白喉毒素突变体CRM197在大肠杆菌中优化表达及人群血清抗体水平分析  

Optimized expression of the diphtheria toxin mutant CRM197 in Escherichia coli and population analysis of serum antibody levels

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作  者:陈晓丽 顾一心[1] 王海瑞 周贵兰 张鑫 刘畅 张建中[1] 邵祝军[1] 张茂俊[1] CHEN Xiao-li;GU Yi-xin;WANG Hai-rui;ZHOU Gui-lan;ZHANG Xin;LIU Chang;ZHANG Jian-zhong;SHAO Zhu-jun;ZHANG Mao-jun(National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区:[1]传染病溯源预警与智能决策全国重点实验室,中国疾病预防控制中心传染病预防控制所,北京102206

出  处:《中国人兽共患病学报》2024年第5期430-434,共5页Chinese Journal of Zoonoses

基  金:国家重点研发计划(No.2021YFC2301000);深圳三名项目(No.SZSM201803081);中国疾病预防控制中心横向课题(No.2023110024000074)。

摘  要:目的 构建白喉毒素突变体CRM197的原核表达载体,在大肠杆菌中表达CRM197蛋白。分析健康人群血清中的CRM197抗体水平。方法 对CRM197基因进行大肠杆菌密码子优化并克隆至pET28a(+)中,经表达条件优化后采用镍柱亲和层析和离子交换层析对重组蛋白CRM197进行纯化,对纯化的CRM197进行Western blot鉴定。最后,经纯化后的CRM197蛋白与健康人群血清进行反应,检测人群血清中针对该蛋白的抗体水平。结果 CRM197蛋白进行了密码子及表达条件优化后,获得了可溶性表达蛋白。经镍柱亲和层析和离子交换层析后其纯度可达95%以上,Western Blot鉴定后能得到单一的特异性条带。经纯化的CRM197蛋白与不同年龄阶段健康人群血清均发生不同程度的抗原抗体反应。结论 利用本研究的策略,CRM197得到高效的可溶性表达,但人群血清中含有高水平的CRM197抗体,这些抗体的存在可能会影响到应用CRM197结合多糖作为抗原进行特异免疫效果的评价以及作为诊断抗原的应用。A prokaryotic expression vector for the mutant diphtheria toxin CRM197 was constructed and expressed in Escherichia coli cells.Anti-CRM197 antibody concentrations were detected in serum samples of healthy volunteers.The crm197 gene was codon-optimized in E.coli and cloned into the plasmid pET28a(+) under optimized expression conditions.CRM197 was purified using Ni-NTA spin columns and ion exchange chromatography,and confirmed by western blot analysis.The purified CRM197 was used to detect specific anti-CRM197 antibody levels in serum samples of different age groups.The results showed that soluble codon-optimized CRM197 was successfully expressed under optimized expression conditions.The purity of CRM197 was more than 95%,as determined with Ni-NTA spin columns and ion exchange chromatography,consistent with the single specific bands obtained by western blot analysis and detection of serum levels of the anti-CRM197 antibody.Collectively,these results confirmed that the proposed expression strategy achieved high-yield production of soluble CRM197,although high levels in human serum may affect evaluation of immune interactions with glycan-CRM197 conjugates for application as a diagnostic antigen.The diphtheria mutant toxin CRM197 is used in many conjugate vaccines.The synthetic crm197 gene with codon optimization in pET28a was transformed into E.coli Origami B(DE3) cells.CRM197 was induced by isopropyl β-d-1-thiogalactopyranoside and high level accumulation of soluble CRM197 was purified using Ni-NTA spin columns and ion exchange chromatography.The purity of the final preparation reached 95%.CRM197 was used to detect the concentrations of the anti-CRM197 antibody in serum samples of healthy volunteers of different ages.The proposed expression strategy yielded high production of CRM197,which could interfere with evaluations of induced immune interactions by glycan-CRM197 conjugates and prohibit application as a diagnostic antigen.

关 键 词:CRM197 可溶性表达 人群血清抗体水平 

分 类 号:R378.2[医药卫生—病原生物学]

 

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