牛蛙皮肤抗菌肽的分离纯化与活性测定

Isolation, Purification and Activity of A Novel Antimicrobial Peptide from Skin of Rana catesbeiana

目的分离纯化牛蛙皮肤抗菌肽,并检测其活性。方法通过刺激与浸提的方法,得到牛蛙皮肤抗菌肽粗提物,测定其抑菌活性及最低抑菌浓度。利用SephadexG-50凝胶过滤和反相高效液相色谱(RP-HPLC)分离纯化粗提物,用质谱仪测定多肽的相对分子质量及氨基酸序列,并进行活性检测。结果两种方法提取的牛蛙皮肤抗菌肽粗提物均具有较强的抑菌活性,纯化后的多肽相对分子质量约为4370,N-端含22个氨基酸,与其他两栖类来源的抗菌肽同源性较低。活性试验表明均无胰蛋白酶水解活性,存在一定的胰蛋白酶抑制剂活性,有轻微的溶血活性,不具有局部出血活性,具有抗凝和溶栓作用。结论已成功分离并纯化了1种新的牛蛙皮肤抗菌肽。

Objective To isolate, purify and determine the activity of a novel antimicrobial peptide from the skin of Rana catesbeiana. Methods The crude extracts of antimicrobial peptide from skin of Rana catesbeiana were obtained by stimulation and lixiviation stimulation of skin respectively and determined for bacteriostatic activity and minimum bacteriostatic concentration, then purified by Sephadex G-50 gel filtration and RP-HPLC. The purified Rana catesbeiana antimicrobial peptide （RCABP） was analyzed for relative molecular mass, amino acid sequence and activity. Results Both the RCABP extracted by lixiviation and stimulation showed strong bacteriostatic activity. The purified RCABP, with a relative molecular mass of about 4 370 and consisted of 22 amino acids at N-terminus, and showed low homology to the antimicrobial peptides from other amphibian origins. It showed anticoagulant and thrombolytic activities, mild hemolytic activity and a certain activity of trypsin inhibitor, but no local hemorrgagic activity or the activity in hydrolysis of trypsin. Conclusion A novel antimicrobial peptide from skin of Rana catesbeiana was successfully isolated and purified.