Purification of antimicrobial factors from human cervical mucus

BACKGROUND: The aim of this study was to separate bactericidal proteins from healthy female cervical mucus. METHODS: Cervical mucus was collected and dissolved in 1 % acetic acid. The antimicrobial activity of acid-soluble extracts was detected by gel overlay assay againstEscherichia coliATCC 43827. The extracts showed consider-able amount of antibacterial activity with a clearly visible band. The bactericidal band was purified by reverse-phase high performance liquid chromatography and the antibacterial activity of the eluate was examined using radial diffu-sion assay. RESULTS: Two antimicrobial proteins were purified and were further characterized by Tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis, N-terminal sequencing and matrix-assisted laser desorption ion-ization time-of-flight mass spectrometry. The proteins were identified as high-mobility group nucleosomal-binding domain 2 (HMG N2) and secretory leukocyte peptidase inhibitor (SLPI). SLPI is an antimicrobial peptide already known in the cervical mucus while HMG N2 in the cervical mucus had not been previously reported. The expression of HMGN2mRNAwas detected in Hela cells and cervical epithelial cells by RT-PCR. Slit hybridization showed abun-dant amounts of the HMG N2 protein in the cervical mucus. CONCLUSIONS: These results suggest that the expression of HMG N2 and SLPI in the healthy female cervical mucus may be relevant to their immune surveillance and defense against potential pathogens in human reproductive system