盤基網柄菌的固定化培養及其優化

生物工程論文編號:SW094  論文字數:13049,頁數:26

目  錄
中文摘要 I
英文摘要 II
目錄 III
1. 緒論 1
 1.1  盤基網柄菌的簡介 1
 1.2  人Fas配體簡介 3
 1.3  盤基網柄菌作為載體 4
 1.4  盤基網柄菌的培養 5
 1.5  盤基網柄菌的固定化 6
2  實驗部分 9
 2.1  菌株 9
 2.2  培養基 9
 2.3  固定化材料 9
 2.4  分析方法 10
3  結果與討論 13
 3.1  AX3-pFH用毛巾作為固定化材料的生長 13
 3.2  AX3-pFH用毛巾內填充聚氨酯泡沫作為固定化材料的生長 14
 3.3  毛巾吸水實驗 14
 3.4  洗脫方法的選擇 15
 3.5  確定最優的毛巾重量 16
 3.6  同濃度懸浮培養液不同質量毛巾載體的吸附能力 17
4  總結與展望 18
致謝 19
參考文獻 20

摘要: 近年來，盤基網柄菌已逐漸發展成為一個極為重要的真核生物表達系統，由染色體外質粒表達復制重組蛋白，培養基成本低廉，適于工業化大量快速培養的重組表達系統，近年來越來越受到人們的重視，但其應用嚴重受到低的生長速度(世代時間為8-12 h)以及低的細胞密度(1-2×107mL-1)的影響。為有效利用盤基網柄菌這一真核表達系統生產異源蛋白諸如人類可溶性Fas配體，有必要實現盤基網柄菌的高密度懸浮培養。本文以可高通量表達人類可溶性Fas配體的重組盤基網柄菌AX3-pFH為研究對象，用毛巾作為固定化材料，來研究盤基網柄菌在固定化中的生長特性，突出固定化的培養優點。討論了毛巾載體在盤基網柄菌培養液中的吸附性質，并根據菌體在載體的吸附特性，選擇兩次用20mL磷酸鹽緩沖液多次擠壓的方式洗脫毛巾內部的盤基網柄菌，同時對毛巾固定化培養的各類參數進行了優化。

關鍵詞: 盤基網柄菌；高密度培養；固定化

Fixed Cultivation and Optimization of Dictyostelium discoideum

Abstract：In recent years, the cellular slime mould Dictyostelium discoideum (Dd) has emerged as a promising eukaryotic system for the expression of recombinant pharmaceutical proteins requiring post-translational modifications. However, its application is seriously affected by slow growth rates (doubling time of 8-10 h) as well as low maximal cell densities (1-2×107 mL-1) in the presence of complex axenic medium. For the production of high-value products like heterogenous proteins in substantial quantities it would represent a considerable advantage, if Dd could be cultivated at high cell density.
  In consequence, some work have been done here which have led – at least partially – to alleviate these two restrictions. The composition of both complex medium HL-5C and synthetic medium SIH was optimized. A Dictyostelium AX3 strain, AX3-pFH was used expressing the soluble human Fas ligand. we studied the adsorption characteristics of towel carrier in the culture, and optimize a series of parameters in towel immobilization. We find the best way to elute Dictyostelium discoideum cells in the towel carrier is to wash and extrude with 20mL phosphate solution twice.
Key words：Dictyostelium discoideum; mass suspension cultivation; stator