Construction and efficacy evaluation of Rb luciferase reporter gene detection system / 西安交通大学学报(医学版)

ABSTRACT

【Objective】 To construct the Rb luciferase reporter gene assay system and detect the activation ability of Rb gene for screening the targeted drugs. 【Methods】 The synthetic Rb gene sequence was annealed to form a double-stranded DNA structure and then inserted into the polyclonal site of pGL6-TA. The junction product was transformed into E.coli DH5α competent cells for expanded culture, and the constructed pGL6-Rb-Luc plasmid and pGL6-TA plasmid were transfected into HEK293 cells. The monoclonal cell line HEK293-Rb-Luc with stable expression was screened by G418, and the activation and inhibition of Rb in HEK293-Rb-Luc were tested by serum and CDK4/6 inhibitor Palbociclib. 【Results】 The sequence of Rb reaction elements in pGL6-Rb-Luc was completely correct. The recovery of serum culture significantly increased the luciferase activity in HEK293-Rb-Luc (P<0.001). Compared with 0 nmol/L, 25, 50, 75 and 100 nmol/L, CDK4/6 inhibitor Palbociclib made the inhibition rate of Rb activity rise to 6.90%, 40.23%, 50.57% and 52.07%, respectively (P<0.05). 【Conclusion】 The Rb luciferase reporter gene detection system HEK293-Rb-Luc was successfully constructed, which can effectively detect the activation level of Rb.