[Study of antmicrobial mechanisms of human cervical mucus: isolation and characterization of antibacterial polypeptides].
OBJECTIVE
To isolate and purify antibacterial polypeptides from human cervical mucus.


METHODS
Human cervical mucus was collected from human healthy subjects and acid-soluble extract was obtained by solving the mucus with 5% acetic acid in the presence of protease inhibitors. The antibacterial components were identified by Agar radial diffusion assay and gel overlay technique. For further purification, Preparative acid-urea gel electrophoresis and Reverse Phase HPLC were performed. The N-terminal sequencing and degenerate PCR-directered cDNA cloning were performed. The E. coli-based recombinant product was prepared and its antibacterial property was determined by minimal inhibitory concentration and minimal bactericidal concentration.


RESULTS
A purified antibacterial polypeptide was obtained. Agar radial diffusion assay showed that the purified polypeptide had antibacterial activities against E. coli ML-35P and Pseudomanas aeruginosa ATCC 27853. The N-terminal amino acid sequence and its full length of cDNA were identical to High Mobility Group Chromosal protein N2 (HMGN2). The purified recombinant HMGN2 was obtained. Its MIC against E. coli ML-35p and P. aeruginosa ATCC27853 were 10.42 microg/ml +/- 3.13 microg/ml and 27.78 microg/ml +/- 8.33 microg/ml respectively, which were equal to human neutrophil defensins HNP, and the MBC were 20.83 microg/ml +/- 6.25 microg/ml and 55.56 microg/ml +/- 16.67 microg/ml respectively.


CONCLUSIONS
HMGN2 may be another antibacterial effecter in the defense mechanisms of human cervical mucus.
Li
Ming
M
Research Unit of Infection and Immunity, Department of Obstetrics and Gynecology, West China Medical Center of Sichuan University, Chengdu 610041, China.
Pan
Xiao-ling
XL
Wang
Li-li
LL
Feng
Yun
Y
Huang
Ning
N
Wu
Qi
Q
Li
Xuan
X
Wang
Bo-yao
BY
D000328:Adult; D000900:Anti-Bacterial Agents / Q000032:analysis / Q000302:isolation & purification*; D002582:Cervix Mucus / Q000737:chemistry / Q000502:physiology*; D005260:Female; D024242:HMGN2 Protein / Q000032:analysis / Q000302:isolation & purification*; D006801:Humans; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*
D000900:Anti-Bacterial Agents; D024242:HMGN2 Protein; D010455:Peptides