分析化学2017，Vol.45Issue(2)：157-162,6.DOI:10.11895/j.issn.0253-3820.160688
基于核酶开关的哺乳细胞内硫胺素焦磷酸荧光生物传感器的建立
Engineering of Thiamin Pyrophosphate Fluorescent Biosensors Based on Ribozyme Switches in Mammalian Cells
摘要
Abstract
Thiamin pyrophosphate (TPP) is a thiamine (vitamin B1) derivative and an essential cofactor in oxidative metabolism of the sugars,fatty acids and amino acids in living cells.By now,numerous TPP-dependent artificial riboswitch systems have been developed to regulate target gene expression but limited in bacteria,fungi or plant cells.Herein,the activating (switch-on) and inhibiting (switch-off) TPP-depended hammerhead ribozyme switches,which are from previous reported structures of prokaryotes screening,were investigated in mammalian cells.These ribozyme switches were inserted into the 3'UTR of the enhanced green fluorescence protein (EGFP) gene to construct the efficient ribozyme-based artificial switches through overlap extension PCR cloning.The HEK293 cells were transfected with the engineered ribozyme switches at increasing concentration of TPP.The EGFP gene-regulatory ability was analyzed with fluorescent microscope and flow cytometry.These TPP-inducible gene regulation devices showed the obvious ligand dose-dependency and excellent specificity.Two "switch-on" and one "switch-off" constructs demonstrated 3.1-fold or 1.9-fold increment and 2.3-fold reduction of EGFP level respectively with 150 μ mol/L TPP.The ligand-responsive ribozyme switches,by tuning the change of TPP concentration into the visual reporter genetic expression in cells,enable an efficient development of label-free,noninvasive and high-specific biosensors in living mammalian cells.关键词
硫胺素焦磷酸核酶开关/增强绿色荧光蛋白/基因表达调控/荧光生物传感器/哺乳细胞Key words
Thiamine pyrophosphate ribozyme switches/Enhanced green fluorescence protein/Gene expression regulation/Fluorescent biosensors/Mammalian cells引用本文复制引用
张媛媛,程慧,孙艳,王金娥,吴正岩,裴仁军..基于核酶开关的哺乳细胞内硫胺素焦磷酸荧光生物传感器的建立[J].分析化学,2017,45(2):157-162,6.基金项目
本文系国家自然基金资助项目(Nos.21628502,21507156,31300096)和江苏省青年基金资助项目(Nos.BK20130357,BK20130351)资助 This work was supported by the National Natural Science Foundation of China (Nos.21628502,21507156,31300096) and the Natural Science Foundation of Jiangsu Province (BK20130357,BK20130351) （Nos.21628502,21507156,31300096）
