白喉毒素突变体CRM197在大肠杆菌中的高效可溶表达、纯化及免疫原性分析  

Expression, purification and characterization of diphtheria toxin mutant CRM197 in Eschrichia coli

作　　者：房婷[1] 陶正红 刘艳红 于长明[1] 职睿智 于蕊[1] Ting Fang;Zhenghong Tao;Yanhong Liu;Changming Yu;Ruizhi Zhi;Rui Yu(Institute of Biotechnology, Academy of Military Medical Sciences, Beij＇ing 100071, China;Department of Biology and Medicine, Beijing City University, Beo＇ing 100083, China)

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]北京城市学院生物医药学部,北京100083

出　　处：《生物工程学报》2018年第4期561-568,共8页Chinese Journal of Biotechnology

基　　金：国家科技重大专项--重大新药创制(No.2013ZX10004001);北京城市学院2016年度实培计划项目资助~~

摘　　要：CRM197(Cross-reacting material 197)是白喉毒素的无毒突变体,在生物制药领域具有非常广泛的应用前景。为了实现CRM197在大肠杆菌中的无标签、可溶表达,以替代现有昂贵、复杂的白喉杆菌分泌发酵工艺,文中对目的蛋白的序列进行优化,转化大肠杆菌经IPTG诱导,目的蛋白获得了可溶性高表达,目的蛋白约占碎菌上清总蛋白的40%。超声破菌后、经阴离子交换、肝素亲和以及脱盐三步柱上层析获得纯度高于95%的CRM197样品。细胞毒性实验证明,CRM197的IC_(50)值是白喉毒素IC_(50)值的2.1×10~7倍,是白喉类毒素IC_(50)值的9.6倍,表明文中表达的CRM197是安全无毒的。随后,比较了高、低剂量的CRM197在小鼠体内的免疫原性,发现2μg组三免后的抗体滴度与20μg组的相当,可达1︰409 600。文中建立了CRM197的无标签、高效、可溶表达的大肠杆菌表达系和快速纯化体系,获得了具有较好免疫原性和安全性的重组蛋白,为该蛋白的进一步应用奠定了基础。CRM197（cross-reacting material 197）,a non-toxic mutant of diphtheria toxin,has wide application potential in biopharmaceuticals.However,it is difficult to express CRM197 in bacteria other than Corynebacterium diphtheriae.Here we proposed a new alternative method to produce soluble CRM197 without label in Escherichia coli.In particular,a synthetic gene coding for CRM197,optimized for E.coli codon usage,was cloned in the p ET32a（＋）vector.Accordingly,the over-expression of the protein was simply induced with IPTG in E.coli BL21（DE3）.The target protein was soluble and accounted for about 40%of the total protein in the supernatant.Following an ultrasonic cytolysis step,the recombinant protein was purified by anion exchange,affinity and desalting chromatography and the purity of the final preparation reached 95%.Cytotoxicity tests showed that the IC_（50） value of CRM197 was 2.1×10~7 times the IC_（50） value of diphtheria toxin,and 9.6 times the IC_（50） value of diphtheria toxoid,telling that the target protein is safe and non-toxic.Subsequently,we found that both the high dose（20μg）and the low dose（2μg）of CRM197 were equally efficient in inducing an immune response against diphtheria toxiod in mice,and the antibodies titer of mice after three immunizations with low dose could reach 1：409 600.In conclusion,our findings provide a highly efficient strategy for the rapid production and purification of unlabeled and soluble recombinant CRM197 in E.coli,with good immunogenicity and safety.

关 键 词：白喉毒素突变体 无标签可溶性表达 纯化 毒性试验 免疫原性 

分 类 号：R392[医药卫生—免疫学]