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

以PCR方法从人脑cDNA基因文库扩增Rac1、Cdc42 cDNA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.

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

Full-length cDNA sequences of Rac1,Cdc42 and GTPase binding domains of their effectors Pak1, N-WASP were amplified from human brain cDNA library by PCR. The amplified cDNA sequences cloned into pECFP-N1 vector, and FRET-based single-molecule probes containing dsRed1, GBD of Pak1 or N-WASP, Rac1 or Cdc42 and ECFP were constructed. Based on these probes, by adding farnesylated motif of CAAM to the C-terminal of dsRed1, the probes containing EGFP,GBD of Pak1,Rac1 or Cdc42, dsREd1 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 3D spatial and temporal imaging of induced activation for Rac1, Cdc42 singaling pathways in living cells, and identify GEF or GAP activities of putative regulatory proteins for Rho GTPases.