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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李昆達 | |
| dc.contributor.author | Hsiu-Chi Lin | en |
| dc.contributor.author | 林修齊 | zh_TW |
| dc.date.accessioned | 2021-06-14T16:44:05Z | - |
| dc.date.available | 2016-08-20 | |
| dc.date.copyright | 2011-08-20 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-12 | |
| dc.identifier.citation | Arulanandam, B. P., Mittler, J. N., Lee, W. T., O'Toole, M., and Metzger, D. W. (2000). Neonatal administration of IL-12 enhances the protective efficacy of antiviral vaccines. J Immunol, 164(7), 3698-3704.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40287 | - |
| dc.description.abstract | 嗜甲醇酵母菌 Pichia pastoris，具有容易進行基因操作，可以進行細胞高密度培養等優點，且 P. pastoris 屬於真核表現系統，可以對蛋白質進行轉譯後修飾，培養成本比動物及昆蟲細胞表現系統便宜；同時 P. pastoris 具有將異源蛋白質分泌至細胞外的能力。本研究之目的在利用 P. pastoris 生產重組人類介白素-12。介白素-12是一個細胞激素，由兩個次單位 ─ p35 及 p40 以雙硫鍵結合而成，介白素-12 可以促進 T helper 1 細胞的分化，對於細胞型免疫扮演著關鍵的角色。輔大蘇睿智老師研究室建構了單鏈人類介白素-12融合基因，本研究則進一步將此單鏈人類介白素-12基因分別建構於嗜甲醇酵母菌 P. pastoris 之誘導型載體 pPICZαC 和持續表現型載體 pGAPZαC，並轉殖至原生型 X33 菌株進行異源表現。結果顯示，GAP 啟動子 (持續表現型) 之介白素-12表現量高於 AOX1 誘導型啟動子 (Dunn’s test，p<0.05)。接著，用人類介白素-12專一性之三明治酵素連結免疫分析法，挑選出高表現量的轉形株於 Hinton 氏搖瓶以 YPG 培養基培養，其產量為 2771 pg/mL，細胞乾重 20.3 g DCW L-1。此外，在 Hinton 氏搖瓶培養中，添加 1 mM phenylmethanesulfonylfluoride (PMSF)、1 mM ethylenediaminetetraacetic acid (EDTA) 蛋白酶抑制劑，或是 1% 蛋白酶受質 casamino acid 來比較對重組人類介白素-12之產量影響。結果顯示，添加蛋白酶受質 casamino acid 可有效減緩重組蛋白質之降解。若從第 0 個小時起每天添加 2% casamino acid，細胞乾重可達 23.7 g DCW L-1，產量可達 4588 pg/mL，分別比未添加 casamino acid 時增加了 20% 和 71%。而在發酵槽中以 BSM 培養基經由細胞高密度培養，其細胞乾重可達 118.4 g DCW L-1，重組人類介白素-12的產量則為 390 ng/L，為在 Hinton 氏搖瓶以 BSM 培養基培養時之 9.9 倍。此外，若於發酵槽中第 0 個小時起每天添加 2% 之 casamino acid，重組人類介白素-12產量則比未添加 casamino acid 時增加了 115%。 | zh_TW |
| dc.description.abstract | Pichia. pastoris has advantages of manipulating genes easily and growing to high density. As a eukaryote, P. pastoris provides post-translational modifications. In addition, it is less expensive than other eukaryotic expression systems, such as CHO cells and insect cells. Meanwhile, P. pastoris has the ability to secrete recombinant proteins into medium. The aim of this study is to produce recombinant human interleukin-12 in P. pastoris. Interleukin-12 (IL-12) is a cytokine, comprising two disulfide-linked subunits, p35 and p40. IL-12 acts as a key regulator of cell-mediated immune responses through the induction of T helper 1 cell differentiation. Dr. Su’s lab constructed a single-chain human IL-12 (schIL-12) fusion gene. The IL-12 gene, we were constructed in pPICZαC and pGAPZαC vectors, and transformed into the P. pastoris X33. Then, we have been expressed in the P. pastoris X33 with the inducible AOX1 promoter and the constitutive GAP promoter. The recombinant human IL-12 expression from GAP promoter was significantly better compared to the AOX1 promoter made by Dunn's test (p<0.05). We selected a highest productivity transformant which is for further research by using a sandwich-ELISA assay specific to human IL-12. By using YPG medium, the productivity of the recombinant human IL-12 was 2771 pg/mL in Hinton’s flasks and dry cell weight reached to 20.3 g DCW L-1. In addition, the effects of different protease inhibitors, 1 mM phenylmethanesulfonylfluoride (PMSF), 1 mM ethylenediaminetetraacetic acid (EDTA), or 1% protease substrate casamino acid, on the productivity of recombinant human IL-12 were compared