中国林蛙(Rana chensinensis)蛙皮抗菌肽的制备及抗菌作用研究

Preparation of Antibiotic Peptide from Skin of Rana Chensinensis and Study of Its Antibacterial Activity

目的从中国林蛙的皮肤中,提取纯化具有抗菌活性的多肽物质,并对其进行抑菌活性的研究。方法用甲醇进行粗提取。对提取的甲醇浓度、甲醇用量、浸提时间和浸提次数,采用L9(34)正交实验,以蛋白含量作为指标,确定最佳提取条件。得到的粗提物经Sephadex G-75、Sephadex G-50和Sephadex G-25凝胶过滤进一步分离纯化获得抗菌肽。对抗菌肽进行氨基酸组成分析,采用杯碟法进行抑菌活性研究。结果提取林蛙抗菌肽的最佳工艺条件:甲醇浓度80%、甲醇体积为蛙皮重量的6倍、提取时间24h,提取次数3次。提取的粗提物经凝胶过滤后得到抗菌肽。抗菌肽的氨基酸组成中,碱性氨基酸占22.1%,酸性氨基酸占13.9%。抗菌肽对细菌的最低抑菌浓度分别为:枯草杆菌73.25μg/ml,金黄色葡萄球菌51.75μg/ml,大肠杆菌51.75μg/ml,铜绿假单胞菌51.75μg/ml。结论经甲醇提取和凝胶过滤可从中国林蛙皮肤得到抗菌肽,该抗菌肽为碱性多肽,对革兰阳性细菌、革兰阴性细菌均有抑制作用。

Objective To isolate the antimicrobial peptide （AMP） from the skin of Rana Chensinensis and study the antibacterial activity of the AMP. Method A crude extract was obtained first by methylalcohol extraction. The extraction conditions concentration and volume of methylalcohol, extraction time and extraction frequency were optimized by using the L9 （3^4 ）orthogonal design. The protein concentration was used as the index. The crude extract was eluted on Sephadex G- 75, Sephadex G - 50 and Sephadex G - 25 chromatographic columns. The amino acid composition of the AMP was analyzed and the antibacterial activity of the AMP was studied by cup method. Results For extracting AMP, the optimum concentration of methyl alcohol was 80%, the optimum volume was 6 times the skin weight of Rana Chensinensis, the extraction time was 24 h and the extraction frequency was 3 times. AMP was obtained by sephadex gel filtration. Basic amino acid was 22.1% and acidic amino acid was 13.9% in the composition of the AMP. The minimal inhibitory concentration （MIC） of AMP on Bacillus subtilis ATCC 6633 was 73.25 μg/ml, on Staphylococcus aureus ATCC 6538 was 51.75 μg/ml, on Pseudomonas aeruginosa ATCC 15442 was 51.75 μg/ml, and on Escherichia coli ATCC 259221 was 51.75 μg/ml. Conclusion The AMP can be obtained by extraction with methyl alcohol and sephadex gel filtration from the skin of Rana Chensinensis. It is a basic polypeptide, having antimicrobial activity against both G^＊ and G^- bacteria.