配体活化多环芳烃受体与二恶英反应元件结合能力的测定  

THE MENSURATION OF BINDING ACTIVITY BETWEEN TRANSFORMED ARL HYDROCAR BON RECEPTOR AND DIOXIN RESPONSE ELEMENTS.

作　　者：孙晞[1] 徐顺清[2] 

机构地区：[1]广东暨南大学食品系,广州510630 [2]华中科技大学同济医学院环境医学研究所,武汉湖北430030

出　　处：《现代预防医学》2005年第3期189-191,共3页Modern Preventive Medicine

基　　金：国家自然科学基金 (2 0 10 70 0 2 ;2 0 3 770 17)

摘　　要：目的 :2 ,3,7,8-四氯化二苯并二恶英 (TCDD)激活细胞溶质中多环芳烃受体后形成的复合物可与含二恶英反应元件的DNA探针结合 ,本研究利用新建立的外切酶保护PCR方法测定此结合亲和力 ,为评估配体的生物或毒效应提供依据。方法 :将 2 0nmol/LTCDD溶液与 10 0 μlSD大鼠肝细胞溶质温育 ,再与 0～ 15nmol/L含二恶英反应元件的寡核苷酸探针作用形成复合物 ,用核酸外切酶Ⅲ和S1核酸酶进行消化后 ,利用SYBRGREEN荧光适明定量PCR测定其诱导产生的结合DNA含量 ,绘制饱和曲线。经过数学变换估算配体—AhR复合物与DNA结合反应的宏观解离常数 (Kd和最大受体结合量 (Bmax)。结果 :绘制出TCDD活化AhR结合DNA的饱和曲线 ,计算得 :宏观解离常数Kd值为 1. 7± 0 . 2 (nmol/L) 2 ,DNA探针的最大受体结合量为 (14 6 7± 12 3)fmol/mg蛋白。结论 :外切酶保护PCR分析可测定配体诱导产生的AhR -DNA结合活性 ,为二恶英作用强度提供信息。Objective:2,3,7,8-tetrachlorodibenzo-p-dioxin activ ated the arl hydrocar bon receptor (AhR)and formed a complex which could bind a DNA probe containing t wo dioxin response elements (DREs)sites .In this study the binding activity betw een transformed AhR and DREs was measured using exonuclease protection PCR assay .Methods:20 nmol/L TCDD dissolved in DMSO,was added into 100 μ1S D rat hepalic cytosol in vitro,and ligand-AhR-DRE complex are formed in additi on of 0～15 nmol/L DNAs probes containing the sequence of DRE.With the digestion of Exonuclease Ⅲand S1 nuclease,free DNAs were digested to oligonucl eotide and binding DNA remained due to protein (AhR)protection.The real-time PCR was used to quant i fy the binding DNA and the saturation binding curve was generated.Through mathe matical transform,the equilibrium dissociation constant (K d)of the TCDD:AhR co mplex binding to DREs and maximal receptor binding capacity (B max )were det e rmined.Results:The equilibrium dissociation constant (K d)here w as 1.7 ±0.2 (mmol/L)2,and the B mas was 1467±123 fmol/mg protein.Conclusion :It demon strated that this method was valid to estimate the affinity of transformed AhR b inding to DREs.

关 键 词：配体 受体结合 DNA探针 反应元件 诱导 解离常数 DNA结合活性 二恶英 多环芳烃 TCDD 

分 类 号：R730[医药卫生—肿瘤] X705[医药卫生—临床医学]