中国生物工程杂志

To construct a expression vector induction by RU486 (mifepristone) and confirm the regulatable effect in vitro.The PRS plasmid vector encoding a GLP65 chimeric regulator and GAL4 hybrid promoter was modified with molecular biological methods. The BGHpolyA fragment was amplified by PCR technique and introduced several enzyme cutting sites. A hCMV promoter replaced the TTR promoter to control the GLP65 regulator, and luciferase reporter gene was inserted into downstream of the GAL4 promoter. To minimize any potential interference, the two transcriptional elements were spaced with a 1.2kb chicken beta-globin insulator. The recombination vector was identified by different restriction endonuclease reactions, sequencing analysis and PCR assay. Dual-Luciferase Reporter Assay was used to demonstrate the activation of this regulatable vector after cotransfection with pRL-TK in HEK293 cells．Forty-eight hours after transfection, cells were harvested to determine luciferase activity by illuminometer. In the presence of RU486 a 40-fold maximum activation of the luciferase reporter gene was observed in cultured cells, whereas in the absence of RU486, no significant activation was observed. There was a positive correlation between the luciferase activation and RU486 concentration in a definite range. The results showed that the RU486-inducible regulatory vector was successfully constructed, which can be used to regulate the expression level of genes of interest in appropriate time by the inducer RU486. This inducible expression vector provides a platform for the research of gene regulation and gene therapy.