基于克隆表达的芳香烃受体系统的二噁[口英]生物检测方法研究

Construction of the dioxin bioassay method based on the clonal expressed aryl hydrocarbon receptor system

目的研究人工克隆表达的芳香烃受体核转位蛋白（ARNT）与天然芳香烃受体（AhR）特异性结合及其对制备的多克隆抗体识别的情况，通过两者结合与二噁[口英]（TCDD）的剂量反应关系，探索TCDD生物检测方法。方法（1）以小鼠肝组织总RNA为模板，通过RT—PCR扩增目的基因AhR—PAS[periodicity （Per）／Aryl hydrocarbon nuclear translocator（ARNT）／single—minded（Sim）]端、AhR羧基末端（AhR—C端）、ARNT—PAS端，构建含有目的基因的pGEX-5XI重组表达质粒，并采用大肠杆菌原核系统进行克隆表达。（2）用上述克隆表达的AhR片段蛋白（AhR—PAS、AhR—C）作为抗原免疫家兔，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复合物，将天然AhR复合物与结合在谷胱甘肽硫转移酶（GST）上面的融合蛋白GST—ARNT—PAS，分别在0．25、0．50、1．00、2．00pmol TCDD存在的条件下进行结合，通过亲和吸附和免疫印迹观察所形成的蛋白复合物的量。结果（1）成功构建含有目的基因AhR—PAS、AhR—C和ARNT—PAS的重组质粒，并通过大肠杆菌原核系统表达出具有结合活性的目的蛋白；（2）免疫家兔制得的多克隆抗体AhR—PAS、AhR-C的检测下限分别为5、1ng；（3）测得该法制备的小鼠肝脏匀浆中总蛋白质的浓度为60．5mg／ml；在TCDD存在的条件下，克隆得到的融合蛋白GST—ARNT—PAS能够与天然AhR复合物特异性地结合，并测得检测下限为0．25pmol，约80pg TCDD。结论成功地建立了基于克隆表达的芳香烃受体系统的TCDD生物检测方法，且检测下限达到pg级别。

Objective To study the specific binding of the artificial clonal aryl hydrocarbon receptor translocator（ARNT） with the natural aryl hydrocarbon receptor（AhR） and the recognization by polyclonal antibody. The dose-response relationship with tetrachlo-rodibenzo-dioxin （TCDD） was also studied to develop TCDD detection method and the binding degree related to dose response. Methods （ 1 ） The target genes including AhR-PAS,AhR-C and ARNT-PAS were amplified by RT-PCR by using the total RNA purified from the liver cells of C57BL/6J mice as templates to construct pGEX-5X1 recombinants. The recombinant plasmids were expressed in E. coli. （2） The rabbits were immuned by the clonal fusion proteins： AhR-PAS, AhR-C to prepare the polyclonal antibody. （3） The natural AhR from the hepatic cytosol of C57BL/6J mice was extracted. The artificial cloning expressed fusion protein： GST-ARNT-PAS and the natural AhR were incubated in different dose of TCDD. The quantity of the heterodimer through affinity adsorption and Western blots were measured. Results （1） The target proteins including AhR-PAS, AhR-C and ARNT-PAS were successfully cloned and expressed in E. coll. （ 2 ） The detection limit of polyclonal antibody AhR-PAS and AhR-C were 5 ng and 1 ng, respectively. （3）The total protein concentration prepared from the liver ceils was60. 5 mg/mL The artificial clonal protein ARNT-PAS could specifically bind to the natural AhR complex with the existence of TCDD. The detection limit of TCDD was 0.25 pmol which was 80 pg approximately. Conclusion A TCDD detection method based on the aryl hydrocarbon receptor system was established and the detection limit might reach pg grade.