摘要
目的:构建分泌型重组人生长激素(recombinant human growth hormone,rhGH)工程菌,优化工程菌表达条件.方法:应用T7启动子与ompA3引导序列构建分泌型表达rhGH工程菌,采用多因素正交优选法初步筛选培养基成分,进而考察葡萄糖对rhGH表达量的影响.在此培养基基础上优化诱导表达条件,分别考察了诱导起始菌密度、诱导温度、诱导剂浓度和诱导表达时间对rhGH表达量的影响.结果与结论:构建了高效分泌型表达rhGH工程菌,在优化培养基中摇瓶培养至D600值约为9.2,37℃进行诱导,IPTG浓度0.5 mmol/L,诱导后继续培养10～12 h,rhGH分泌表达量可达到 140～160 mg/L培养液,目的蛋白占周质总蛋白的30%～40%.
Objective: To construct engineering bacteria expressing secretion type recombinant human growth hormone( rhGH) and optimize expression conditions for the bacteria. Methods: The engineering bacteria expressing rhGH in secretion type was constructed by using T7 promoter and ompA 3 leader sequence. The effect of phosphate, yeast extract and lactose on expression level of rhGH was determined by orthogonal test, and the influence of different starting glucose concentrations on rhGH expression level was also studied to optimize culture medium compositions. Induction stage, induction temperature, IPTG concentration and expression time were studied to optimize secretory expression of rhGH in engineering bacteria. Results and Conclusion: The engineering bacteria secreting high-level expression of rhGH was constructed, and the culture medium compositions were optimized. When the bacteria were cultured in optimized medium, under the conditions of induction starting about 9.2 of D 600 value, induction temperature at 37℃, induction concentration of IPTG at 0.5 mmol/L and culture continuing for 10-12 h after induction, the maximum secretion level of 140-160 mg rhGH in 1L culture solution was obtained in shaking flasks, and the object protein acounted for about 30%-40% of the periplasmic total proteins.
出处
《军事医学科学院院刊》
CSCD
北大核心
2005年第1期55-59,共5页
Bulletin of the Academy of Military Medical Sciences
关键词
重组人生长激素
周质
分泌型表达
表达量
rhGH
periplasmic space
secretory expression
expression level
