【作者】 王卓；

【导师】 张万起； 吴蕴棠；

【作者基本信息】 天津医科大学 ， 营养与食品卫生学， 2007， 硕士

【摘要】 目的采用分子生物和生物化学技术,依据芳香烃受体(AhR)在二恶英的活化作用下能与芳香烃受体核转位蛋白(ARNT)特异性结合的分子机制。本课题旨在探索一种基于芳香烃受体系统的快速、简便、经济且特异性很强的二恶英生物检测方法。方法(1)以小鼠肝组织中提取的总RNA为模板,采用RT-PCR的方法扩增目的基因,DNA测序分析验证后,利用pGEX-5X-1表达质粒,构建AhR和ARNT全长及相应片段AhR-PAS、AhR-C和ARNT-PAS共5个蛋白的重组表达质粒。酶切鉴定后将重组质粒转化入大肠杆菌BL21(DE3)的感受态细胞中,在该原核系统中大量表达目的蛋白质,采用谷光甘肽琼脂糖凝胶(Glutathione SepharoseTM 4B)亲和层析纯化GST-融合蛋白或利用凝血因子Xa对融合蛋白的进行酶切获得目的蛋白质。(2)利用AhR-PAS和AhR-C这两个特异性片段蛋白免疫新西兰大白兔制备多克隆抗体。(3)从C57BL/6J纯系小鼠肝组织中提取具有结合活性的天然AhR,采用Bradford法对总蛋白进行定量,计算出其中AhR的含量。(4)将天然AhR与GST-ARNT、GST-ARNT-PAS以等摩尔的比例混合,在室温下与不同剂量的TCDD进行温育结合。将结合了的GST-ARNT-AhR复合物用Glutathione Sepha-roseTM 4B珠子进行亲和吸附,并用结合缓冲液洗去非特异性结合蛋白。最后采用免疫印迹(Western Blot)等方法,以AhR-PAS或AhR-C两种蛋白制得的多克隆抗体作为检测所需的一抗,研究AhR与融合蛋白ARNT、ARNT-PAS结合的条件、程度及剂量反应关系,从而对TCDD进行定量分析。结果(1)成功地克隆了目的基因并在原核系统中表达了相应蛋白；(2)免疫家兔获得2种高效价的多克隆抗体,免疫印迹的检测下限分别是1 ng和5ng；(3)从C57BL/6J纯系小鼠肝组织中提取出具有结合活性的天然AhR,其浓度为3-7.2pmol/ml cytosol;(4)克隆蛋白与天然AhR在TCDD存在的前提下温育结合,免疫印迹足以检测到0.25 pmolTCDD；(5)在克隆蛋白与天然AhR充足的情况下,免疫印迹中的一抗所识别AhR的量随着TCDD剂量的增加而增加(呈剂量反应关系)。结论本课题应用免疫学和分子生物学等相关技术,为建立基于芳香烃受体系统的痕量二恶英生物检测方法提供了重要的研究基础与相关材料。更多还原

【Abstract】 Objective By the mechanism of the aryl hydrocarbon receptor (AhR) binding aryl hydrocarbon receptor translocator (ARNT) with the interaction of TCDD. This project is aiming at establishing one convenient, rapid and specially dioxins detection method.Methods (1) Amplified the genes encoding AhR and ARNT by RT-PCR using the total RNA purified from the liver of C57BL/6J mice as templates. The fidelity of PCR was determined by agarose electrophoresis and DNA sequence analysis. Ligated the DNA fragments purified from the agarose into expression vector pGEX-5X-1 to construct 5 recombinants. Then transfered the identified recombinant into the competent cells of E.coli BL21(DE3).To explore the condition of induction and the intracellular location of the prokaryotic expression system,and affinity purify the fusion proteins by Glutathione SepharoseTM 4B beads or blood coagulation factor Xa. (2) Prepared the polyclonal antiserum of AhR-PAS and AhR-C through immunizing the obtained fusion proteins to rabbits. (3) Prepared the hepatic cytosol of C57BL/6J mice, and then quantify the total proteins by Bradford to calculate the content of AhR. (4) Incubated AhR and GST-ARNT-PAS which molar ratio is 1:1 with different TCDD graded concentrations and precipitated the mixtures with Glutathione SepharoseTM 4B beads. Study the binding condition, binding degree and the dose response relation of the two proteins by Western Blot.Results (1) Acquire the aimed genes and express fusion proteins. (2) Acquire the polyclonal antiserum through immunizing the rabbits. (3) Purify the actived AhR from C57BL/6J mice hepatic cytosol. (4) The Incubation of two proteins with the min TCDD quantity is 0.25 pmol. (5) If the proteins for interaction is abundant, the Western Blot of TCDD shows positive correlation between the two proteins.更多还原