具有长期感应尿酸浓度功能的稳定细胞系的构建与鉴定

Construction and Identification of a Stable Cell Line as a Long-Term Uric Acid Sensor System

目的:利用合成生物学方法构建具有尿酸感应作用的基因回路,稳定转染细胞系,构建长期自发稳定感应尿酸浓度的细胞系p-Huc O8-SEAP-m UTs。方法:以耐辐射型球菌R1基因组中转录抑制物基因huc R及其结合位点基因huc O为基本元件,人工合成优化的转录抑制物基因m UTs及其结合位点的8串联结构基因huc O8,将m UTs和huc O8作为已知功能的生物模块,用于完成基因回路的构建,并稳定转染Hep G2细胞;通过实时定量PCR检测稳定细胞系中目的基因表达的稳定性,通过检测分泌型碱性磷酸酶(SEAP)的表达量来验证对尿酸的感应作用回路及其长期可逆性。结果和结论:构建了p-Huc O8-SEAP-m UTs稳定细胞系,稳定株不同代数之间目的基因的表达稳定;p-Huc O8-SEAP-m UTs稳定细胞系能有效地反映环境中的尿酸浓度,且比瞬时转染细胞系更加敏感,可逆性良好,没有明显的"记忆效应"。

Objective： To establish the Hep G2 stable cell line as a long-term uric acid responsive sensor by auric acid mediated gene circuit constructed with synthetic biology approach. Methods： Based on the transcriptionalinhibitor huc R and its binding site huc O in the genome of Deinococcus radiodurans R1, synthesize optimized tran-scriptional inhibitor gene m UTs and its binding site 8-series structure（huc O8） chemically to construct the circuit.Transfect Hep G2 stably, verify thee stability of target gene by real time PCR, and characterize its reaction to uricacid and its long-term reversibility by assaying the expression of secreted alkaline phosphatase（SEAP）. Results Conclusion： The stable cell line p-Huc O8-SEAP-m UTs is successfully established, target gene expression remainstable in different algebra, it can reflect the uric acid concentration of the circumstance effectively, more sensitivethan the transient transfection cell line,have good reversibility without showing obvious expression＂memory effect＂.