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结核分枝杆菌可以产生11种丝氨酸/苏氨酸蛋白激酶,其中蛋白激酶G(PknG)对于结核分枝杆菌在巨噬细胞内以“持留”状态长期存活有着重要作用。本研究以结核分枝杆菌基因组DNA为模板,在大肠杆菌中克隆表达了MTBPknG蛋白,并分离纯化得到PknG纯酶。本研究还采用三步级联反应方法测定了PknG酶活性,建立和优化了PknG抑制剂高通量筛选模型。利用此模型共筛选发酵液样品2120个,化合物样品2300个,筛选得到阳性化合物1个,阳性发酵液13个,阳性率0.32%。
Mycobacterium tuberculosis can produce 11 serine / threonine protein kinases, of which protein kinase G (PknG) plays an important role in long-term survival of Mycobacterium tuberculosis in macrophages. In this study, Mycobacterium tuberculosis genomic DNA as a template, cloned and expressed in E. coli MTBPknG protein, and isolated and purified PknG pure enzyme. In this study, the PknG activity was measured by a three-step cascade reaction and a high-throughput screening model of PknG inhibitor was established and optimized. A total of 2120 fermentation broth samples and 2300 compound samples were screened by this model. One positive compound was screened, and 13 positive fermentation broths were obtained, the positive rate was 0.32%.