【作者】 赵娜；

【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， 2005， 硕士

【摘要】 为建立二恶英生物检测体系,本课题在分析与研究AhR与ARNT两个蛋白质特征的基础上,克隆表达出AhR与ARNT两个全长蛋白及AhR的两个特征性多肽片段,并用后者免疫动物制备多克隆抗体。这些蛋白与抗体为检测体系的建立及相关研究奠定了重要的基础。 本研究以从C57BL/6J纯系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,通过琼脂糖凝胶电泳和DNA测序分析验证扩增产物的正确性后,将其定向克隆入pGEX-5X-1表达质粒中,构建了AhR和ARNT全长及片段AhR-PAS、AhR-C、ARNT-PAS共五个蛋白的pGEX-5X-1-融合蛋白重组表达质粒。将酶切鉴定结果正确的重组质粒转化入大肠杆菌BL21（DE₃）的感受态细胞中,通过诱导前后的SDS-PAGE分析,筛选出目的蛋白表达阳性的细菌克隆,通过划板或冻存菌液的方式保存好菌种。 然后,针对用于制备AhR多克隆抗体的两个片段蛋白AhR-PAS和AhR-C进行蛋白表达诱导条件的优化和大量提纯。通过对表达蛋白的细胞内定位分析,发现表达产物AhR-C大部分为存在于胞质中的可溶性蛋白,而AhR-PAS的表达产物主要以包涵体的形式存在于沉淀中,且通过优化诱导条件无法使其以可溶性蛋白的形式表达。鉴于此,我们分别采取了Glutathione Sepharose^TM 4B珠子亲和吸附与包涵体纯化的方法来提纯这两个片段蛋白的表达产物。然后利用收获的融合蛋白,采用2002年Cell Research介绍的一种快速制备多克隆抗体的新方法免疫新西兰大白兔制备AhR的特异性抗体。更多还原

【Abstract】 The objective of this project is to establish one rapid and specially dioxin detection method.This dissertation mainly contained two parts.The first part is the amplification of DNA sequence and the reconstruction of expression plasmid.The second part is the preparation of polyclonal antibody special to AhR. The content of this study provide the basis for establishment of dioxin bioassay system and related research on AhR.In this paper,the genes encoding AhR and ARNT were amplified by RT-PCR using the total RNA purified from the liver of C57BL/6J mice as templates and the oligonucleotide primers with restrictive sites at both ends for cloning purposes. The fidelity of PCR was determined by agarose electrophoresis and DNA sequence analysis. We ligated the DNA fragments purified from the agarose into expression vector pGEX-5X-1 by T₄ DNA ligase. The positive recombinant were validated by digestion of restrictive endonuclease. Then we transfered identified recombinant into competent cells of E.coli BL21（DE3）. Screening the positive clones which could express fusion proteins with predicted molecular weight after adding IPTG through SDS-PAGE analysis. Preserved the positive clones for future using.In order to prepared polyclonal antiserum especially to AhR, we plan to express and purify the two fragments of it in the first place. We found that most of the expressed AhR-C located in cytoplasm in the form of soluble protein,whereas the induced protein AhR-PAS mainly existed in the inclusion body. So we purified AhR-C by affinity adsorption of Glutathione Sepharose? 4B,but acquired the protein of AhR-PAS through purification of inclusion bodies.Then we prepared the polyclonal antiserum to AhR through immunizing the obtained fusion proteins to rabbits.更多还原