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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李昆達(Kung-Ta Lee) | |
dc.contributor.author | Wu-Chang Lin | en |
dc.contributor.author | 林武璋 | zh_TW |
dc.date.accessioned | 2021-06-15T01:26:02Z | - |
dc.date.available | 2010-01-11 | |
dc.date.copyright | 2010-01-11 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-23 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42852 | - |
dc.description.abstract | 為有效提供植物來源之重組塵蟎過敏原Der p 2,以發展用於減緩氣喘之可食用性疫苗,本實驗室先前已建立可生產重組Der p 2 (rDer p 2) 的菸草懸浮細胞株。但由於在生物反應器的培養中,菸草細胞有明顯的褐化現象,使得細胞生長受抑制,降低了rDer p 2的產量。本研究為了提升rDer p 2的產量,首先經由通氣量與細胞生長量的關係,確定可提供充足溶氧且不會使細胞褐化的通氣速率為0.06 vvm。接著在搖瓶培養,發現採用3% 蔗糖作為初始碳源進行批次培養 (batch culture) 時,具有最高的rDer p 2的生成率。以5 L生物反應器進行批次培養時,rDer p 2產量在第19天達最高,為4.85 mg/L,是搖瓶批次培養的2.3倍;而以批次饋料進行培養時,生物反應器內之rDer p 2在第14天達最高之 7.00 mg/L,為搖瓶批次饋料培養的1.4倍。此外,菸草懸浮細胞之蛋白質粗抽液,經硫酸銨分劃、Sephacryl S-100膠體過濾,與DEAE-sepharose離子交換處理後,可獲得以rDer p 2 為主要蛋白質之溶液,其純化倍數為2.1倍,回收率為8.1 %。 | zh_TW |
dc.description.abstract | To efficiently provide recombinant protein of the major house dust mite allergen Der p 2 from a transgenic plant system for development of an edible vaccine to reduce asthma allergy, clones of recombinant Der p 2 (rDer p 2) producing tobacco suspension cell lines had been established in our previous study. However, the browning of tobacco cells caused the low cell growth as well as low rDer p 2 production yield in the culture period. In this study, by examining the relationship between aeration rate and the biomass accumulation, the aeration rate at 0.06 vvm was considered adequate for cell growth without browning of cells. Based on the data revealed from flask cultures, evaluating the ratio of rDer p 2 to carbon source, 3% sucrose was used as the initial carbon source. In batch cultures, the rDer p 2 production yield in the 5 L bioreactor reached 4.85 mg/L on the 19th day, which is 2.3 times the yield of flask batch cultures. In fed-batch cultures, the rDer p 2 production yield in the 5 L bioreactor reached 7.00 mg/L on the 14th day, which is 1.4 times the yield of fed-batch cultures in flasks. Furthermore, the rDer p 2 produced by tobacco suspension cells was purified approximately 2.1-fold by ammonium sulfate fractionation, gel filtration through Sephacryl S-100 and ion-exchange on DEAE-sepharose, with the recovery yield about 8.1%. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:26:02Z (GMT). No. of bitstreams: 1 ntu-98-R96B47113-1.pdf: 2402723 bytes, checksum: 85e054ceb094a7592c744830187f1c2a (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 口試委員審定書……………………........…………...……………………………….......I
謝誌………………...………...……………………...……………………………….......II 中文摘要……………..……...……………………...……………………………….......III Abstract……...……………………..…………………………….……………..….…...IV Abbreviation……………………………………………………………………...….......V Index /專有名詞 中英文對照表……………………………………………...…......... VI Contents………………………………………………………………...………........…VII Contents of tables and figures………………………………………………..…..........IX Chapter I. Introduction…………………….....………………………………..…...1 1.1 House dust mite allergens…………………………………….......…………..……….2 1.2 Expression of recombinant allergens in different hosts………………………...……..5 1.3 Transgenic plant cell cultures………………………………………………..………...7 1.4 Downstream process of rDer p 2 from tobacco suspension cells…………........……12 1.5 History of this study………................................……………………….……………12 1.6 Aim of this study…………………………………….....……………….……………13 Chapter II. Materials and Methods……………………………….......………..14 2.1 rDer p 2 expressing suspension cells……………………...........................................15 2.2 Detection and quantification of rDer p 2………………………....………………….20 2.3 Purification of rDer p 2………………………........................................................…22 Chapter III. Results……………………………………….......…………………….25 3.1 Standard curve of sugars…………………………………......................……………26 3.2 Confirmation of Der p 2 gene and protein expression……………......……………...26 3.3 Shake-flasks cultures………………………………………………..............……….26 3.4 Bioreactor cultures…………………..............................................................……….28 3.5 Purification of rDer p 2…………….......................................................…………….31 Chapter IV. Discussion………………………...........................................………...33 4.1 High cell-density culture……..............................................................................……34 4.2 Foaming………………............................................................................…………...35 4.3 Aeration and browning…………………............