Optimization of prokaryotic expression conditions of mouse β-defensin 3 and detection of its antibacterial activity
10.13200/j.cnki.cjb.004705
- VernacularTitle:小鼠β防御素3原核表达条件的优化及其抗菌活性检测
- Author:
Qiang CHEN
- Publication Type:Journal Article
- Keywords:
Mouse β-defensin 3(MBD3);
Prokaryotic expression;
Orthogonal design;
Antibacterial activity
- From:
Chinese Journal of Biologicals
2026;39(05):599-605+612
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo optimize the prokaryotic expression conditions of mouse β-defensin 3(MBD3) through orthogonal design, and detect its antibacterial activity, in order to provide a basis for the study of MBD3 function.MethodsThe fusion protein MBD3(fMBD3) recombinant plasmid pET22b(+)/fMBD3 was constructed and transformed into competent E. coli Rosetta-gami 2(DE3), and the effects of induction time(2, 4, 6, 8, 10 h), induction temperature(28, 31, 34, 37, 40 ℃),IPTG final concentration(0. 1, 0. 25, 0. 5, 0. 75, 1. 0 mmol/L), culture medium(LB, SOB, TB, SB, 2 YT), and EDTA final concentration(0, 0. 1, 0. 25, 0. 5, 0. 75 mmol/L) on the expression of fMBD3 were investigated via single-factor experiments.Four factors with significant effects on the expression of fMBD3 were selected, and the appropriate range of each factor was determined to conduct a 4-factor and 3-level orthogonal test(nine groups in total). With the expression levels of fMBD3 as the evaluation indicator, the optimal induction conditions were analyzed using range analysis and variance analysis. Under the optimal induction conditions, fMBD3 was expanded and cultured, and purified by Ni Sepharose~(TM)6 Fast Flow chromatography column. Western blot and mass spectrometry analysis were performed, and the antibacterial activity was assessed using the microdilution method.ResultsSingle-factor experiments determined that the four factors, induction time(4, 6, 8 h), IPTG final concentration(0. 1, 0. 25, 1. 0 mmol/L), induction temperature(31, 37, 40 ℃) and culture medium(SOB, TB, SB), had different degrees of influence on the expression of fMBD3, and EDTA had an inhibitory effect on the expression. Orthogonal test range analysis showed that the influence degree of four factors was as follows: induction time > induction temperature >culture medium > final concentration of IPTG. Variance analysis showed that the induction time had a significant effect on the expression level(F = 5. 945, P < 0. 05). The optimal induction conditions were determined as follows: SB medium, IPTG final concentration of 0. 25 mmol/L, induction at 40 ℃ for 4 h. The expression level of fMBD3 reached(24. 91 ± 1. 68)%after scale-up cultivation, and the purity after purification was more than 90%. It exhibited specific binding to anti-His tag antibody, and the relative molecular mass was 5 796. 96. The minimum inhibitory concentration of fMBD3 against Staphylococcus aureus was 12. 5 ??g/mL, and the half inhibitory concentration against E. coli and Candida albicans was both 12. 5 ??g/mL.ConclusionThe prokaryotic expression conditions of fMBD3 were optimized by orthogonal test, yielding fMBD3 with high purity and good antibacterial activity, laying a foundation for further function study of MBD3.
