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

作　　者：袁龙[1,2] 郑幽 范杰 梁剑青 牛祖彪[2] 高丽华 黄红艳[3] 陈昭烈 管静芝[4] 孙强[2] 

机构地区：[1]山西医科大学第二临床医学院,山西太原030001 [2]军事医学科学院生物工程研究所,北京100071 [3]首都医科大学附属北京世纪坛医院肿瘤内科肿瘤治疗性疫苗北京市重点实验室,北京100038 [4]解放军第309医院肿瘤内科,北京100091

出　　处：《生物技术通讯》2017年第4期405-409,共5页Letters in Biotechnology

基　　金：国家自然科学基金(81572799;31671432);国家重点研发计划(2016YFC1303303)

摘　　要：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.

关 键 词：PEST序列 基因编辑 蛋白酶体 绿色荧光蛋白 

分 类 号：Q78[生物学—分子生物学]