Prokaryotic expression and purification of antimicrobial peptide LL-37 and the inhibiting effect against Candida albicans
10.3760/cma.j.issn.0529-567x.2016.02.008
- VernacularTitle:抗菌肽LL-37的原核表达纯化及对假丝酵母菌的抑制作用
- Author:
Yan HUO
;
Fang WANG
;
Bei SUN
;
Lirong YIN
;
Pingping ZHANG
;
Yujuan ZHANG
;
Bumei ZHANG
- Publication Type:Journal Article
- Keywords:
Candidiasis,vulvovaginal;
Cathelicidin;
Recombinant fusion proteins;
Prokaryotic expression
- From:
Chinese Journal of Obstetrics and Gynecology
2016;(2):120-125
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the inhibitory effect of antimicrobial peptide LL-37 on Candida albicans through its ability to promote the secretion of immune factors by vaginal epithelial cells. Methods (1) LL-37 prokaryotic expression vector pET-Duet/LL-37 was constructed and its expression was induced in Escherichia coli M15. The expressed LL-37 fusion protein was purified and identified by western blot. Antifungal activity of the purified protein was initially identified by Kirby-Bauer (K-B) method. (2) Purified LL-37 protein was added to human vaginal epithelial cells co-cultured with Candida, and inhibitory effect on Candida growth was determined by the glucose consumption method. Interferon γ (IFN-γ), interleukin 10 (IL-10) concentration and IFN-γ/IL-10 ratio were measured by ELISA at different time points. Results (1) LL-37 fusion protein was purified to 96% purity at a concentration of 433.92 μg/ml, and was shown to possess anti-fungal activity confirmed by the K-B method. (2) A Candida-vaginal epithelial cells co-culture system was successfully constructed. LL-37 recombinant protein inhibited the growth of Candida with absorbance values significantly higher in the treatment group compared to the control group at all measured time points (12-hour:3.008±0.003 versus 2.967±0.003, 24-hour:2.941±0.003 versus 2.601±0.003, 48-hour:2.893 ± 0.004 versus 2.409 ± 0.003; all P<0.01). Furthermore, the rate of decrease was also much slower compared to the control group. In both control and experimental groups, IFN-γ and IL-10 secretion levels were observed to rise at first peaking at 24 hours and subsequently decrease. For each time period, IFN-γconcentration in the experimental group was significantly higher at 24 hours compared to the control group [(104.00 ± 1.07) versus (85.17 ± 0.28) pg/ml, P<0.01]. In contrast, IL-10 concentrations were significantly lower than the control group at all time points (P<0.01). IFN-γ/IL-10 ratio was also observed to be significantly higher than the control group at all measured time points (P<0.01). Conclusions (1) Recombinant protein LL-37 could significantly inhibit the growth of Candida. (2) By influencing the secretion of immune factors such as IFN-γ, IL-10, etc, recombinant protein LL-37 is able to adjust vaginal epithelial cells local immunity, and enhance resistance to Candida infection.