….…………………………………36 4.4 Continuous culture potential……………………....…………………………………37 4.5 Purification of rDer p 2……………………………………............…………………38 Chapter V. Conclusion and perspective…………...........……………………...39 5.1 Conclusion……………………………………...............................…………………40 5.2 Perspective...................……………………………………............…………………40 Tables………………………………………………………….....……….……………...43 Figures………………………………………………………….....….…………………..47 References………………………………………………………………......……………72 Appendices……………………………………………………………......……………...78 Contents of tables and figures Table 1 House dust mite allergens…………........………….…….…...…….………..….43 Table 2 Examples of recombinant allergens expressed in various systems….….…........44 Table 3 Data of rDer p 2 production in different cultivation modes……....................….45 Table 4 Purification scheme of rDer p 2 from tobacco suspension cells…………..…....45 Table 5 Proteins expressed in transgenic tobacco suspension cell cultures…….....….... 46 Fig. 1 X-ray structure of Der p 2, the major house dust mite allergen…….……….…....47 Fig. 2 How Agrobacterium genetically transforms plants…………………………….....48 Fig. 3 The construction of T-DNA region…………………………………………..……49 Fig. 4 High performance liquid chromatography of authentic sugars…………………...50 Fig. 5 HPLC standard calibration curve of authentic sugar concentration……………....51 Fig. 6 Western blotting of rDer p 2……………………………........................................52 Fig. 7 Effect of sucrose concentration on growth of transgenic tobacco suspension cells in flask batch cultures.............................................................................................….53 Fig. 8 Effect of sucrose concentration on medium conductivity in flask batch cultures.………………………............................................................…………....54 Fig. 9 Effects of sucrose concentration on rDer p 2 production and ratio of rDer p 2 to sucrose…………………...............................................................................…..…55 Fig. 10 Time course of biomass, rDer p 2 content and rDer p 2 production in shake-flask fed-batch cultures………………………………………………...........…………56 Fig. 11 Residue sugars in shake-flask fed-batch cultures………………..................…....57 Fig. 12 Time course of conductivity and pH in shake-flask fed-batch cultures................58 Fig. 13 Effect of aeration rate on cell growth in the 5 L bioreactor………………...…...59 Fig. 14 Time course of cell mass (by dry cell weight), rDer p 2 content and rDer p 2 production in the 5 L bioreactor batch culture……........…………................…...60 Fig. 15 Time course of conductivity and pH of the 5 L bioreactor batch culture……….........................................................................................……..….61 Fig. 16 Residue sugars in the 5 L bioreactor batch culture…………………………………………...........................................……62 Fig. 17 Foaming of the bioreactor fed-batch culture (No antifoam reagent added)…………………………………………….............................………....63 Fig. 18 Time course of rDer p 2 content and production in the 5 L bioreactor fed-batch culture.……………………..……….................................................…….…….64 Fig. 19 Time course of biomass, pH and medium conductivity in the 5 L bioreactor fed-batch culture…………….............……...................................................…..65 Fig. 20 Residue sugars in the 5 L bioreactor fed-batch batch culture.…….......……..….66 Fig. 21 Ammonium sulfate fractionation of rDer p 2…………………......………….…67 Fig. 22 Gel filtration chromatography……………………………………......……….…68 Fig. 23 Ion exchange chromatography………………..................................................…69 Fig. 24 Overview of purification on SDS-PAGE………..................................................70 Fig. 25 Effect of antifoam reagent Y-30 on growth of tobacco suspension cells…………………………………....……………......................................…71 | |
dc.language.iso | en | |
dc.title | 菸草懸浮細胞高密度培養生產重組塵蟎過敏原Der p 2 | zh_TW |
dc.title | High cell-density cultures of transgenic tobacco suspension cells for rDer p 2 production | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇遠志,黃鵬林,江伯倫,劉祖惠 | |
dc.subject.keyword | 家居塵蟎,過敏原,Der p 2,菸草懸浮細胞,高細胞密度培養,批次饋料培養, | zh_TW |
dc.subject.keyword | house dust mite allergen,Der p 2,tobacco suspension cells,high cell-density cultures,fed-batch cultures, | en |
dc.relation.page | 81 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-23 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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