肝靶向干扰素(IFN-CSP)融合基因原核表达条件的优化  

Optimization of Expression Conditions of an Induction Strategy for Improving Liver Targeted Interferon(IFN-CSP)Production in E.coli

作　　者：黄演婷[1,2] 卢雪梅[1,2] 汪洁 金小宝[1,2] 朱家勇 

机构地区：[1]广东药学院药用生物活性物质研究所 [2]广东省生物活性药物研究重点实验室,广州510006

出　　处：《生物医学工程学杂志》2014年第2期432-438,共7页Journal of Biomedical Engineering

基　　金：国家科技部新药创制重大专项资助项目(2013ZX09103003-003)

摘　　要：本研究目的是优化肝靶向干扰素(IFN-CSP)在大肠杆菌中的诱导表达条件,以提高蛋白表达量。通过考查培养基pH值、诱导剂IPTG浓度、诱导温度、诱导时间和起始菌浓度等五个因素对IFN-CSP融合基因原核表达量的影响,在此基础上,从上述影响因素中各选取4个水平进行正交实验作进一步分析。结果表明,起始菌浓度和诱导温度为影响表达的主要因素,最佳组合诱导条件为OD600为0.8时开始诱导、诱导4h、诱导温度37℃、诱导剂IPTG浓度0.4mmol/L、培养基pH值为6.0,融合蛋白的表达量高达74.3%。通过表达优化后,IFN-CSP的产量为688.8mg/L,提高了1.2倍。这为进一步制定生产工艺和评价IFN-CSP的生物活性奠定基础。Expression conditions of induction strategies for the cytoplasmic inclusion bodies （IBs） production of liver targeted interferon IFN-CSP by recombinant Escherichia coli （E. coli） BL21（DE3） were optimized in shake-flask cultures in this study. The factors of the optimized protocol included in the present study were pH, inducer IPTG （i- sopropyl β-D-thiogalactoside） concentration, culture growth temperature, incubation time and induction point. The effects of those factors were investigated by ＇ single variable at a time＇ method, aimed to analyze characterization of the recombinant strain. Orthogonal experimental design was further used to optimize the above critical factors for IFN-CSP production. According to the expression optimization result, it was confirmed that the main influence fac- tors were cell density and induction temperature. The IFN-CSP gene expression optimized conditions were： pH value of the culture medium was 6.0, culture temperature 37℃, adding IPTG to final concentration 0.4 mmol/L when the recombinant strain growth density OD600 achieved 0.8 and induction time 4 h. At this point, the IBs represented 74.3 % of the total cellular protein. Compared with the non-optimized condition, IFN-CSP production obtained in op- timized induction strategies were increased by approx. 1.2-fold. The optimized induction strategy yielded 688.8 mg/ L of IFN-CSP, providing experimental data to study the biology activity and productive technology of IFN-CSP.

关 键 词：肝靶向干扰素 大肠杆菌 优化表达 正交设计实验 

分 类 号：Q786[生物学—分子生物学]