家蝇幼虫分泌物抗菌肽的分离及其部分特性

Isolation and Purification of Antibacterial Peptides from the Larvae Secretion of Housefly and the Characteristics

目的分离家蝇(Musca domestica)幼虫分泌物抗菌肽并研究其部分理化特性。方法用超滤、固相萃取、反相高效液相色谱(RB-HPLC)分离纯化家蝇幼虫分泌物抗菌肽;测定抗菌肽对多株革兰氏阴性菌及革兰氏阳性菌的最低抑菌浓度(MIC)和最低杀菌浓度(MBC);比较其在不同pH值(pH 6．0～9．0)、不同阳离子浓度(Mg2+:(0.5×10-3～10．0×10-3)mol／L, Na+、K+:(10×10-3～100×10-3)mol／L)及不同小鼠血清浓度(12．5％～75％)环境中抗菌活性的变化。结果超滤分离获得的活性蛋白相对分子质量为Mr 3000～30000,该蛋白经20％、30％、70％及80％乙腈洗脱,其组分均有活性,其中以70％乙睛洗脱组分活性较强且稳定。进一步用RP-HPLC从70％洗脱组分中纯化出两个抗菌活性肽。抗菌肽对大肠埃希菌、绿脓杆菌、金黄色葡萄球菌及枯草芽孢杆菌的MIC值分别为32．738、16．369、65．475及32．738μg/ml。在培养基pH值为6～9、不同阳离子浓度及不同小鼠血清浓度的环境中,各实验组的A570增加量均小于0．05,而对照组的A570增加量多为0.3以上,各实验组与对照组比较,其差异均具有统计学意义(P值均<0．01)。结论用固相萃取等方法从家蝇幼虫分泌物中可快速分离出具有较强活性的抗菌肽。该抗菌肽在不同条件下均具有较好的抗菌效果。

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 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 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 （Mg^2＋： （0.5×10^-3-10.0×10^-3）mol/L, Na^＋, K^＋：（10×10^-3-100×10^-3）mol/L）, serum content（12.5%-75%）. Results Molecular weight of the peptides was about Mr 3 000-30 000 after uhrafihrat The fractions eluted with 20%, 30%, 70%, and 80% of acetonitrile （ACN） all showed antibacterial activity by s phase extraction. The fractions eluted with 70% ACN showed strongest and stablest antibacterial activity which further purified by RP-HPLC. Two sub-fractions appeared at around RT 15.5 min and 18.5 min were obtained antibacterial activity. The MIC to those standard Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis was 32.7380, 16.3688, 65.4750 and 32.7380μg/ml respectively. In the nutritive medium of pH 9.0, different divalent cations and serum content, the increment of A570 in experiment groups was less than 0.05, w that of the control group was greater than 0.3 （P〈0.01）. Conclusion SPE and RP-HPLC have been effective purifying the antibacterial peptides which show strong activity in different conditions.