基于报告基因和干扰素-α信号通路的药物筛选模型的建立

目的　建立基于报告基因和干扰素 α(Interferon α)信号通路的药物筛选模型,用于筛选具有IFN α样活性的小分子化合物。方法　构建带有ISRE(IFN αstimulatedresponseelement)靶序列和报告基因的诱导性表达载体,并将其转染入血管内皮细胞 (ECV304 )中,筛选报告基因碱性磷酸酶(SEAP)表达受IFN α诱导的阳性克隆。选取相关的细胞因子干扰素 β、干扰素 γ和生长因子甲状旁腺素(PTH)进行模型特异性考察。结果　ECV304细胞中SEAP的表达受IFN α的诱导并呈现剂量依赖关系,IFN α的最高诱导表达率是IFN β最高诱导表达率的 9 0倍、IFN γ的 8 8倍、PTH的 9 1倍,具有相对特异性。IFN α对ECV304细胞无增殖作用。本方法的Z' 因子为 0 8,说明此模型具有很好的稳定性。结论　本模型的SEAP基因表达水平能够被其特异性配体强诱导表达,利用此模型在 96孔板上用化学发光法测定SEAP基因的诱导表达水平可筛选具有IFN α样活性的小分子化合物。

Development of drug screening model based on reporter gene and the signal transduction of interferon-α

Aim To develop a method of drug screening ba sed on reporter gene and the signal transduction of interferon-α system in order to scre en the small molecular compounds with Interferon-α-like activity.Meth ods A recombinant vector pTAL/ISRE-SEAP was constructed by inserting a synthetic sequence composed of five IFN-α bounded elements in front of promot er of pTAL/SEAP vector. pTAL/ISRE-SEAP was then transfected into ECV304 cells. HygromycinB(500 mg·L -1) was added 48 h after transfection to select positive clones that can be induced by IFN-α to express reporter gene SEAP. Stably tra nsfected cell clones were isolated and used to screen compound banks for the com pounds with Interferon-α-like activity.The speciality was tested by the cytokines of same family (IFN-β;IFN-γ) and growth factor (eg parathyroid factor). Results Stably transfected clones were obtained.The expression of reporter gene SEAP from one of the cloned cells was induced by IFN-α in do se-dependent manner.The expression level of induction by IFN-β,IFN-γ and PT H was low, indicating the high speciality of the developed method.The Z'-facto r(0.8) showed that the method is very stable .Conclusion Stabl y transfected positive clone cells line transfected with recombinant vector pTAL /ISRE-SEAP were obtained.The expression of SEAP was specially induced by IFN- α The cloned cells can be used to screen for compounds with interferon-α-lik e activity by testing luminescent value of expressed SEAP in wells of microlitr e plate.