组氨酸激酶(YycG)在筛选肺炎链球菌抑制剂中的应用

Application of Histidine Kinase (YycG) in Screening Inhibitor of Streptococcus pneumoniae

为了获得具有体外活性的肺炎链球菌组氨酸激酶YycG并利用其筛选寻找新的抑制剂。原核表达组氨酸激酶YycG的激酶功能域,经SDS-PAGE,Western blot鉴定及镍层析柱纯化后,采用激酶试剂盒检测其激酶活性;利用对其激酶活性的抑制作用从105种候选化合物中筛选有效的抑制剂,并通过实验验证抑制剂的抗菌作用。原核表达得到约35kDa的目的蛋白激酶域片段YycG′,其纯度达95%,并具有体外水解ATP的激酶活性;利用其活性筛选得到数种不同抑制效果的小分子化合物,且体外验证具有较好的抑菌效果。通过肺炎链球菌组氨酸激酶YycG活性筛选找到的小分子抑制剂可为进一步研发与该菌相关的药物或消毒剂提供基础。

To obtain a protein fragment of Streptococcus pneumoniae histidine kinase YycG with in vitro ATP - dependent kinase activity for screening novel bacterial inhibitors, the genomic DNA encoding the kinase domain of YycG was expressed in E. coli BL21 （DE3）. The product was identified by SDS-PAGE and Westem blot assays, purified by a His-tagged Ni affinity column, and determined for kinase activity. It was about 35 kDa, reaching a purity of 95%, and has dose - dependent kinase activity of hydrolyzing ATP in vitro. Based on the inhibitory action on the catalyzing function of the purified kinase protein, several effective inhibitors were screened out from 105 candidate compounds available, one of which was selected out and verified overt bactericidal effects in vitro. Collectively, a fragment of histidine kinase YycG expressed in vitro was successful used to screen kinase inhibitors that may be important micro-molecule compound for developing antibacterial drugs or disinfectant.