋白質電泳分析之進行.............................21 10 西方墨點法(Western blot)與免疫點漬法(Immuno-dot blot)........21 11. 蕃茄轉型、篩選與再生...........................................22 12. 蕃茄轉植株的分子檢測...........................................24 12.1 植物基因組DNA(genomic DNA)之萃取.........................24 12.2 Genomic DNA之定量........................................25 12.3 以PCR確認蕃茄轉植株.......................................25 13 南方氏墨點法(Southern blot)..................................26 14.植物表現重組人類IL-12的測量.....................................28 14.1 植物可溶性蛋白的粗萃取.....................................28 14.2 蛋白質的定量..............................................29 14.3 酵素連結免疫吸附分析法(ELISA).............................29 15.統計方法......................................................30 結果................................................................31 一、單鏈人類IL-12基因之構築.......................................31 1、帶有連接子(Linker)片段的人類p40與p35質體之構築..............31 2、重組質體pCRII-schIL12的構築................................31 二、大腸桿菌表現單鏈人類IL-12蛋白.................................32 三、表現單鏈人類IL-12的植物重組質體之構築..........................32 四、蕃茄植株的轉型作用...........................................33 五、擬轉殖植物之分子檢測.........................................34 六、基因轉殖蕃茄葉片中單鏈人類IL-12蛋白之表現......................35 七、基因轉殖蕃茄果實中單鏈人類IL-12蛋白之表現......................36 八、培養瓶中擬轉殖蕃茄表現單鏈人類IL-12蛋白之情形..................36 討論..............................................................38 單鏈人類介白素-12基因的構築.....................................38 植物表現重組質體的構築..........................................40 蕃茄植株的轉型作用 .........................................40 人類IL-12於植物表現的情形.......................................42 轉殖IL-12基因對植物的可能影響...................................44 未來的工作....................................................45 參考文獻..........................................................46 附錄一、本實驗使用的菌種與植物名稱和特性..............................81 附錄二、本實驗使用之質體............................................82 附錄三、本實驗使用的引子與PCR程式....................................83 附錄四、細菌生長之培養基............................................84 附錄五、SDS-聚丙烯胺蛋白質電泳的藥品成分..............................85 附錄六、植物組織培養的培養基.........................................86 附錄七、植物馴化的培養基............................................87 附錄八、單鏈老鼠IL-12基因的構築流程..................................88 附錄九、hIL-12基因在人類與蕃茄細胞的condon usage之比較................89 作者簡歷...........................................................90
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