Genetic detoxification of pertussis toxin S1 subunit
10.13200/j.cnki.cjb.004424
- VernacularTitle:百日咳毒素S1亚基的基因脱毒
- Author:
LIU Hongbo
- Publication Type:Journal Article
- Keywords:
Bordetella pertussis CS strain;
Genetic detoxification;
Pertussis toxin(PT);
1B7;
CHO cell agglutination
- From:
Chinese Journal of Biologicals
2025;38(02):143-148+154
- CountryChina
- Language:Chinese
-
Abstract:
Objective To edit the gene of pertussis toxin(PT)-S1 subunit in order to complete the genetic detoxification of Bordetella pertussis CS strain.Methods Electroporation competent cells of Bordetella pertussis CS strain were prepared and electro-transfected with the linear recombinant DNA fragments carrying Kana resistance genes, which was then exchanged with the PT-S1 subunit through recombination of the homologous arms on both sides. The recombinant strains were screened with Kana antibiotics, and the recombinant sequence was determined by gene sequencing. The PT protein was obtained from the culture supernatant of the recombinant strains for ELISA, Western blot and in vitro toxicity analysis.Results S1-R9K/E129G double mutant strain FE3 and S1-R9K single mutant strain FE16 were obtained by genome and mRNA sequencing.The growth rate of FE3 and FE16 decreased slightly, but the maximum bacterial concentration increased. The content of PT protein in shake flask culture supernatant of strain FE3 and FE16 was 796. 8 and 185. 9 ng/mL, respectively, and the PT proteins could be recognized by S1 subunit monoclonal antibody 1B7. The in vitro toxicity of PT proteins from strain FE3 and FE16 decreased to 0. 001 6% and 0. 008 1%, respectively, compared with that of PT standard.Conclusion Gene editing can be performed on Bordetella pertussis CS strain using electroporation of linear DNA fragments and Kana antibiotic screening, and result in genetically detoxified pertussis strains with PT protein of significantly reduced toxicity, which lays a foundation for further research on pertussis vaccines.
