中药五谷虫抗菌物质的分离纯化及抗菌机制的实验研究  

Study of Antibiotic Purification and Antibacterial Mechanism of Wuguchong

作　　者：王寿宇[1] 张振[1] 李旭东[1] 吕德成[1] 

机构地区：[1]大连医科大学附属第一医院骨科,辽宁大连116011

出　　处：《中华中医药学刊》2013年第9期1843-1845,I0001,共4页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金资助项目(81270052;30901950);大连市科学技术基金资助项目(2010J21DW022)

摘　　要：目的:分离纯化五谷虫的有效抗菌物质,同时研究五谷虫抗菌物质对大肠杆菌的杀伤作用,初步探讨其可能的抗菌机制。方法:将五谷虫与大肠杆菌共培养使五谷虫获得二次免疫。用DEAE-Sepharose Fast Flow离子交换层析柱和sephacryls-200HR凝胶过滤层析柱分离纯化,浊度法检测,获得五谷虫抗菌物质。用荧光探针、琼脂糖凝胶电泳技术分析五谷虫的抗菌机制。结果:五谷虫粗提物浓度>1.0 mg/mL时对大肠杆菌的抗菌效果较强,分子量在10KD左右。荧光探针技术示20 mg/mL的抗生素变化率为35.4%,50 mg/mL的抗生素变化率为40.0%,100 mg/mL的抗生素变化率为42.3%,2 mg/mL的粗提物变化率为1223.1%,80 mg/mL的粗提物变化率为1300.0%。琼脂糖凝胶电泳图示含2 mg/mL和80 mg/mL粗提物的泳道无质粒DNA条带。结论:用DEAE-Sepharose Fast Flow离子交换层析柱和sephacryls-200HR凝胶过滤层析柱可以纯化得到有效的五谷虫抗菌成分,呈单一蛋白条带。其抗菌机制可能是破坏了大肠杆菌的细胞膜以及影响了质粒DNA的复制。Objective ： To isolate and purify the antimicrobial substances of Wuguchong extracts and investigate the an- tibacterial mechanism of Wuguchong. Method： To separate the crude extract of maggot by ion exchange chromatography （ DEAE - Sepharose Fast Flow） and then to purify and detect the most active crude extract by gel filtration chromatogra- phy （sephacryls -200HR）. To analyze the antibacterial mechanism of antimicrobial by fluorescence probe and agarose gel electrophoresis. Result ： There was a better antiblastic effect to E. coli when the density of crude extract exceeded 1.0 mg/ mL, and the molecular weight of the substance was about 10 KD. The fluorescent probe showed that the change rate of antibiotics at 20 mg/mL was 35.4% , at 50 mg/mL was 40.0% , at 100 mg/mL was 42.3% , at the same time, the data showed that the change rate of crude extract at 2 mg/mL was 1223.1% and at 80 mg/mL is 1300.0%. Agarose gel elee- trophoresis diagrams containing 2 mg/mL and 80 mg/mL crude extract the lane without plasmid DNA strip. Conclusion： Ion exchange chromatography（ DEAE -Sepharose Fast Flow） and gel filtration chromatography（ sephacryls -200HR） are effective to isolate and purify antibacterial substance of Wuguehong. The mechanism is to destroy the cell membrane of E. coli and inhibit the replication of the plasmid DNA.

关 键 词：五谷虫 二次免疫 抗菌肽 分离 纯化 抗菌机制 

分 类 号：R285.5[医药卫生—中药学]