Drug Screening Based On Reporter Gene And The Signal Transduction Of Interferon-α

OBJECTIVE Interferon-α is a recombinant protein widely used in the therapy of viral infection. However, IFN-α is unstable in vitro and hydrolyzed easily in vivo .Therefore, it is very important to develop a method of drug screening model and to find compounds with Interferon-α-like activity. As we all knew, research and development of new drugs is a complicated and systemic project which include several indispensable steps, among them the finding of lead compound is a key step. In our study, cell-based screening model was established by the advanced methods and technology of molecular biology and cell biology, and then the small molecular compounds were screened for finding novel molecules with Interferon-a-like activity.METHODS A recombinant vector pTAL/ISRE-SEAP was constructed by inserting a synthetic sequence composed of five IFN-α bounded elements ISRE in front of promoter of pTAL/SEAP vector. pTAL/ISRE-SEAP was then transfected into ECV304 cells. Hygromycin B(500μg·ml^-1) was added 48h after transfection to select positive clones that can be induced by IFN-α to express reporter gene SEAP. Stably transfected cell clones were isolated, and the speciality of the model was examined by the cytokines of other interferon (IFN-β; IFN-γ) and growth factor (e.g. parathyroid factor), and the stability and sensitivity of this model were evaluated, and then the model was used to screen 300 compounds to test whether those compounds have IFN-α-like activity or not. The "hit" compound NO.258 was studied in antiviral model in vitro, and the mechanism of possible anti-HBV were explored by RT-PCR.RESULTS The recombinant pTAL/ISRE-SEAP plasmid was confirmed by restriction enzyme; Stably transfected clones were isolated; The expression of reporter gene SEAP from one of the cloned cells was induced by IFN-a in a dose-dependent manner. The expression level of induction by IFN-β, IFN-γ and PTH was low, indicating the high speciality of the developed method. The Z'-factor of this model is 0.8 suggesting it is very stable. The signal transduction of IFN-a. can be activated by compound NO.258, DNA copies of HBV in HepG2.2.15 cells were treated by this compound were lower than cell controls, OAS3 gene in ECV304 cells...