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目的通过检测重组酵母中报告基因的表达产物来反映环境雌激素通过雌激素受体介导而产生的拟雌激素作用。方法用乙酸锂法将本实验室已构建成功的全长人雌激素受体酵母表达载体pGAD_hER和雌激素效应元件(ERE)调控的酵母报告载体 pLacZi_2ERE导入酵母细胞中 ,得到重组酵母YM_ADER_ERE。结果与对照DMSO相比 ,加入17β_雌二醇(E2)后YM_ADER_ERE中报告基因lacZ的表达产物 β_半乳糖苷酶活性显著升高。用终浓度为10-15～10-7mol/L的E2 进行实验 ,E2 在YM_ADER_ERE中最大效应EAmax 为94 31U ,EC50 为0 34nmol/L。结论重组酵母中报告基因的表达是雌激素配体依赖的 ,符合环境雌激素测评系统的设计思想和要求 ,初步表明重组酵母测评系统是成功的
Objective To detect the effect of estrogen on estrogen receptor-mediated estrogen production by detecting the expression product of the reporter gene in recombinant yeast. Methods The recombinant yeast YM_ADER_ERE was obtained by introducing the yeast reporter vector pLacZi_2ERE which was successfully constructed in our laboratory into pGAD_hER and estrogen responsive element (ERE). Results The β-galactosidase activity of lacZ gene was significantly increased in YM_ADER_ERE after addition of 17β-estradiol (E2) compared with control DMSO. Experiments were performed with E2 at a final concentration of 10-15-10-7 mol / L. The maximal effect of E2 in YM_ADER_ERE was 94 31U with an EC50 of 0 34 nmol / L. Conclusion The expression of reporter gene in recombinant yeast is estrogen ligand-dependent and conforms to the design idea and requirements of environmental estrogen evaluation system, which initially indicates that the recombinant yeast evaluation system is successful