天然抗菌肽分离纯化方法的建立

Establishment of a separation and purification method for natural antimicrobial peptide

目的寻找高效、广谱的新型天然抗菌肽，建立稳定、有效的分离纯化方法。方法家蝇免疫诱导后制成组织匀浆，经超速离心、分子筛过滤、SephadexG50层析、反相高效液相色谱法（RP-HPLC）、透析浓缩等技术进行分离纯化，用K-B法鉴定抗菌活性。结果组织提取液经超速离心后，用pH6．8Tris-HCI尿素缓冲液作流动相，SephadexG50层析，收集到有明显抗菌活性的成分。再用pH6．8磷酸盐缓冲液作流动相，经RP-HPLC半制备柱进一步纯化，各成分分离效果良好，保留时间为18．008min处的谱峰，有明显的抗菌活性。用RP-HPLC分析柱显示其为单一成分，达到了色谱纯。该抗菌肽对大肠埃希菌ATCC25922、铜绿假单胞菌ATCC27853和耐甲氧西林金黄色葡萄球菌（methicillin-resistantS．aureus，MRSA）均有抗菌活性。结论建立了有效的家蝇抗菌肽分离纯化方法，纯化的家蝇抗菌肽有广谱抗菌活性。

Objective To seek for a broad spectrum and high effective natural antibacterial peptide, and to establish a steady and effective separation and purification method for it. Methods Musca domestica was induced by injury-infection and made into homogenate in pH 6.8 tris-HCl-urea buffer, and antibacterial peptide was separated and purified with high speed centrifugation grade sedimenta- tion, centrifugation ultrafiltration, Sephadex G50 gel filtration and reverse phase high performance liquid chromatography （RP-HPLC） techniques. The antibacterial activity was examined by plate （K-B） method. Results Musca domestica tissue ultrafiltrate was purified by Sephadex G50 chromatography, pH 6.8 tris-HCl-urea buffer as mobile phase. The components with antibacterial activity were collect- ed. With pH 6.8 phosphate buffer as mobile phase, the antibacterial components were analysed further by semi-separated RP-HPLC. The components were separated. The sample with 18. 008 min time-point peak was acquired and purified by analytic RP-HPLC. There was only a chromatographic peak left in chromatogram, and a chromatograpic pure antibacterial peptide was acquired, which could obviously inhibit E. coli ATCC25922, P. aeruginosa ATCC27853 and methicillin-resistant S. aureus （MRSA）. Conclusion A steady separation and purification method is established for musca domestica antibacterial peptide. The antibacterial peptide has significantly broad antimicrobial activity, and exerts effects to both gram negative bacilli and positive coccus.