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

硫胺素焦磷酸(Thiamine pyrophosphate,TPP)是维生素B1在细胞内的主要活性形式,也是糖、脂肪酸和氨基酸氧化代谢中重要的辅助因子。在细胞内,利用TPP适配体与天然核酶组装成的人工核酶开关调节靶基因表达,目前仅局限于原核、真菌或植物细胞。本实验将原核生物中筛选的"Switch-on"与"Switchoff"的两种类型的TPP核酶开关,运用重叠延伸PCR的方法构建于增强绿色荧光蛋白(EGFP)报告基因的3'非翻译区(UTR),转染人胚肾上皮细胞(HEK293),通过荧光显微镜和流式细胞仪分析,观察了不同浓度TPP对EGFP表达能力的调控。结果表明,构建的两种"Switch-on"和一种"Switch-off"核酶开关均表现出明显的TPP浓度依赖性,且具有良好的特异性,在150μmol/L TPP时分别将EGFP的荧光强度提高3.1倍、1.9倍和降低2.3倍。这种构建通过TPP与核酶开关中其适配体的特异性作用直接将TPP浓度的变化转化为报告基因表达的改变,利于通过荧光检测方法实现对哺乳活细胞内代谢物或因子的无标记、无损伤、可视、高效的检测。

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 TPPdependent 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 μmol/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.