改进型重组基因酵母TR-GRIP1检测化合物甲状腺激素干扰活性

应用酵母双杂交技术构建重组TR(甲状腺激素受体)基因酵母，用以检测类/抗甲状腺激素化合物及环境样品的甲状腺激素干扰活性. 提取并纯化含有TR基因的酵母表达质粒pGBT9-TR以及含有TR共激活因子GRIP1基因的酵母表达质粒pGAD424-GRIP1，将pGBT9-TR和pGAD424-GRIP1同时转化至酵母细胞Y187，以营养缺陷型培养基(SD/-Trp/-Leu)筛选阳性菌落，构建TR-GRIP1双杂交酵母. 考察该酵母与天然甲状腺激素——T₃(三碘甲状腺原氨酸)的结合情况，确定最佳暴露时间，建立剂量－效应关系曲线. 结果表明:TR-GRIP1双杂交酵母能够与T₃结合诱导β-半乳糖苷酶活性，选择暴露时间为2 h，T₃诱导酶活性的EC₅₀值为1.1×10-7 mol/L；构建的重组基因酵母TR-GRIP1提供了检测化合物甲状腺激素干扰活性的新方法. 

Use of Modified Recombinant Gene Yeast TR-GRIP1 to Screen Chemicals for Thyroid Disrupting Activity

The recombinant thyroid hormone receptor(TR) gene yeast constructed using a two-hybrid yeast technique can be used to test the thyroid hormone disrupting effects of chemicals with TR antiagonistic activity and environmental samples.The two-hybrid TR-GRIP1 yeast was constructed by extracting and purifying two yeast expression plasmids,including pGBT9-TR coding for TR and pGAD424-GRIP1 coding for GRIP1,followed by co-transforming pGBT9-TR and pGAD424-GRIP1 into Y187,and selecting by growth on a synthetic medium(SD/-Trp/-Leu).The binding ability of TR-GRIP1 yeast to a natural ligand,3,3’,5-triiodo-L-thyronine(T3) was tested,and the best exposure time and the dose-effect relationship were also determined.The results show that T3 could induce the β-galactosidase activity of two-hybrid TR-GRIP1 yeast,and the best exposure time was 2 h.The EC50 value of the T3 was 1.1×10-7 mol/L.All the results proved that the recombinant gene yeast TR-GRIP1 assay provides a new method to screen chemicals for thyroid disrupting activity.