王文杰_hEGF毕业论文(二改).doc

第 1 页 共 23 页人表皮生长因子人表皮生长因子 hEGF 在大肠杆菌中的高效表达工艺研究在大肠杆菌中的高效表达工艺研究王文杰广州大学生命科学学院摘要摘要 目的：目的：为了提高 hEGF（human epidermal growth factor）的产量，本次实验主要从培养基优化，IPTG 诱导浓度和诱导时间的摸索等方面来提高重组大肠杆菌hEGF 表达量，并进行了 5 L 发酵罐的小试发酵研究方法：方法：采用摇瓶培养方式，先通过单因子实验从甘油，乳糖，葡萄糖三种碳源中选出两种效果显著的碳源作为混合碳源从玉米浆，蛋白胨，酵母膏三种氮源中选出两种效果显著的碳源作为混合氮源通过“四因素三水平”正交实验确定最佳的培养基配方并在此优化的培养基基础上进行 IPTG 浓度和诱导时间摸索结果：结果：单因子实验确定了乳糖和甘油作为有效的混合碳源，蛋白胨和酵母膏作为有效混合氮源，“四因素三水平”正交实验确定最佳的培养基配方（甘油 1.5%，乳糖 2%，蛋白胨 0.5%，酵母膏 1.5%，氯化纳 1%）时表达量达到 14.8 %同时，最佳的 IPTG 浓度为 1.0 µm/ml 和最佳诱导时间为 4h。

5 L 发酵罐的小试发酵放罐时菌重（湿重）为 42.3 g/L，表达量为 15.7 %关键词关键词 人表皮生长因子（hEGF）；大肠杆菌表达系统；培养基优化；发酵第 2 页 共 23 页High-level expression of recombination human EGF in E. coliWang wen jieSchool of Life Sciences，Guangzhou UniversityABSTRACT Objective: In order to improve the yield of hEGF, which was expessed in recombinant Escherichia coli. Studies include optimization of culture medium, searching for optimization concentration of IPTG and induction time, were carried out. Also, a small-scale fermentation was carried out in 5 L fermentor. Methods: To study the optimal cultivation condition, single factor and orthogonal experiment had been combined to research by shake-flask cultivate. Two kinds of carbon source and nitrogen source was selected from lactose, glycerol, glucose, corn steep liquor, peptone and yeast extract by single factor screen experiment as mixed carbon and nitrogen source. Then, the optimized formula of the culture medium was confirmed through the 4-factors and 3-levels orthogonal experiment. Based on this optimization of culture medium, the optimized concentration of IPTG and induction time was investigated. Results: Lactose and glycerol were selected as mixed carbon source, peptone and yeast extract were selected as mixed nitrogen source. The expression of hEGF reached 14.8 % at the optimized formula of the culture medium (glycerin 1.5 %, lactose 2 %, peptone 0.5 %, yeast extract 1.5 %, NaCl 1%). Meanwhile, the result showed the optimized concentration of IPTG is 1.0 mmol/L and the optimized induction time is 4 h. Finally, wet cell weight of recombinant E.coli in 5 L fermentor was high as 42.3 g/L, and the expression of hEGF reached 15.7 %.KEY WORDS hEGF; E. coli expression system; Medium optimization；Fermentation第 3 页 共 23 页目录1 前 言.................................................................................................................................42 材料与方法.......................................................................................................................52.1 实验材料.....................................................................................................................52.1.1 实验仪器...............................................................................................................52..1..2 实验试剂............................................................................................................62..1..3 培养基及试剂配制.............................................................................................62.2 实验方法.....................................................................................................................82.2.1 高表达菌株的筛选...............................................................................................82.2.2 培养基筛选..........................................................................................................92.2.3 诱导物 IPTG 浓度对 hEGF 表达的影响 .........................................................112.2.4 诱导时间对 hEGF 表达的影响........................................................................112.2.5 5L 发酵罐小试 ...................................................................................................113 结果与分析.....................................................................................................................123.1 高表达菌株的筛选...................................................................................................123.2 培养基筛选...............................................................................................................133.2.1 碳源筛选............................................................................................................133.2.2 氮源筛选............................................................................................................143.2.3 正交实验............................................................................................................143.3 诱导物 IPTG 浓度对HEGF 表达的影响.................................................................173.4 诱导时间...................................................................................................................173.5 5 L 发酵罐小试 .........................................................................................................184 讨论.................................................................................................................................205 参考文献.........................................................................................................................22致谢..........。