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

发布时间：2018-01-14 12:41

本文关键词：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　出处：《分析化学》2017年02期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Thiamine pyrophosphate (TPP) is the main active form of vitamin B1 in cells, and it is also a sugar. In the cell, artificial ribozyme switch composed of TPP aptamer and natural ribozyme is used to regulate target gene expression, which is limited to prokaryotic expression. In this study, two types of TPP ribozymes of "Switch-on" and "Switchoff" were selected in prokaryotes. PCR was constructed from the 3'untranslated region of the enhanced green fluorescent protein (EGFP) reporter gene and transfected into human embryonic renal epithelial cells (HEK293). The regulation of EGFP expression by different concentrations of TPP was observed by fluorescence microscope and flow cytometry. The two kinds of "Switch-on" and one "Switch-off" ribozyme switch were constructed in a dose-dependent manner and had good specificity. The fluorescence intensity of EGFP was increased by 3.1 times at 150 渭 mol/L TPP. By 1.9-fold and 2.3-fold reduction, the construction directly transformed the change of TPP concentration into the change of reporter gene expression by the specific action of its aptamer in TPP and ribozyme switch. It is helpful for the detection of metabolites or factors in lactation living cells by fluorescence detection without marking, injury, visualization and high efficiency.
【作者单位】：安徽医科大学生命科学学院;中国科学院合肥物质研究院;中国科学院苏州纳米技术与纳米仿生研究所;
【基金】：国家自然基金资助项目(Nos.21628502,21507156,31300096) 江苏省青年基金资助项目(Nos.BK20130357,BK20130351)资助~~
【分类号】：O657.3;TP212.3
【正文快照】： 1引言核酶开关是近些年出现的一种人工基因调控开关。常见的核酶开关由锤头状核酶和适配体组成[1]。作为一种顺式作用元件,在特异识别适配体的配体作用下,无需蛋白质辅助,即可通过调节自身裂解反应,调控mRNA的翻译,已应用于多种细胞的基因调控[2~6]。迄今,随着指数富集配基

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