用生物化学方法从植物内生真菌提取液中高通量筛选细菌β-内酰胺酶抑制物

Biochemical high throughput screening of bacterial β-lactamase inhibitors from endophytic fungal extracts

目的采用生物化学方法从植物内生真菌代谢物中筛选细菌β-酰胺酶抑制剂。方法克隆β-内酰胺酶KPC-2、TEM-10和NDM-1基因至表达载体pET28；纯化重组蛋白质，以此为药靶，从植物内生真菌代谢产物库中高通量筛选β-内酰胺酶抑制物；检测效应物对β-内酰胺类药物的增效效应。结果成功克隆了3个目的基因，纯化了重组蛋白质至纯度80．95％；从3752种真菌提取液中筛选确定了5个对3种纯化的β-内酰胺酶活性均有抑制的效应物，其中效应物PF000，212能够使美罗培南对3株携带NDM—l基因的临床分离“超级细菌”菌株的最低抑菌浓度降低8～16倍。结论以β-内酰胺酶为药靶，以植物内生真菌代谢物为材料，应用生物化学方法进行高通量筛选，是获得新型β-内酰胺酶抑制物的一条有效途径。

Objective To screen for bacterial [Mactamase inhibitors from extracts of plant endophytic fungi by biochemical assays. Methods Genes encoding bacterial β-1actamase KPC-2, TEM-10 and NDM-1 were cloned into expression vector pET28. The recombinant proteins were purified and used as target to identify β-ractamase inhibitors from a plant endophytic fungal extract library in high-throughput biochemical screening. The hit extracts were tested for their potentiating effect on inhibiting bacterial growth by β-ractam antibiotics. Results The target genes were successfully cloned and the recombinat proteins were purified to an purity of 80-95%. Five extracts were identified from 3752 fungal extracts as having inhibitory effect on multiple purified β-lactamases. Among them, extract PF000,212 was able to decrease the minimum inhibitory concentration of meropenem by 8-16 folds on three clinical isolates of ＂superbacteria＂ carrying the NDM-1 gene. Conclusions High-throughput biochemical screening using purified β-lactamase as target and endophytic fungal metabolites as candidates is an effective approach to identify novel inhibitors of β-lactamases.