低稳定性绿色荧光蛋白EGFP-PEST的载体构建及表达验证

Constructing Vector for the Expression of EGFP-PEST, a Modified Enhanced Green Fluorescent Protein with Low Stability

目的:构建含有蛋白降解基序PEST序列的增强型绿色荧光蛋白(EGFP)融合基因表达载体pQCXIP-EGFPPEST-N1,并检测其对蛋白稳定性的调节。方法:以NFATx6-mPro-EGFP-PEST-YES为模板,PCR扩增EGFP-PEST序列,克隆入逆转录病毒载体pQCXIP-EGFP-N1构建pQCXIP-EGFP-PEST-N1,病毒包装后感染293FT细胞获得稳定细胞系;用蛋白酶体抑制剂MG-132处理细胞,Western印迹检测EGFP的表达变化。结果:构建获得逆转录病毒载体pQCXIP-EGFP-PEST-N1,感染293FT细胞后,与对照组相比,EGFP表达显著降低,并且该降低可被MG-132处理逆转。结论:构建了EGFP-PEST蛋白的表达载体,PEST可以介导EGFP蛋白发生蛋白酶体依赖的降解。为实现基因编辑效果的可视化筛选奠定了基础。

Objective： To construct retroviral vector pQCXIP-EGFP-PEST-N1 for the expression of EGFP-PEST protein, and examine the effects of PEST motif on EGFP stability. Methods： DNA fragment containing EGFP- PEST was amplified by PCR from NFATx6-mPro-EGFP-PEST-YES as template, and then cloned into the retrovi- ral expression vector pQCXIP-EGFP-N1 to generate pQCXIP-EGFP-PEST-N1, which was packaged into retrovirus to infect 293FT cells. Western blot was employed to examine changes at protein level upon treatment of protea-some inhibitor（MG-132）. Results： Compared with pQCXIP-EGFP-N1 virus, retroviral vector of pQCXIP-EGFP- PEST-N1 produced significantly lower EGFP in 293FT cells, and MG-132 treatment reverted the expression of EGFP-PEST but not EGFP, suggesting effective PEST-mediated degradation via proteasome. Conclusion： A vector has been constructed to express EGFP-PEST, and PEST motif has been proved to effectively mediate EGFP degra- dation in a proteasome-dependent manner. This work set a basis for visualing target screen in gene edition.