以SecA为靶点的新型抗绿脓杆菌药物细胞水平筛选模型的建立和应用

The Establishment and Application of Anti-Pseudomonas aeruginosa Cell-based Screening Model Targeting to SecA

摘要:

Sec途径(即分泌途径 secretion pathway)是蛋白质转运的主要途径。其中, 最为关键的组分之一是SecA ATP酶, 是蛋白质转运途径中的“动力泵”, 通过ATP的水解循环驱使蛋白质前体穿过细菌内膜, 在细菌中是不可缺少的。我们推测抑制SecA ATP酶活性的化合物, 必然会在一定程度上抑制蛋白质的转运和分泌。通过绿脓杆菌与大肠杆菌SecA蛋白的互补作用, 利用本实验室构建的高效表达SecA蛋白的基因工程菌, 建立了SecA蛋白ATP酶活性抑制剂的细胞水平筛选模型。利用所纯化的绿脓杆菌SecA蛋白的ATP酶活测定体系, 验证了所建立的细胞水平筛选模型具有一定的特异性。研究结果表明其中两个酯相组分在细胞水平和蛋白水平均具有活性, 值得进行深入的研究。

Abstract:

The most of secreted proteins are exported by Sec translocase (secretion pathway). SecA ATPase is one of the most important subunit in the Sec translocase, which is preprotein translocase nanomotor that undergo membrane insertion and deinsertion to drive preprotein across the bacterial inner membrane, and SecA is indispensable to bacteria. It should be presumed that the compound which inhibits the activity of SecA ATPase probably can be used as the candidate of bactericide. A secA gene from Pseudomonas aeruginosa PAO1 was amplified and expressed in Escherichia coli BL21.19 (secA13). It has been shown that the wild-type SecA of Pseudomonas aeruginosa could fully complement the E. coli amber (secA13) mutant at the non-permissive temperature. So a cell level screening model targeting on SecA was established based on the above result. The inhibition of PaSecA ATPase activity was applied to validate the specificity of the cell-based method. Two positive samples based on both of cell and enzyme activities will be further studied.