[Isolation and purification of antibacterial peptides from the larvae secretion of housefly and the characteristics].

OBJECTIVE

To isolate and purify the antibacterial peptides from larvae secretion of housefly (Musca domestica) and study their partial characteristics.

METHODS

Protein isolation and purification were performed by routine process, namely, ultrafiltration, solid phase extraction (SPE) and reversed-phase high-performance liquid chromatography (RP-HPLC). The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the antibacterial peptides were examined. The antibacterial effect of peptides was studied in nutritive medium with different pH value(pH 5.0-10.0), divalent cations (Mg2+: (0.5 x 10(-3) - 10.0 x 10(-3))mol/L, Na+, K+: (10 x 10(-3) - 100 x 10(-3))mol/L), and serum content (12.5%-75%).

RESULTS

Molecular weight of the peptides was about Mr 3 000-30 000 after ultrafiltration. The fractions eluted with 20%, 30%, 70%, and 80% of acetonitrile (ACN) all showed antibacterial activity by solid phase extraction. The fractions eluted with 70% ACN showed strongest and stable st antibacterial activity which was further purified by RP-HPLC. Two sub-fractions appeared at around RT 15.5 min and 18.5 min were obtained with antibacterial activity. The MIC to those standard Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis was 32.7380, 16.3688, 65.4750 and 32.7380 microg/ml respectively. In the nutritive medium of pH 6.0-9.0, different divalent cations and serum content, the increment of A570 in experiment groups was less than 0.05, while that of the control group was greater than 0.3 (P<0.01).

CONCLUSION

SPE and RP-HPLC have been effective in purifying the antibacterial peptides which show strong activity in different conditions.