Construction and identification of plasmid with luciferase reporter gene for detection of T-bet expression activity / 中华微生物学和免疫学杂志

ABSTRACT

Objective To construct a T-bet response reporter gene, for the detection of T-bet tran-scriptional activity and application in high-throughput screening for the functional genomies. Methods The cis-acting DNA element, ThRE, based on CNS-22 T box site of IFN-γ gene, was recombined into a reporter vector pLUC-MCS. The reporter gene was transfected into HEK 293T cells to detect its response to T-bet. And the binding of T-bot to TbRE was identified with electrophoretic mobility shift assay(EMSA). Results ThBE was successfully cloned into pLUC-MCS, named as TbRE-LUC. Using a luciferase assay, expression of the reporter gene is found to be induced by T-bet in a dose dependent manner and correlate with T-bet ex-pression positively with activation up to 20 folds. Moreover, the binding specificity of T-bet to TbRE is vali-dated by EMSA. Conclusion We successfully constructed a T-bet response reporter gene, ThRE-LUC, which responds to T-bet keenly and specifically. TbRE-LUC will be a useful tool in high-throughput screen-ing for human genes associated with transcription activity of T-bet.