家蝇抗菌肽分子结构及抗菌谱研究

Study on the molecular structure and antimicrobial spectra of Musca domestica antibacterial peptide

目的研究家蝇抗菌肽的分子结构和抗菌谱,寻找广谱高效天然抗菌肽。方法家蝇被损伤感染诱导免疫后制成组织匀浆,用蛋白质纯化技术分离纯化抗菌肽,测定抗菌活性及抗菌谱,纳升电喷雾飞行时间质谱测定氨基酸序列。结果家蝇组织提取液经蛋白质纯化,得到一纯化的抗菌活性成份,纳升电喷雾飞行时间质谱测序,得到两个肽段的氨基酸序列:肽段1.QPNLYYNK和肽段2.PNNVYYTK。通过NCBInr蛋白质数据库检索,未发现有与之序列相匹配的蛋白质。该抗菌肽对11种临床标本分离菌株铜绿色假单胞菌、大肠埃希菌、伤寒沙门菌、普通变形菌、异形柠檬酸杆菌、肺炎克雷伯菌、阴沟肠杆菌、福氏痢疾杆菌、表葡菌、耐甲氧西林金葡菌(MRSA)和白念珠菌的MIC分别为:3.89、2.22、4.44、6.67、3.33、6.67、5.0、6.67、4.44、4.44和13.33μmol/L。结论本文得到的抗菌肽是一个新型抗菌肽,有广谱抗菌活性,对革兰阴性菌和革兰阳性菌均有一定的抑制作用。

Objective To investigate the broad spectrum and high effective natural Musca domestica antibacterial peptide, and study its molecular structure and antibacterial spectra. Methods Muscadomestica was induced by injury-infection and subject to homogenate, and antibacterial peptide was separated, and the amino acid sequenced by Nano-ESI-Q-TOF2-MS/MS techniques. The antibacterial activity and spectra was examined by plate （KB） and dilution method. Results Musca domestica tissue homogenate was purified by protein purification system, and an antibacterial component obtained, it was hydrolyzed by trypsin, the amino acid sequence of tryptic hydrolysis fragments were 1. QPNLYYNK and 2. PNNVYYTK respectively, then queried NCBInr database, there was no any known proteins and polypeptides to match the antibacterial peptide. The 11 clinical strains （P. aeruginosa, E. coli, S. typhi, P. vulgaris, C. diversus, K. pneumoniae, E. cloacac, S. flexneri, S. epidermidis, Methecillin Resistant Staphylococcus and C. albicans） could obviously be inhibited by the anlibacterial peptide, and MICs （μmol/L） were 3.89μmol/L, 2.22μmol/L, 4.44μmol/L, 6.67μmol/L, 3.33μmol/L, 6. 67μmol/L, 5.0μmol/L, 6.67μmol/L, 4.44μmol/L, 4.44μmol/L and 13.33μmol/L respectively. Conclusions It is a new antibacterial peptide, has significantly broad antimicrobial activity, and display inhibition against Gram negative bacilli, positive coccus and C. albicans.