研究报告
李剑,饶凯锋,马梅,王子健.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[J].环境科学学报,2011,31(1):33-39
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
- 基金项目：国家自然科学基金项目(No.20737003);中国科学院知识创新工程重要方向项目(No.KZCX2-YW-Q02-05)
- 作者
- 单位
- 李剑
- 北京师范大学, 水科学研究院, 地下水污染控制与修复教育部工程研究中心, 北京 100875
- 饶凯锋
- 中国科学院生态环境研究中心环境水质学国家重点实验室, 北京 100085
- 马梅
- 中国科学院生态环境研究中心环境水质学国家重点实验室, 北京 100085
- 王子健
- 中国科学院生态环境研究中心环境水质学国家重点实验室, 北京 100085
- 摘要：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.
- Abstract：Arecombinant estrogen-related receptor (ERR) yeast strain was constructed using two-hybrid yeast technique and was used to screen the ERRanti/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-OHTcould 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. Amethod 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 BPAitself neither induced nor inhibited the enzyme activity. However,when exposed together with 4-OHT,BPAcould suppress the ERRγ-GRIP1 yeast inhibition activity induced by 4-OHT. Furthermore,the activity of BPAwas strengthened after metabolic activation. Our results demonstrated the suitability of the constructed ERRγ-GRIP1 yeast for screening endocrine-disrupting chemicals.
- Key words：two-hybrid yeast estrogen-related receptor recombinant gene yeast endocrine disrupting chemicals
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