小鼠小肠抗生素肽的初步分离

Isolation of antimicrobial peptides from small intestine of mouse

目的初步分离小鼠小肠抗生素肽。方法将12只健康ICR小鼠剖腹切取从屈氏韧带到回盲部的小肠,以10%乙酸溶液冲洗肠腔收集灌洗液,并以30%乙酸溶液制备小肠组织匀浆,反复离心,聚乙二醇浓缩后透析,冰冻干燥,进行AU-PAGE、Tricine-SDS-PAGE、考马斯亮蓝R-250染色或银染显示多肽条带,然后进行凝胶成像分析和杀菌试验。结果小鼠小肠组织匀浆和灌洗液中均含有可杀灭细菌、真菌的抗生素多肽,其中组织匀浆中所含的此类多肽较多;小鼠小肠抗生素肽的杀菌作用具有剂量依赖关系,且对不同细菌、真菌的杀灭作用也不同;小鼠小肠抗生素肽的分子量主要集中于3.0kD～14.3kD和29.0kD。结论小鼠小肠含有多种抗生素肽,具有广谱的杀灭微生物的作用。

【Objective】To isolate antimicrobial peptides from small intestine of mouse.【Methods】The intestine was removed from 12 adult ICR mice killed by cervical dislocation,and the lumen of each resected intestine was rinsed with 10% acetic acid,and the washed intestine was disrupted thoroughly in 30% acetic acid.After being stirred,the perfusate and homogenates were clarified by centrifugation,then concentrated by polyethylene glycol before dialysis,lyophilized and stored at-20℃.The samples were eletrophoresed on 12.5% AU-PAGE and Tricine16.5% SDS-PAGE,and the peptide bands were visualized with Coomassie R-250 and by silver-staining separately.The antimicrobial activity of samples was measured.【Results】①There were many antimicrobial peptides in the perfusate and homogenate of small intestine of mouse,which can kill bacteria or fungus.And the quantity of antimicrobial polypeptides in homogenate was more than that in perfusate.② The effect of antimicrobial polypeptides in small intestine was dose-dependent and was different for different bacteria or fungus.③ Molecular weights of antimicrobial peptides were 3.0 kD～14.3 kD,29.0 kD.【Conclusions】There are many sorts of antimicrobial peptides in small intestine of mouse,which have the broad-spectrum antimicrobial activity.