臺灣博碩士論文加值系統

中文摘要
質體在細菌及酵母菌細胞中可以當成一個基因載體表現目標基因，亦可應用於人體做為基因治療與基因疫苗，近年來基因治療與疫苗技術發展日益成熟，因此質體的大量生產及純化是十分值得的課題。本文將質體pEGFP-C1分別轉殖進入E.coli BL21(DE3)、Top 10、DH5α及JM 109等四株宿主細胞中，並培養於LB、SDCAS及Defined三種培養基中，以JM 109於Defined培養基中培養所得的質體含量最高且質體中超螺旋體構型的比例也最高，因此選擇了JM 109與Defined為生產質體用的宿主細胞與培養基。利用發酵槽大量培養細胞生產質體，其中所添加的抗生素(Kan)會在菌體培養的過程中持續降解造成質體的漏失，而持續添加抗生素培養菌體雖可維持質體的含量但卻不利於菌體的生長。帶正電之polyethylenimine(PEI)高分子電解質固定化於多孔性高分子薄膜上，利用靜電交互作用，可快速、大量的純化高純度的質體(A260/280=1.8~2.1)，且可去除RNA，避免RNase的使用。PEI薄膜可以多次重複的使用，純化能力在6成以上，且所得質體純度可做為細胞轉殖上的應用。

Abstract
Plasmid pEGFP-C1 was transformed into various Escherichia coli (E. coli) hosts: BL21 (DE3), Top 10, DH5α and JM109, respectively for evaluates a best host for plasmid production. The growth curves, plasmid content and the ratio of supercoil structure of plasmid in those transformants were studied by cultivating in LB, SDCAS and Defined mediums, respectively. E.coli JM 109 growing in Defined medium resulted in a best plasmid yield. JM 109 and defined medium were employed for plasmid production in a fermentor. The presence of Kanamycin shows a significant influence on plasmid stability. Batch fermentation by intermittent feeding Kanamycin can produce 2.98 mg/L plasmid. Polyethylenimine (PEI) is a positive polymer, which can be covalently bound on an activated microspores membrane. Using this PEI membrane can efficiently and fast recover plasmid via electrostatic interaction. About 60% of plasmid in the crude extract of 3ml cell culture can be recovered by using a single sheet (25mm diameter) of PEI membrane. The purity of the recovered plasmid is A260/280=1.8~2.1. The purity is also comparable with that brained by using BioRed mini kit as shown in agarose gel electrophoresis. The PEI membrane can be reused at least for 5 times without losing its purification capability.

目錄
中文摘要………………………………………………………………..Ⅰ
英文摘要……………………………………………………………… Ⅱ
目錄……………………………………………………………………..Ⅲ
圖目錄…………………………………………………………………..Ⅵ
表目錄…………………………………………………………………..Ⅷ
第一章 緒論……………………………………………………………..1
1.1 前言………………………………………………………………….1
1.2 質體DNA……………………………………………………………2
1.3 質體的應用………………………………………………………….5
1.4 研究內容簡介……………………………………………………….9
第二章 文獻回顧………………………………………………………11
2.1 質體穩定度………………………………………………………...11
2.2質體的純化…………………………………………………………15
第三章 實驗材料、設備與方法………………………………………22
3.1 實驗材料…………………………………………………………...22
3.1.1 菌株………………………………………………………………22
3.1.2 質體pEGFP-C1………………………………………………….22
3.1.3 酵素………………………………………………………………24
3.1.4 DNA操作試液套件組……………………………………………24
3.1.5 標準分子量溶液…………………………………………………24
3.1.6 試藥………………………………………………………………24
3.2 實驗設備…………………………………………………………...27
3.3 實驗方法…………………………………………………………...29
3.3.1 平板培養基配製…………………………………………………29
3.3.2 培養基配製………………………………………………………29
3.3.3 質體轉殖至E.coli………………………………………………..31
3.3.4 菌種保存…………………………………………………………33
3.3.5 緩衝液配製………………………………………………………33
3.3.6 質體之純化………………………………………………………34
3.3.7 限制酶反應………………………………………………………37
3.3.8 瓊脂凝膠電泳……………………………………………………37
3.3.9 發酵槽批式培養…………………………………………………38
3.3.10 PEI薄膜純化質體……………………………………………….42
第四章 結果與討論……………………………………………………45
4.1 決定最適培養基與宿主細胞……………………………………...45
4.1.1 質體的確認………………………………………………………45
4.1.2 最適培養基………………………………………………………45
4.1.3 最適宿主細胞……………………………………………………46
4.2 質體穩定度………………………………………………………...54
4.2.1 平板培養基放置時間對質體穩定度的影響……………………54
4.2.2 發酵生產質體及質體在發酵槽中的穩定度……………………54
4.2.2.1 批式發酵(未加抗生素)………………………………………..54
4.2.2.2 批式發酵……………………………………………………….58
4.2.2.3批式發酵(持續添加抗生素)……………………………………64
4.3 PEI薄膜純化質體…………………………………………………..69
4.3.1 RNA隨時間降解情形……………………………………………69
4.3.2 PEI薄膜純化條件………………………………………………...70
4.3.3 固定化PEI濃度對純化能力的影響…………………………….70
4.3.4 PEI薄膜的重複使用……………………………………………...74
4.3.5 PEI薄膜純化所得質體與市售純化組所得質體之比較………...74
4.3.6 轉殖確認PEI薄膜純化之質體………………………………….74
第五章 結論與建議……………………………………………………78
5.1 結論………………………………………………………………...78
5.2 建議………………………………………………………………...80
參考文獻………………………………………………………………..81

