白喉毒素突变体CRM197在白喉杆菌中的表达纯化.pdfVIP

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN 年第 期 2014 3 白喉毒素突变体 CRM197 在 白喉杆菌中的表达纯化 朱涛  邓立功  段磊  常云松  邵忠琦  毛慧华  宇学峰 （天津科技大学，天津 300457） 摘 要 ： CRM197 是一种白喉毒素突变体，第52 位的甘氨酸突变为谷氨酸，作为载体蛋白广泛用于疫苗开发。将阐述一种 新的生产CRM197 方法。将合成的CRM197 基因片段克隆到表达载体pCKM4.1 中，表达质粒pCKM5.1 电转化至大肠杆菌E. coli S17-1 中，通过结合转移转化至白喉杆菌 （ATCC® 270 10TM 的白喉杆菌）中，载体上的导肽序列可以使得CRM197 作为可溶性蛋白 分泌到胞外表达，CRM197 蛋白可占到菌体总蛋白的70%。增强对铁的调控，进一步优化培养基及发酵条件以提高产量。经过Q 膜、 硫酸铵沉淀、阴离子交换纯化步骤获得纯度达到95% 的CRM197 样品，提高了蛋白得率，节约了纯化时间和成本。 关键词 ： CRM197 白喉杆菌 接合转移 可溶蛋白 铁调控 阴离子柱 Expression and Purification of the Diphtheria Toxin Variant CRM197 in Corynebacterium diphtheriae Zhu Tao Deng Ligong Duan Lei Chang Yunsong Shao Zhongqi Mao Huihua Yu Xuefeng （Tianj in University of Science and Technology ，Tianj in 300457 ） Abstract:  The CRM197 protein is a variant of the diphtheria toxin （DTx, 58 kD ）characterised by a single mutation （i.e. a glycine- glutamic acid substitution in position 52 ）that reduces its toxicity. The protein nonetheless retains the same inflammatory and immunostimulant properties as thediphtheria toxin, and it is widely used as a safe carrier in conjugatedvaccines. A new and alternative procedure for the production of full-length CRM197 in Corynebacterium diphtheriae was proposed. The gene of CRM197 was synthesized and then expressed in Corynebacterium diphtheriae. Expression vector pCKM5.1 was first electrotransformed into Escherichia coli-s-17, reisolated, and subsequently transferred into the C. diphtheri