盘基网柄菌的固定化培养及其优化

文档编号:SW094  文档字数:13049,页数:26

摘要: 近年来，盘基网柄菌已逐渐发展成为一个极为重要的真核生物表达系统，由染色体外质粒表达复制重组蛋白，培养基成本低廉，适于工业化大量快速培养的重组表达系统，近年来越来越受到人们的重视，但其应用严重受到低的生长速度(世代时间为8-12 h)以及低的细胞密度(1-2×107mL-1)的影响。为有效利用盘基网柄菌这一真核表达系统生产异源蛋白诸如人类可溶性Fas配体，有必要实现盘基网柄菌的高密度悬浮培养。本文以可高通量表达人类可溶性Fas配体的重组盘基网柄菌AX3-pFH为研究对象，用毛巾作为固定化材料，来研究盘基网柄菌在固定化中的生长特性，突出固定化的培养优点。讨论了毛巾载体在盘基网柄菌培养液中的吸附性质，并根据菌体在载体的吸附特性，选择两次用20mL磷酸盐缓冲液多次挤压的方式洗脱毛巾内部的盘基网柄菌，同时对毛巾固定化培养的各类参数进行了优化。

关键词: 盘基网柄菌；高密度培养；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

目  录
中文摘要 I
英文摘要 II
目录 III
1. 绪论 1
 1.1  盘基网柄菌的简介 1
 1.2  人Fas配体简介 3
 1.3  盘基网柄菌作为载体 4
 1.4  盘基网柄菌的培养 5
 1.5  盘基网柄菌的固定化 6
2  实验部分 9
 2.1  菌株 9
 2.2  培养基 9
 2.3  固定化材料 9
 2.4  分析方法 10
3  结果与讨论 13
 3.1  AX3-pFH用毛巾作为固定化材料的生长 13
 3.2  AX3-pFH用毛巾内填充聚氨酯泡沫作为固定化材料的生长 14
 3.3  毛巾吸水实验 14
 3.4  洗脱方法的选择 15
 3.5  确定最优的毛巾重量 16
 3.6  同浓度悬浮培养液不同质量毛巾载体的吸附能力 17
4  总结与展望 18
致谢 19
参考文献 20

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