重组免疫毒素IL-6_(T23)-PE38KDEL在大肠杆菌中自诱导表达条件的优化  

Optimization of condition for auto-inducing expression of recombinant immunotoxin IL6_(T23)-PE38KDEL in E. coli

作　　者：丛楠[1] 卢士英[1] 任洪林[1] 李岩松[1] 翟新新[1] 柳增善[1] 

机构地区：[1]吉林大学动物医学学院人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《中国生物制品学杂志》2013年第5期717-722,共6页Chinese Journal of Biologicals

摘　　要：目的优化重组免疫毒素IL-6(T23)-PE38KDEL在大肠杆菌中的自诱导表达条件,以提高菌体浓度及可溶性目的蛋白的表达量。方法采用摇瓶培养,利用单因素和正交试验对工程菌自诱导的培养条件(接种量、诱导时间、诱导温度、装瓶量)和培养基组分(有机氮源、无机氮源、碳源、有机酸)进行优化,检测菌体浓度、可溶性目的蛋白的表达量及质粒稳定性。结果确定最适自诱导培养条件为装瓶量20 ml/250 ml、接种量2%,28℃诱导25 h;最适自诱导培养基组分为:蛋白胨2%、酵母浸粉0.5%、Na2HPO425 mmol/L、KH2PO425 mmol/L、硫酸铵25 mmol/L、葡萄糖0.05%、甘露醇1%、乳糖0.6%、苹果酸钠20 mmol/L、MgSO40.5 mmol/L。此时可溶性目的蛋白的表达量和菌体浓度分别为LB培养基(IPTG诱导)的1.76和2.12倍,质粒稳定性由87%提高至98%。结论优化了重组免疫毒素IL-6(T23)-PE38KDEL在大肠杆菌中的自诱导表达条件,为其工业化生产奠定了基础,也为自诱导在其他重组蛋白药物生产中的应用提供了参考。Objective To optimize the condition for auto-inducing expression of recombinant immunotoxin IL6（T23）PE38KDEL in E.coli so as to increase bacterial concentration and expression level of soluble target protein.Methods Recombinant E.coli was cultured in shake flask,of which the culture condition（inoculum amount,time and temperature for induction,filling volume）and medium composition（organic nitrogen source,inorganic nitrogen source,carbon source and organic acid） were optimized by single factor and orthogonal tests,and bacterial concentration,expression level of soluble target protein and stability of plasmid were determined.Results The optimal filling volume,inoculum amount, temperature and time for auto-induction were 20 ml/250 ml,2%,28℃ and 25 h respectively.The optimal medium consisted of 2% tryptone,0.5% yeast extract powder,25 mmol/L disodium hydrogen phosphate,25 mmol/L potassium dihydrogen phosphate,25 mmol/L ammonium sulfate,0.05% glucose,1% mannitol,0.6% lactose,20 mmol/L sodium malate and 0.5 mmol/L magnesium sulfate.Under the optimal condition,the expression level of soluble target protein and bacterial concentration were 1.76 and 2.12 times of of those in LB medium under induction of IPTG respectively.The plasmid stability increased from 87% to 98%.Conclusion The condition for auto-inducing expression of recombinant immune toxin IL6（T23）-PE38KDEL in E.coli was optimized,which laid a foundation of industrial production of the immunotoxin,and provided a reference for application of auto-induction in production of other recombinant protein drugs.

关 键 词：免疫毒素 大肠杆菌 自诱导 表达 优化 

分 类 号：Q786[生物学—分子生物学]