title: [Prokaryotic expression and purification of antimicrobial peptide LL-37 and the inhibiting effect against Candida albicans].
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-γ concentration 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.
authors.affiliation: Department of Family Planning, the Second Hospital of Tianjin Medical University, Tianjin 300211, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Huo
authors.forename: F
authors.initials: F
authors.lastname: Wang
authors.forename: B
authors.initials: B
authors.lastname: Sun
authors.forename: L R
authors.initials: LR
authors.lastname: Yin
authors.forename: P P
authors.initials: PP
authors.lastname: Zhang
authors.forename: Y J
authors.initials: YJ
authors.lastname: Zhang
authors.forename: B M
authors.initials: BM
authors.lastname: Zhang
mesh_terms: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D015153:Blotting, Western; D002176:Candida albicans / Q000187:drug effects / Q000235:genetics*; D002177:Candidiasis; D054804:Cathelicidins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D018920:Coculture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells; D004926:Escherichia coli; D005260:Female; D006801:Humans; D007371:Interferon-gamma; D016753:Interleukin-10 / Q000235:genetics* / Q000378:metabolism; D011387:Prokaryotic Cells; D011994:Recombinant Proteins
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D054804:Cathelicidins; C508609:IL10 protein, human; D011994:Recombinant Proteins; D016753:Interleukin-10; D007371:Interferon-gamma