遵义小钝吻鮠体表黏液抗菌肽的分离纯化

Separation and purification of antimicrobial peptide from skin mucus of Leioxassis microcrassirostris Zunyiensis Xiao sp. nov.

【目的】从遵义小钝吻鮠(Leioxassis microcrassirostris Zunyiensis Xiao sp.nov.)体表黏液中获取高质量的抗菌肽,为提高其先天免疫能力及病害防治效果奠定基础。【方法】采用物理方法收集遵义小钝吻鮠的体表黏液,以5%乙酸进行浸提,采用Sephadex G-50和Sephadex G-25两步凝胶层析技术对抗菌粗提物进行分离纯化,结合Tricine-SDS-PAGE电泳法进行分离鉴定,并运用牛津杯法和挖孔法进行抑菌试验。【结果】遵义小钝吻鮠体表黏液经乙酸法浸提处理并冷冻干燥,其粗提物呈白色粉末,稀释液蛋白质含量为1755μg/m L;经Sephadex G-50凝胶层析分离,其纯化产物抑菌活性峰主要在峰Ⅱ和峰Ⅲ,抑菌圈直径11.66 mm;再经Sephadex G-25凝胶层析分离纯化后出现单一活性峰,其产物为分子量小于4.1 k D的抗菌肽,抑菌圈直径12.85 mm。【结论】遵义小钝吻鮠无需诱导刺激,可直接采用物理方法收集其体表黏液,以5%乙酸浸提后经Sephadex G-50和Sephadex G-25两步凝胶层析便可分离出纯度较高的抗菌肽。

[Objective ]In order to lay the foundation for enhancing congenital immune ability of Leioxassis microcras- sirostris Zunyiensis Xiao sp.nov and improving its disease control effect, the high-quality antimicrobial peptides had ob- tained from skin mucous of L. microcrassirostffs Zunyiensis Xiao sp. nov. [Method]The skin mucus of L. microcras- sirostr/s Zunyiensis Xiao sp.nov was collected by using physical method, and which was extracted with the concentration of 5% acetic acid. Then the crude extracts were separated and purified by two-step column chromatography technique （Sephadex G-50 and Sephadex G-25）. Combining with Tricine-SDS-PAGE technique, the purified products were iden- tified, and then its antibacterial activity was detected by Oxford Cup and digging hole method. [Result]After the skin mucus extracted with acetic acid had been freeze-dried, its crude extracts were found to be white powder, the protein content of its diluent was 1755 μg/mL. Using column chromatography of Sephadex G-50, the antibacterial activity peak of purified product was in peak I1 and peak 111, its bacteriostatic circle diameter was 11.66 mm. Then using column chro- matography of SephadexG-25, the purified product showed single activity peak, which was less than 4.1 kD with antimi- crobial circle diameter of 12.85 mm. [Conclusion]The skin mucus of L. microcrassirostns Zunyiensis Xiao sp.nov can be col- lected by using physical method without induction and stimulation. The high-purity antibacterial peptide can be separated with 5% acetic acid combined with two-step column chromatography of Sephadex G-50 and Sephadex G-25.