利用唐鱼雌激素受体构建环境雌激素重组酵母测评系统的研究

Recombinant yeast assay system for environmental estrogens mediated by estrogen receptor from Tanichthys albonubes

利用唐鱼雌激素受体基因片段构建重组酵母,用以筛选环境雌激素类化学物质.实验先将唐鱼雌激素受体terα基因片段插入载体pGADT7中构建表达质粒p GADT7/TERα,同时将雌激素效应元件(ere)片段插入pMP206载体中构建报告质粒pMP206/ERE-Lac Z;然后把表达质粒和报告质粒共转化到酵母AH109中,经筛选成功构建了由唐鱼雌激素受体调控的表达lac Z基因的重组酵母AHpTERα/ERE.结果表明,所构建的重组酵母AHpTERα/ERE在不同浓度17β-雌二醇(E2)的诱导下,β-半乳糖苷酶的活性呈现出明显的剂量-效应关系,其EC50为(0.521±0.700)nmol·L-1.与DMSO对照组相比,重组酵母在17β-雌二醇诱导下有明显的β-半乳糖苷酶活性增强的现象.在不同浓度17α-乙炔基雌二醇(EE2)、壬基酚(NP)及其混合物、双酚A(BPA)、17β-雌二醇(E2)(阳性对照)的诱导下,重组酵母均呈现出剂量-效应关系,且灵敏度大小为E2>EE2>NP>BPA.结果表明,本研究成功构建了重组基因酵母测评系统,初步判定该重组酵母可应用于环境雌激素的筛选.

Yeast with recombinant estrogen receptor fragment of Tanichthys albonubes( terα) was used to screen the environmental estrogen-like compounds. terα and estrogen responsive element( ere) were cloned into pGADT7 and pMP206 respectively to obtain receptor( pGADT7 / terα) and reporter( p MP206 / ERE-Lac Z) constructs. The recombinant yeast AHpTERα / ERE was obtained by co-transformation of the receptor and reporter constructs,in which the expression of lac Z is regulated by the activity of terα. The assay sensitivity was evaluated by different concentrations of 17β-estradiol( E2) treatment. The results showed that the activity of beta-galactosidase induced by E2 was dose-dependent,and was significantly higher than that of DMSO control. The EC50 of E2 was( 0. 521 ± 0. 700) nmol·L- 1. The assay system is also sensitive to other estrogen compounds like 17α-ethynylestradiol( EE2),Bisphenol A( BPA) and nonylphenol( NP) technical mixtures dose-dependently,and the sensitivity of different chemicals ranked as E2> EE2> NP > BPA. The results showed that recombinant yeast assay system for environmental estrogens could be applied to screen the estrogens-like compounds in the environment.