沼水蛙抗菌肽brevinin-2GHa1分离纯化及结构分析

目前,已自青蛙皮肤分泌物中分离获得多种具有较强抗菌活性的多肽.本文利用电刺激法自沼水蛙背腺和耳后腺获得其皮肤分泌物,利用凝胶过滤色谱(Sephadex G-50)和反相高效液相色谱(reverse-phase high performance liquid chromatography,RP-HPLC)分离纯化,获得一种新型抗菌肽,命名为brevinin-2GHa1.抑菌实验显示,该抗菌肽对革兰氏阳性菌和革兰氏阴性菌均有抑制作用,对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和沙门氏菌的最小抑制浓度分别为:7.8、3.9、2.0μg/mL和250.0μg/mL.该抗菌肽在水中为无规卷曲结构,在浓度为10 mmol/L SDS水溶液和不同浓度三氟乙醇水溶液中则呈α-螺旋结构,该抗菌肽结构的研究对阐明其抑菌机制具有重要作用.

Purification and Structure Analysis of an Antibacterial Peptide Brevinin-2GHa1 from Skin Secretion of Hylarana guentheri

Numerous antimicrobial peptides have been isolated from frog skin secretions. In this study, we purified an antibacterial peptide of 33 amino acids, brevinin 2GHa1, from the skin secretion of Hylarana guentheri. The skin secretion from the dorsal skin glands of young adult frogs was obtained by 10-Volt electrical stimulation. Sephadex G-50 size exclusion gel filtration, followed with reverse-phase high performance liquid chromatography (RP-HPLC) was used for purification. Brevinin 2GHa1 inhibited the growth of both Gram-positive and Gram-negative bacteria. The minimum inhibitory concentrations (MICs) against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Salmonella were 7.8, 3.9, 2.0, and 250 μg/mL. The secondary structure of brevinin 2GHa1 was analyzed using circular dichroism spectroscopy. High level of random coils was indicated when brevinin-2GHa1 was dissolved in deionized water, whereas increased ordered α-helix conformation in SDS (10 mmol/L) or different concentrations of TFE was observed. The results provided useful clues to elucidate the antibacterial mechanism of brevinin-2GHal.