文档介绍：人表皮生长因子hEGF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: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: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(%,乳糖2%,%,%,氯化纳1%) %。同时, 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) g/L, %。
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
High-level expression of bination human EGF in E. coli
Wang wen jie
School of Life Sciences,Guangzhou University
ABSTRACT Objective: In order to improve the yield of hEGF, which was expessed in binant 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 bined 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 % at the optimized formula of the culture medium (glycerin %, lactose 2 %, peptone %, yeast extract %, NaCl 1%). Meanwhile, the result showed the optimized concentration of IPTG is mmol/L and the optimized induction time is 4 h. Finally, wet cell weight of binant i