respectively in Hinton’s flasks. For these experiments, we concluded that addition of protease substrate casamino acid, could reduce proteolytic degradation. By adding 2% of casamino acid per day from 0 hour, the biomass reached to 23.7 g DCW L-1 and the production of recombinant human IL-12 was 4588 pg/mL in Hinton’s flasks. These results were 20% and 71% higher compared to the control which was without casamino acid respectively. Through a high cell density culture in the Bioflo110 fermentor by BSM medium, dry biomass reached to 118.4 g DCW L-1 and the production of IL-12 was 390 ng/L, which were 9.9 times more than the production in Hinton’s flasks by BSM medium. When adding 2% of casamino acid every day in the fermentor from 0 hour, the production of recombinant human IL-12 was 115% higher than those in medium without casamino acid. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T16:44:05Z (GMT). No. of bitstreams: 1 ntu-100-R98b47107-1.pdf: 2759807 bytes, checksum: d3adf7e692908299c8b2801bd01812d7 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 口試委員審定書 I
謝誌 II 中文摘要 III Abstract IV 縮寫表 VI 專有名詞 中英文對照表 VII 目錄 VIII 圖表目錄 XI 第一章 前言 1 1.1 介白素-12之結構 2 1.2 介白素-12在免疫上扮演之角色 2 1.3 介白素-12之功能 3 1.3.1 抗腫瘤 3 1.3.2 增強對病源菌之抵抗力 4 1.3.3 抑制過敏性氣喘 5 1.3.4 做為疫苗佐劑 6 1.4 嗜甲醇酵母 Pichia pastoris 表現系統 7 1.4.1 嗜甲醇酵母 Pichia pastoris 簡介 7 1.4.2 P. pastoris菌株簡介 8 1.4.3 啟動子 9 1.4.4 P. pastoris 表現載體 9 1.5 P. pastoris 表現重組醫藥用蛋白質之發展 11 1.5.1 N型醣基化 11 1.5.2 以 P. pastoris 表現醫藥用蛋白質 11 1.6 蛋白質降解 12 1.6.1 降解成因 12 1.6.2 抑制蛋白質降解之策略 13 1.6.3 蛋白酶抑制劑 13 1.7 異源表現介白素-12之相關研究 14 1.8 研究動機與目的 15 1.9 研究大綱 16 第二章 材料與方法 17 2.1 微生物及載體 18 2.2 P. pastoris 表現載體之建構 18 2.2.1 置換限制酶切位 18 2.2.2 E. coli 轉形及篩選 19 2.2.3 持續表現型及誘導型載體之建構 19 2.2.4 挖膠純化 20 2.3 酵母菌之轉形 20 2.3.1 質體 DNA 抽取 20 2.3.2 線性質體之製備 21 2.3.3 酵母菌之電穿孔法 22 2.3.4 以 PCR 確認轉形株 23 2.4 酵母菌Genome之抽取 23 2.5 IL-12 之 mRNA 確認 24 2.5.1 抽取Total RNA 24 2.5.2 去除殘留DNA 24 2.5.3 反轉錄 PCR (Reverse transcription PCR) 25 2.6 選擇適當的表現菌株 26 2.6.1 篩選高拷貝數之轉形株 26 2.6.2 小量培養篩選高表現量之菌株 26 2.7 Hinton 氏搖瓶小量培養 26 2.8 蛋白酶抑制劑之比較 27 2.9 發酵槽培養 27 2.10 三明治酵素連結免疫分析法 28 2.11 SDS-聚丙醯胺膠體電泳 29 2.12 西方墨點轉漬法 29 第三章 結果 30 3.1 P. pastoris 之IL-12基因轉形株建立 31 3.1.1 中間載體 yT&A-IL12 之建構 31 3.1.2 表現載體 pPICZαC-IL12 和 pGAPZαC-IL12 之建構 31 3.1.3 IL-12轉形株之確認 31 3.2 IL-12 之 mRNA 確認 32 3.3 篩選高表現量菌株 32 3.4 啟動子 AOX1 及 GAP 之比較 33 3.5 西方墨點轉漬法檢測結果 33 3.6 不同培養基於 Hinton 氏搖瓶之比較 34 3.7 蛋白酶抑制劑之比較 34 3.8 Casamino acid添加次數及濃度之最適化 34 3.9 發酵槽培養 35 第四章 討論與結論 37 4.1 討論 38 4.2 結論 43 圖表 44 參考文獻 64 附錄 74 | |
| dc.language.iso | zh-TW | |
| dc.subject | 抑制劑 | zh_TW |
| dc.subject | 畢赤酵母菌 | zh_TW |
| dc.subject | 重組人類介白素-12 | zh_TW |
| dc.subject | 蛋白酶 | zh_TW |
| dc.subject | 細胞高密度培養 | zh_TW |
| dc.subject | Pichia pastoris | en |
| dc.subject | High cell density culture | en |
| dc.subject | Protease inhibitor | en |
| dc.subject | Recombinant human IL-12 | en |
| dc.title | 以 Pichia pastoris 生產重組人類介白素-12 | zh_TW |
| dc.title | Production of recombinant human Interleukin-12 using Pichia pastoris | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇遠志,黃健雄,江伯倫,蘇睿智 | |
| dc.subject.keyword | 畢赤酵母菌,重組人類介白素-12,蛋白酶,抑制劑,細胞高密度培養, | zh_TW |
| dc.subject.keyword | Pichia pastoris,Recombinant human IL-12,Protease inhibitor,High cell density culture, | en |
| dc.relation.page | 79 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-08-14 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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