酵母双杂交技术构建重组雌激素受体相关受体(ERR)基因酵母

Using two-hybrid yeast technique to construct the recombinant estrogen-related receptor (ERR) yeast

应用酵母双杂交技术构建了重组雌激素受体相关受体(ERR)基因酵母,用以筛选环境中具有ERR干扰活性的化合物.实验先提取并纯化含有ERRγ基因的酵母表达质粒pGBT9-ERRγ,称为诱饵质粒;再提取并纯化含有ERRγ协同激活因子GRIP1基因的酵母表达质粒,形成靶质粒pGAD424-GRIP1,然后将诱饵质粒和靶质粒同时转化到酵母细胞Y187中,于营养缺陷型培养基(SD/-Trp/-Leu)上筛选阳性菌落,构建ERRγ-GRIP1双杂交酵母.同时,考察了ERRγ-GRIP1酵母与典型的雌激素拮抗剂——4-羟基他莫西酚(4-OHT)的结合情况.结果表明,4-OHT能够抑制ERRγ-GRIP1酵母酶活性,并存在显著的剂量-效应关系,半数抑制浓度(RIC50)值为7.9×10-6mol·L-1.最后,进一步建立了检测环境化合物诱导/抑制ERRγ-GRIP1酵母β-半乳糖苷酶活性的实验方法,用于检测典型的内分泌干扰物.双酚A(BPA)实验结果表明,BPA不能诱导/抑制ERRγ介导的酶活性,但BPA与4-OHT共同暴露时,能够修复4-OHT的抑制活性;经体外代谢活化后,BPA的修复活性得到加强,证实了该重组基因酵母用于环境污染物内分泌干扰效应筛选的可行性.

A recombinant estrogen-related receptor （ERR） yeast strain was constructed using two-hybrid yeast technique and was used to screen the ERR anti/agonistic activity of environmental pollutants. Two yeast expression plasmids were extracted and purified,including pGBT9-ERRγ and pGAD424-GRIP1. The constructed two-hybrid ERRγ-GRIP1 yeast was obtained by co-transforming the yeast cells Y187 with the pGBT9-ERRγ and pGAD424-GRIP1 plasmids,then selected by growth on synthetic dextrose （SD） medium （lacking tryptophan and leucine） in agar. The binding ability of the ERRγ-GRIP1 yeast with its antagonist,4-hydroxytamoxifen （4-OHT） was tested. The result showed that 4-OHT could inhibit the activity of recombinant gene yeast in a concentration-dependent manner with the concentration causing a 50% inhibition of the maximum effect （RIC50） of 7.9×10-6 mol·L-1. A method for testing ERRγ anti/agonistic activity with the constructed yeast was established and was used to detect the anti/agonistic activity of bisphenol A （BPA）,a typical environmental endocrine disruptor. The results showed that BPA itself neither induced nor inhibited the enzyme activity. However,when exposed together with 4-OHT,BPA could suppress the ERRγ-GRIP1 yeast inhibition activity induced by 4-OHT. Furthermore,the activity of BPA was strengthened after metabolic activation. Our results demonstrated the suitability of the constructed ERRγ-GRIP1 yeast for screening endocrine-disrupting chemicals.