以蛋白激酶G为靶点的抗结核药物筛选模型的建立和初步应用

The Establishment and Application of Anti-tuberculosis Drugs Screening Model Targeting to PknG

摘要:

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MTB PknG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%。

Abstract:

Mycobacterium tuberculosis (MTB) PknG, one of 11 Ser/Thr protein kinases produced by MTB, plays an essential role in the residing of MTB in the macrophages as persistent state. In this work, we amplified the pknG gene from the genome of H37Rv, and constructed a recombinant plasmid pET-pknG to express the PknG protein in the E. coli. Using purified PknG, we established and optimized a high-throughout screening model to screen for PknG inhibitors. Among 2120 samples of microbial fermentations and 2300 samples of compounds tested, one positive compound and 13 positive fermentation samples were identified with inhibitory effect on activity of PKnG, resulting in a 0.32% of positive rate.