白喉毒素突变体CRM197在大肠杆菌中表达纯化及性质研究  

Expression,Purification and Characterization of CRM197 in Escherichia coli

作　　者：赵雪娜 董相陈 侯登勇 孙丽霞 王晶翼 王庆民 

机构地区：[1]齐鲁制药有限公司药物研究院,济南250100

出　　处：《生物技术通报》2010年第1期153-156,共4页Biotechnology Bulletin

摘　　要：CRM197是一种白喉毒素突变体,作为载体蛋白广泛用于疫苗开发。将合成的CRM197基因片段克隆到表达载体pET25b中,转化大肠杆菌BL21(DE3),经IPTG诱导,CRM197获得高效表达,达到菌体总蛋白的20%。目的蛋白主要以包涵体形式存在,变性、复性后经DEAE阴离子交换、S-100分子筛纯化获得纯度高于95%的CRM197样品,用该蛋白样品免疫新西兰大白兔,免疫兔血清中检测到特异性抗体应答。急性毒性试验中,每只豚鼠皮下注射200μgCRM197纯化样品,未出现明显毒性反应症状,与之相比较,注射后48h内,20ng白喉毒素阳性对照组动物全部死亡。结果表明,利用本试验的表达策略,CRM197得到高效表达,并且具有良好的免疫原性和安全性,为其进一步生产及应用奠定基础。CRM197（cross reacting material 197） ,as diphtheria toxin mutant,is being used as vaccine carrier more and more commonly. In this research ,the gene of CRM197 was synthesized and then expressed in Escherichia coli BL21 （ DE3 ） driven by T7 promoter. The recombinant protein was over expressed mainly as inclusion body and accounted for around 20% of the total cell proteins. After denaturation and renaturation,CRM197 was purified by DEAE anion-exchange column and S-100 molecular sieve. The purified protein was used to immunize New Zealand white rabbits ,and specific antibody response against CRM197 was observed. In acute toxicity experiment 200 μg purified CRM197 was subcutaneously injected into guinea pigs. The recombinant CRM197 was still safe to test animals even the injected dose was 10000 times of diphtheria toxin. These results demonstrated that CRM197 had been expressed successfully in E. coli at high level,and also possessed excellent immunogenicity and high safety. Given these characteristics, the CRM197 expressed in E. coli is suitable for further research.

关 键 词：CRM197 白喉毒素 包涵体免疫原性 急性毒性 

分 类 号：Q78[生物学—分子生物学]