监测活细胞中诱导激活的Rac、Cdc42时空图像的FRET单分子探针的构建

Construction of FRET-based Single-molecule Probes for Monitoring Spatial and Temporal Imaging of Induced Activation of Rac,Cdc42 in Living Cells

以PCR方法从人脑cDNA基因文库扩增Rac1、Cdc42cDNA全序列及其效应蛋白基因Pak1、N-WASP的GTP酶联结区域(GBD)序列,从dsRed1-N1质粒扩增红色荧光蛋白dsRed1cDNA全序列。将cDNA序列依次定向克隆至pECFP-N1质粒载体,获得基于FRET原理,包含dsRed1,Pak1或N-WASP的GBD,Rac1或Cdc42,ECFP编码序列的单分子探针。在dsRed1的C末端加入一段CAAM法尼基化基序,构建包含EGFP,Pak1的GBD,Rac1或Cdc42,dsRed1-CAAM的质膜特异表达的单分子探针。采用这两种探针,可用于监测活细胞中诱导激活的Rac1、Cdc42信号转导通路的3D时空图像,检测待测蛋白分子的GEF或GAP活性。

Full-length cDNA sequences of Racl, Cdc42 and GTPase binding domains of their effectors Pakl, N-WASP were amplified from human brain cDNA library by PCR. The amplified eDNA sequences cloned into pECFP-N1 vector, and FRET-based single-molecule probes containing dsRedl, GBD of Pakl or N-WASP, Racl or Cdc42 and ECFP were constructed. Based on these probes, by adding farnesylated motif of CAAM to the C-terminal of dsRedl, the probes containing EGFP, GBD of Pak1 , Racl or Cdc42, dsREdl and CAAM motif, which can be specifically expressed in plasm membrane, were constructed. These two kinds of probes could be used to localize and trace the 3 D spatial and temporal imaging of induced activation for Racl, Cdc42 singaling pathways in living cells, and identify GEF or GAP activities of putative regulatory proteins for Rho GTPases.