作者：黄杰, 李松, 孙俊, 赖艺, 何刚, 魏鑫, 侯文礼

作者单位：成都康华生物制品股份有限公司研发中心, 四川 成都 611130

关键词：白喉毒素突变体;蛋白纯化;结合疫苗;鉴定

摘要：

为了制备高纯度的白喉毒素无毒突变体CRM197，可作为载体蛋白用于细菌性结合疫苗开发，对CRM197基因进行大肠杆菌密码子优化，并克隆至表达载体pET28a(+）中，将鉴定正确的重组质粒pET28a-CRM197转化到大肠杆菌BL21(DE3)，经IPTG诱导表达，并分析其表达形式及表达条件的优化。再对表达的重组CRM197蛋白进行纯化，最后对纯化的CRM197进行WB、纯度、分子量和圆二色谱等检测项目的初步鉴定分析。PCR酶切和测序鉴定结果表明重组质粒pET28a-CRM197构建成功，将其转化到大肠杆菌BL21(DE3)中，获得重组工程菌(E.coli(DE3/p28a/197)。该重组工程菌发酵的最佳接种量为5%~10%(v/v)，且主要以包涵体形式表达，收获的菌体在高压均质机破碎压力为1000 bar的条件下进行破碎，然后利用6 mol/L盐酸胍进行溶解变性，复性及经30 kD超滤膜包超滤后上纯化系统AKTA pure150 M，经一步阴离子交换层析进行纯化，其纯度可达98%以上，WB、分子量及圆二色谱等鉴定结果均与标准品一致。综上所述，成功建立大肠菌表达系统CRM197制备方法，具有无标签、产量高及纯度高等特点，可用于该蛋白的规模化生产。

Production and identification of high purity diphtheria toxin mutant CRM197
HUANG Jie, LI Song, SUN Jun, LAI Yi, HE Gang, WEI Xin, HOU Wenli
Research and Development Center, Chengdu Kanghua Biological Products Co., Ltd., Chengdu 611130, China
Abstract: To product highly purified diphtheria toxin mutant cross reacting material 197(CRM197), it can be used as a carrier protein for the development of bacterial conjugate vaccines. In this current work, the CRM197 gene was codon-optimized in Escherichia coli and cloned into pET28a(+), and the correctly identified recombinant plasmid pET28a-CRM197 was transformed into Escherichia coli BL21(DE3). The recombinant plasmid was induced by IPTG, and its expression form was analyzed and optimized expression conditions. Then the expressed recombinant CRM197 protein was purified, and finally purified CRM197 was analyzed by western blotting WB, purity, molecular weight and circular dichroism(CD). The results showed that the recombinant plasmid pET28a-CRM197 was successfully constructed by PCR digestion and sequencing, and it was transformed into Escherichia coli BL21 (DE3) to obtain recombinant engineering bacteria E.coli (DE3/p28a/197). The optimal inoculum amount for fermentation was 5%-10% (v/v), and it was mainly expressed in a form of inclusion body. The harvested cells were crushed under the condition of crushing pressure of 1000 bar, and then dissolved with 6mol/L guanidine hydrochloride, refolded and ultrafiltered by centrifugal filters (30 kD), and then purified by AKTA pure150 M system, and purified by one-step anion exchange chromatography. The purity of CRM197 was more than 98%, and the identification results of WB, molecular weight and circular dichroism are all consistent with the standard product. The results indicated that CRM197 expression system was established, which has no label, high yield and purity, and can be used for the large-scale production of the protein.
Keywords: diphtheria toxin variant;protein purification;conjugate vaccine;identification
2023, 49(11):176-183  收稿日期: 2023-04-10;收到修改稿日期: 2023-05-26
基金项目: 四川省科技创新人才项目(2022JDRC0048)
作者简介: 黄杰(1987-),男,四川遂宁市人,助理研究员,硕士,主要从事疫苗研发工作。
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