參考文獻
蔡有癸，國立台灣科技大學化學工程研究所碩士論文，高密度發酵基因重組大腸菌生產，D-Amidohydrolase的研究，2000年。
楊佩芬，國立台灣科技大學化學工程研究所碩士論文，以NusA融合方式增進D型醯胺基水解酶溶解度及其活性結構之研究，2001年。
黃麗華，基因治療~二十一世紀的醫療新科技，NTU BioMed Bulletin., No1., May.,(2000)
Grunwald, A. G., and Shields, M. S., “Plasmid purification using membrane-based anion-exchange chromatography”, Analytical Biochemistry. 296, 139-141, (2001)
Mazus’, B., Krysiak, C., and Buchowicz, J., “Tungsten Particle-Induced Nicking of Supercoiled Plasmid DNA”, Plasmid. 44, 89-93, (2000)
Beuken, E., Vink, C., and Bruggeman, C., “One-step procedure for screening recombinant plasmids by size”, Biotechniques. 24, 748-750, (1998)
Bieber, T., and Elsasser, H. P., “Preparation of a low molecular weight polyethylenimine for efficient cell transfection”, Biotechniques. 30, 74-81 (January 2001)
Birmboin, H. C., and Doy, J., “A rapid alkaline extraction procedure for screening recombinant plasmid DNA”, Nucleic Acids Res. 7: 1513-1523, (1979)
LaMarca, C., Lenhoff, A. M., and Dhurjati, P.,”Partitioning of host and recombinant cells in agueaus two-phase polymer systems” Biotechnol. Bioeng. 36, 484-492, (1990)
Daniel, H., Bohuslav, R., Jan, S., Alena, S., Jiri, L., and Milan, J. B., “Properties of RNase A Immobilized on Magnetic Poly (2-hydroxyethyl methacrylate) Micro spheres”, Biotechnol. Prog. 17, 447-452, (2001)
Davis, B. D., Dulbeco, R., Eisen, H. H., Microbiology, 3rd ed Philadelphia, PA, Harper and row, (1980)
Inloes, D. S., Simith, W. J., Taylor, D. P., Cohen, S. N., Michales, A. S., and Robertso, C. R.,”Hollow-fiber membrane bioreactors using immobilized E.coli for protein synthesis.” Biotechnol, Bioeng. 25, 2653-2681, (1983)
Godwin, D., and Slater, J. H., “The influence of the growth environment on the stability of a drug resistance plasmid in Escherichia coli K12”, J. Gen. Microbiol. 111, 201-210, (1979)
Henry, K. L., Davis, R. H., and Taylor, A. L., “Continuous recombinant bacterial fermentations utilizing selective flocculation and recycle ”, Biotechnol.Prog. 6, 7-12, (1990)
Helinski, D., “Bacterial plamids: autonomous replication and vehicles for gene cloning”, CRC Crit Rev Biochem. 7, 83-101, (1979)
Gerdes, K., “The Parb (HOK/SOK) Locus of Plasmid R1: a general purpose plasmid stabilization system”, Bio/Technology. 6, 1402-1405, (1988)
Mosbach, K., Brinbaum, S., Hardy, K., Davies, J., and Bulow, L., ”Formation of proinsulin by Bacillus subtilis.” Nature. 302, 543-545, (1983)
Kleman, G. L., Strohl, W. R., “Devlopments in high cell density and high productivity microbial fermentation”, Curr. Opin. Biotech. 5, 180-186, (1994)
Lee, S. Y., “Review: High cell-density culture of Esherichia coli”, Tibtech. 14, 98-105, (1996)
Schmidt, M. T., and Flaschel, E.,” Plasmid DNA for pharmaceutical applications” Dev Biol. Basel, Karger. 44, 25-31, (2000)
PerSeptice Biosystems, Purification of plasmid DNA, Application note.
Kumar, P. K. R., Maschke, H-E., Friehs, K., and Schugerl, K.,”Strategies for improving plasmid stability in genetically modified bacteria in bioreactors.”
Tibtech. 9, 279-284, (1991)
Qiagen Plasmid Handbook. Qiagen, Inc., Chatsworth, CA.
O’Kennedy, R. D., Baldwin, C., Keshavarz-Moore, E., “Effects of growth medium selection on plasmid DNA production and initial processing steps”, Journal of Biotechnology. 76, 175-183, (2000)
Garrett, R. H., and Grisham, C. M., Principles of biochemistry with a human focus.,A Harcourt Higher Learning Company. (2000)
Riesenberg, D., Schulz, V., Knorre, W. A., Pohl, H. —D., Korz, D., Sander, E. A., Roβ, A. and Deckwer, W. —D., “High cell density cultivation of Escherichia coli at controlled specific growth rate”, J. Biotechnol. 20, 17-28, (1991)
Riesenberg, D., “High-cell-density cultivation of Escherichia coli”, Curr. Opin. Biotech. 2, 380-384, (1991)
Sambrook, J., Fritsch, E. F., and Maniatis, T.: Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (1989)
Schmidt, T., Friehs, K., Schleef, M., Voss, K., Flaschel, E., “Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis”, Analyt Biochem. 274, 235-240, (1999)
Summers, D. K., “The Biology of Plasmids.” Oxford, Black well Science, PA, (1996)
Turkova, J., Bioaffinity Chromatography, 2nd ed.; Elsevier Amsterdam, (1993)