β-内酰胺酶抑制剂快速筛选方法的建立

摘要

为建立用于抗耐药菌的β-内酰胺酶(β-1actamase)抑制剂快速筛选方法,本研究采用双层平板法,以2％的琼脂为底层,1％的含碘试剂为上层,以青霉素为β-内酰胺酶的底物,克拉维酸作为体系验证的阳性对照,通过比较透明圈形成大小,优化反应体系,并对190个微生物发酵产物进行了初步筛选.研究最终确定的筛选条件为,pH7.0的PBS缓冲液体系,底物青霉素(160万单位)浓度为50 mg/mL,β-内酰胺酶浓度为10 U/mL,40℃孵育40 min.通过测量反应体系透明圈形成大小来筛选具有活性的β-内酰胺酶抑制剂.结果证实该方法可以为耐药菌β-内酰胺酶抑制剂的筛选提供快速简单、易行、稳定、可靠的研究平台,并初步筛选获得了具有较高β-内酰胺酶抑制活性的化合物.%In order to establish a rapid screening method for β-lactamase inhibitor,the penicillin was used as β-lactamase substrate,clavulanic acid was used as positive control for the reaction system validation,and a double-plate method including 2％ agar for bottom and 1％ agar containing iodine reagents for the upper was established for screening sensitive drugs against drug-resistant bacteria by comparing transparent ring sizes.After optimizing reaction conditions,the screening method was used preliminary to screen β-lactamase inhibitor from 190 metabolites from microbial fermentation.The result showed the optimal screening method system was as following:pH was 7.0 PBS buffer systems,the substrate concentration of penicillin (1.6 million units) was 50 mg/mL,β-lactamase enzyme concentration was about 10 per milliliter U.The transparent ring size was measured formed by the reaction system to screen the active β-1actamase inhibitor.The present method could provide a simple,stable and reliable research platform for the screening of β-lactamase inhibitor.Finally,a composition with a high β-lactamase inhibitory activity was obtained.