TrkA与snapin蛋白的直接相互作用

Direct Interaction between TrkA and Snapin

目的确认TrkA与snapin蛋白间的直接相互作用。方法采用DNA重组技术,构建增强型荧光蛋白表达载体pECFP-TrkAICD(TrkA膜内区)和pEYFP-snapin,共转染HEK 293T细胞后以激光扫描共聚焦显微镜观察并进行荧光共振能量转移(fluorescence resonance energy transfer,FRET)分析。结果成功构建了snapin和TrkA的重组质粒,共转染细胞后激光扫描共聚焦显微镜分析表明两种蛋白分布在细胞质同一层面,荧光共振能量转移(FRET)分析表明能量转移效率>5%,与对照相比有显著区别(P<0.05)。结论激光扫描共聚焦及FRET实验结果都证明了TrkA膜内区与snapin两个蛋白之间存在着直接的相互作用。

To prove the presence of the direct interaction between TrkA and snapin. Methods By using DNA recombi- nant technique, the plasmids, pECFP - TrkAICD and pEYFP - snapin were constructed for expressing fused enhanced cyan fluorescent protein - TrkAICD and fused enhanced yellow fluorescent protein - snapin. After co - transfection of HEK 293 cell with these two plas- mids, the study was performed with confocal laser scanning microscopy and fluorescence resonance energy transfer （FRET）. Results It was successful to construct the fused fluorescent protein - expressing plasmids related with TrkAICD and snapin. After co - transfection of cells with them, confocal laser scanning microscopy analysis showed that these two protein were contributed to the same faultage in the cy- toplasma. Fluorescence resonance energy transfer analysis showed that the energy transfer efficiency 〉 5% , and it was significant to com-pare with negative control, with the P 〈 0. 05. Conclusion Confocal laser scanning microscopy analysis and fluorescence resonance ener- gy transfer analysis prove respectively that there is a direct interaction between TrkA and snapin.