Optimized expression of the diphtheria toxin mutant CRM197 in Escherichia coli and population analysis of serum antibody levels
10.3969/j.issn.1002-2694.2024.00.073
- VernacularTitle: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
- Author:
Xiao-Li CHEN
1
;
Yi-Xin GU
;
Hai-Rui WANG
;
Gui-Lan ZHOU
;
Xin ZHANG
;
Chang LIU
;
Jian-Zhong ZHANG
;
Zhu-Jun SHAO
;
Mao-Jun ZHANG
Author Information
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
- Keywords:
CRM197;
soluble expression;
antibody concentrations in serum
- From:
Chinese Journal of Zoonoses
2024;40(5):430-434
- CountryChina
- Language:Chinese
-
Abstract:
A prokaryotic expression vector for the mutant diphtheria toxin CRM197 was constructed and expressed in Esch-erichia coli cells.Anti-CRM197 antibody concentrations were detected in serum samples of healthy volunteers.The crm 197 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 puri-fied 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.Collec-tively,these results confirmed that the proposed expression strategy achieved high-yield production of soluble CRM197,al-though high levels in human serum may affect evaluation of immune interactions with glycan-CRM197 conjugates for applica-tion as a diagnostic antigen.The diphtheria mutant toxin CRM197 is used in many conjugate vaccines.The synthetic crm 197 gene with codon optimization in pET28a was transformed into E.coli Origami B(DE3)cells.CRM197 was induced by isopro-pyl 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 prepara-tion reached 95％.CRM197 was used to detect the concentra-tions 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.
