Immunogenicity and expression of a soluble CRM197 mutant of diphtheria toxin in Escherichia coli
10.3969/j.issn.1002-2694.2025.00.118
- VernacularTitle:白喉毒素突变体CRM197重组蛋白在大肠杆菌中可溶性表达及免疫原性分析
- Author:
Zeyue ZHANG
1
;
Biao MA
;
Jiali LI
;
Mengqi XU
;
Mingzhou ZHANG
Author Information
1. 中国计量大学生命科学学院,国家市场监督管理总局重点实验室,微生物计量检测与生物制品质量安全,杭州 310018
- Publication Type:Journal Article
- Keywords:
CRM197 protein;
soluble protein;
immunogenicity;
purification;
Escherichia coli;
conjugate vaccine
- From:
Chinese Journal of Zoonoses
2025;41(8):816-823
- CountryChina
- Language:Chinese
-
Abstract:
This study investigated the expression of the soluble nontoxic mutant CRM197 of diphtheria toxin,to prepare a safe and effective recombinant CRM197 protein.Codon-optimized CRM197 gene sequences were cloned into the pCold Ⅱ and pET-28a(+)prokaryotic expression vectors.The successfully cloned recombinant plasmids were screened and transformed into Escherichia coli BL21(DE3)competent cells.After induction of expression,the protein expression type was determined through SDS-PAGE analysis.The conditions for expression of the solublerecombinant protein were then optimized.The recombinant CRM197 protein was purified through two rounds of Ni-NTA affinity chromatography.The purified protein was used to immunize mice,and antibody levels in the se-rum were measured with ELISA.The codon adaptation index(CAI)of the optimized sequence increased from 0.72 to 0.93.The recom-binant plasmids pET-28a(+)-CRM197 and pCold Ⅱ-CRM197 were successfully constructed,as confirmed through colony PCR and double digestion.Expression analysis revealed that pET-28a(+)-CRM197 was expressed primarily as inclusion bodies,whereas pColdⅡ-CRM197 was expressed predominantly in soluble form.The conditions for soluble protein expression via pCold Ⅱ-CRM197 were optimized.When the inoculum was 3%and the IPTG concentration was 0.4 mmol/L,induction at 20 ℃ for 24 h significantly increased the expression of the soluble target protein.The pCold Ⅱ-CRM197 recombinant protein was purified from the supernatant with Ni-NTA affinity chromatography,thus resulting in a target protein with a purity greater than 98%.ELISA after three rounds of immuniza-tion indicated that the levels of IgG,IgM,IgG1,and IgG2a antibodies in the serum in immunized mice were significantly higher than those in the control group.In summary,the CRM197 recombinant protein was successfully prepared with the pCold Ⅱvector and exhib-ited high soluble expression,high purity,and favorable immunogenicity.
