【作者】 欧俊杰；

【导师】 耿信笃；

【作者基本信息】 西北大学 ， 分析化学， 2003， 硕士

【摘要】 本文包括六个章节，主要对重组人干细胞因子（recombinant human stem cell factor，rhSCF）的生产工艺进行研究，包括重组大肠杆菌的培养基、摇瓶培养条件和发酵罐间歇培养的发酵生产，以及目标蛋白的分离纯化。 1．发酵培养基的研究 通过摇瓶发酵对用大肠杆菌（E.coli）DH5α/pBV220生产rhSCF的发酵培养基进行了研究。首先从几种常用的培养基中筛选出M9培养基是最适合工程菌生长和rhSCF表达的培养基，然后又采用单因子实验方法对M9培养基中的碳源和氮源进行了逐个筛选，结果表明，最适合工程菌DH5α/pBV220生长的碳源为葡萄糖，氮源为蛋白胨和酵母粉，最后设计正交试验，对培养基配方进行了优化，确定出较适合的培养基，同时在培养基中添加了Mg²⁺以加速菌体的生长。 2．摇瓶发酵工艺的研究 系统地考察了培养温度、诱导时机、诱导维持时间对菌体生长和rhSCF表达的影响，也对发酵液初始pH值、溶氧、种子菌龄、接种量等工艺条件进行了研究，选择出最佳的摇瓶培养条件。结果显示，大肠杆菌适宜在30℃进行培养，发酵液初始pH值为7.2-7.4，在300mL的摇瓶中装入50mL培养液，摇床转速控制在180r/min左右，在对数生长前期升温至42℃诱导表达3h，然后转入35℃再培养，可获得较高的菌体产量和rhSCF的表达量。此时rhSCF表达量为24.6％，rhSCF产量是0.276g/L。另外，在优化的条件下对整个摇瓶培养过程的深层发酵特性进行研究，表明摇瓶中溶氧不足是限制菌体生长和rhSCF表达的主要原因。 3．在5L发酵罐上的间歇培养 以摇瓶发酵的研究结果为基础，在5L发酵罐中，用大肠杆菌DH5α/pBV220生产rhSCF的间歇培养工艺条件进行了研究，确定出最优培养条件为：通过调节通气量和搅拌速度控制溶氧大于50％，采用10％-15％的接种量，在OD₆₀₀达2.0时诱导可获得最高的thSCF产量，达到0.6269几，是摇瓶中产量的2.3倍。也进行了整个间歇培养过程的深层发酵特性研究，发现在培养结束时培养液中仍含有丰富的营养物质，因此可以推测如果采用分批补料培养可以进一步提高rhSCF产量。4. rhSCF的分离纯化研究 用不同的提取液对rhSCF包含体进行粗提，然后用高效疏水作用色谱和高效离子交换色谱分离纯化rhSCF。经过高效疏水作用色谱一步纯化，:hSCF的纯度可达95%，其质量回收率为32.6%。同时用高效离子交换色谱对提取液进行纯化，其纯化效率不高，仍需要对提取工艺、色谱条件等进行优化。更多还原

【Abstract】 The thesis consists of four sections. A production technology of recombinant human stem cell factor (rhSCF) was mainly studied, including the studies of the culture media of recombinant E.coli, culture conditions being in shaking-flask, batch culture in 5-liter-fermenter, separation and purification of the rhSCF. It was summarized as following.1. Investigation of culture mediaThe culture media of fermentation for the recombinant E.coli DH5a/pBV220 to produce the rhSCF were studied with shaking-flask method. Firstly, the M9 medium was selected from several kinds of basic culture media, and adapted to the growth of bacteria and expression of rhSCF. Secondly, With a single factor experiment, the substances of carbon and nitrogen sources were selected. The results showed that glucose is one of the best carbon sources, and tryptone and yeast extract are good in many nitrogen sources. Thirdly, the recipe of optimized culture medium was obtained by orthogonal test. In addition, the growth of bacteria was found to become fast when magnesium(II) was added in culture broth.2. Optimization of the fermentation conditions with shaking-flaskThe effects of culture temperature, time of inducing, expression times on the growth of E.coli and expression of rhSCF were systematically investigated. Also the process conditions, such as initial pH of culture broth, dissolved oxygen, inoculum age, inoculum volume and so on were optimized. The results showed the optimum conditions : temperature 30℃, initial pH 7.2-7.4, the volume of culture broth in shaking-flask 50mL, approximate rotation speed 180r/min. It was suitable for inducing cultivation at 42℃ when the growth of bacteria was in the initial logarithm growthphase and expressing 3 hours, then shifted to 35℃. Under these culture conditions, the expression and yield of rhSCF in shaking-flasks were 24.6% and 0.276g/L, respectively. Additionally, the fermentative characteristics in shaking-flasks under these optimum conditions were studied and indicated that the insufficient of dissolved oxygen was the mainly reason for the restricting growth of bacteria and expression of rhSCF in the whole fermentation process.3. Batch culture with 5-liter-fermenterBased on the results of shaking-flask cultivation, the batch culture conditions of the rhSCF in 5-liter-fermenter were investigated. The optimum conditions were found as following: the level of dissolved oxygen no less than 50% by adjusted inhalant volume and rotation speed, inoculum volume 10-15%, induced at OD600 2.0. Then the yield of 0.626g/L of rhSCF was obtained, which was enhanced 130% more than the yield of 0.276g/L in shaking-flask. The studies on fermentative characteristics of the rhSCF in the whole batch fermentation process showed that nutrients were sufficient in the end of fermentation, so we can expected that the yield of rhSCF may be further added in fed-batch culture.4. Separation and purification of rhSCFThe inclusion bodies of rhSCF were extracted by different solutions, which were directly injected into high performance hydrophobic interaction chromatography (HPHIC) and high performance ion exchange chromatography (HPIEC). With only one HPHIC step, the purity of rhSCF obtained was 95%, and its mass recovery was 32.6%. Additionally, compared to HPHIC, the purity of rhSCF was lower when the extract was separated by HPIEC.更多还原