Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90042
DC FieldValueLanguage
dc.contributorYau-Heiu Hsuen_US
dc.contributor徐堯煇zh_TW
dc.contributor.author江閔超zh_TW
dc.contributor.authorMin-Cho Jiangen_US
dc.contributor.other生物科技學研究所zh_TW
dc.date2014zh_TW
dc.date.accessioned2015-12-09T02:07:01Z-
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dc.identifier.urihttp://hdl.handle.net/11455/90042-
dc.description.abstract干擾素γ (interferon gamma, IFNγ) 是一種具有抗病毒、抗腫瘤和免疫調節作用的細胞激素,當細胞受到病毒感染時,由活化的T細胞和NK細胞所產生,進而達到抑制病毒複製與細胞生長的作用,為重要的醫療蛋白之一。因此本研究之主要目的為利用菸草作為醫療蛋白的生產工廠,發展不同的病毒載體來生產重組人類干擾素γ,而為了有效從植物中純化出目標蛋白,更試著建立一套植物外泌蛋白的系統。在本實驗中,首先利用四種不同的植物病毒,包含竹嵌紋病毒 (Bamboo mosaic virus, BaMV)、狐尾草嵌紋病毒 (Foxtail mosaic virus, FoMV) 、菸草嵌紋病毒 (Tobacco mosaic?virus, TMV) 以及馬鈴薯病毒X (Potato virus X, PVX) 作為病毒載體,將各病毒的外鞘蛋白基因序列置換成 IFNγ 序列,所構築的載體利用農桿菌接種於菸草,分析接種葉,發現BaMV載體可生產出最高量的 IFNγ,進一步以 Northern blot 證實所表現出來的 IFNγ並不會影響 chimeric BaMV 之mRNA的累積。為了取得更好的 IFNγ 多株抗體,我們以大腸桿菌進行大量表現 IFNγ,我們將 IFNγ N端外泌訊號去除掉20個胺基酸,建構出 Nd20IFNγ,結果發現不僅在大腸桿菌可以大量表現出 IFNγ,在BaMV感染的菸草中所表現的 IFNγ 也有顯著的增加。另外,為了建立一套植物蛋白的外泌系統,以利 IFNγ 純化與生產,我們將 Nd20IFNγ 接上菸草的外泌訊號胜?,並以 BaMV作為病毒載體,建構出 pKBdCSSNd20IFNγ,並在 IFNγ基因的 C 端融合 arabinogalactan-protein (AGP) ,以穩定蛋白質的結構,所構築的質體 pKBdCSSNd20IFNγ(SP)10,利用農桿菌接種於菸草,發現 IFNγ 會形成不同片段大小的形式,我們推測是由於菸草的外泌訊號胜?將蛋白送到植物胞器內進行修飾所造成的。未來我們希望建立出菸草懸浮培養細胞來表現帶有菸草外泌序列的 IFNγ ,透過回收細胞培養液,來證實 IFNγ 可以外泌至細胞外,以利純化並生產出高價值的重組人類 IFNγ。zh_TW
dc.description.abstractHuman Interferon gamma (IFNγ) is an important therapeutic cytokine, and plays a significant role in anti-viral, anti-tumor growth, and immunoreactions. In order to produce IFNγ protein, we generated viral-based expression system to express candidate protein in Nicotiana benthamiana plants or suspension cell line. By replacing coat protein gene of Bamboo mosaic virus (BaMV), Foxtail mosaic virus (FoMV), Tobacco mosaic?virus (TMV), and Potato virus X (PVX) with IFNγ sequence, four independent viral expression cassettes namely pKBdCIFNγ, pKFdCIFNγ, pKTdCIFNγ and pKPdCIFNγ were generated. These expression cassettes were transformed into Agrobacterium and infiltrated into N. benthamina leaves. The transient expression results showed that BaMV based vector produce the most high-level protein accumulation in infiltrated leaves. The expression of IFNγ does not cause deleterious effect on replication ef?ciency of BaMV genome. To improve the IFNγ production, the N terminal secretory sequence of IFNγ was truncated, named as Nd20IFNγ. The Nd20IFNγ not only expressed efficiently in E. coli, but also significantly increased IFNγ accumulation in N. benthamiana. In order to produce IFNγ large-scale with low cost, we developed secretory expression vectors carry signal peptides fused at N terminal of Nd20IFNγ, named as pKBdCSSNd20IFNγ. Furthermore, to increase Nd20IFNγ stability, 10 repeats of the AGP glycomodule Ser-Pro (SP) were fused at C terminal and generated as pKBdCSSNd20IFNγ(SP)10. Western blot analysis revealed that IFNγ formed incremental shifts in molecular weight, suggesting the results of secretion and glycosylation. In the future, the pKBdCSSIFN γ (SP)10 transformed N. benthamiana plants and stable transgenic cell lines to produce recombinant IFNγ protein secreted into cell-suspension medium will be established. This method will be facilitate the high-level production of IFNγ.en_US
dc.description.tableofcontents中文摘要 i 英文摘要 ii 壹、前言 1 一、人類干擾素 1 (一) 人類干擾素的發現 1 (二) 干擾素的分類與功能 1 (三) 干擾素γ之生物活性 2 二、植物分子農場 2 (一) 植物病毒載體 (plant virus-based vector) 3 (二) 植物蛋白的外泌系統 4 三、竹嵌紋病毒 5 (一) 竹嵌紋病毒簡介 5 (二) 竹嵌紋病毒的基因體 5 (三) 竹嵌紋病毒的病毒載體 7 貳、材料與方法 8 一、提高干擾素γ的產量之病毒載體 8 (一) pCFdCIFNγ質體之構築 8 (二) pKBdCNd20IFNγ質體之構築 9 二、植物蛋白的外泌系統之載體構築 10 (一) pKBdCSSNd20IFNγ(SP)10質體之構築 10 (二) pKBdTCSSNd20IFNγ(SP)10質體之構築 11 三、勝任細胞製備及轉形作用 13 (一) 大腸桿菌勝任細胞製備 13 (二) 轉形作用 13 四、電穿孔轉形作用 (Electroporation) 14 (一) 農桿菌勝任細胞的製備 14 (二) 電穿孔轉形作用 14 五、Nd20IFNγ多株抗體之製備 15 (一) pET28aNd20IFNγ之構築 15 (二) pET28aNd20IFNγ之篩選 15 (三) 大量表現 Nd20IFNγ 15 (四) Nd20IFNγ多株抗體之備製 16 六、植物總量及干擾素γ 蛋白分析 16 (一) 萃取總量蛋白 16 (二) 聚丙醯胺膠體電泳 (SDS-Polyacrylamide Gel Electrophoresis) 16 (三) 西方墨點分析 (Western blot analysis) 17 七、北方墨點分析 (Northern blot analysis) 18 (一) 核酸探針建築及其製備 18 (二) 植物總量RNA之萃取 18 (三) RNA 電泳分析及轉漬 19 (四) 北方雜配分析 19 八、純化干擾素γ蛋白 20 (一) 植物葉片中純化干擾素γ蛋白 20 (二) 菸草懸浮細胞培養液中純化干擾素γ蛋白 20 參、結果 22 一、提高干擾素-γ的產量 22 (一) 干擾素γ (Interferon gamma) 之選殖與構築 22 (二) 不同病毒載體表現干擾素-γ於菸草 22 (三) 雙聚體干擾素γ 之確認 23 (四) 表現干擾素γ對於病毒複製效率之影響 23 (五) 以 E. coli 表達干擾素γ 24 (六) 表現 Nd20 IFNγ於菸草 25 (七) 表現病毒外鞘蛋白互補缺失性病毒載體 25 (八) 表現靜默抑制子 (silencing suppressor) 來提升干擾素γ 蛋白之產量 26 (九) 純化干擾素γ蛋白 27 二、建立一套植物蛋白的外泌系統 28 (一) 建構一套植物蛋白外泌系統之載體 28 (二) 植物蛋白外泌系統之載體表現干擾素γ於菸草 28 (三) 建立 Minimum vector 表現干擾素γ於菸草 29 (四) 表現靜默抑制子提高Minimum vector 表現干擾素γ 30 (五) 確立菸草訊號胜? (signal peptide) 之功能 31 (六) 純化外泌之干擾素γ蛋白 32 肆、討論 33 一、重組干擾素γ之生物活性 33 二、干擾素γ對於病毒RNA累積之影響 34 三、缺失病毒外鞘蛋白的病毒載體對於病毒複製之影響 34 四、提升干擾素γ於菸草的產量 35 五、以菸草葉片純化干擾素γ蛋白 36 六、以植物蛋白的外泌系統之載體表現干擾素γ蛋白 37 伍、參考文獻 39 陸、圖表與附錄 48zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2017-08-31起公開。zh_TW
dc.subject干擾素zh_TW
dc.subject植物病毒載體zh_TW
dc.subjectinterferonen_US
dc.subjectplant viral vectoren_US
dc.titleEfficient production of human IFNγ protein in Nicotiana benthamiana plant and suspension cells by viral vectorsen_US
dc.title應用不同的病毒載體來提高人類干擾素γ重組蛋白在菸草植物及細胞中的產量zh_TW
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2017-08-31zh_TW
dc.date.openaccess2017-08-31-
item.openairetypeThesis and Dissertation-
item.fulltextwith fulltext-
item.cerifentitytypePublications-
item.grantfulltextrestricted-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1zh_TW-
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