高通量筛选雌激素类化合物重组绿色荧光蛋白酵母细胞测评体系

Bioassay of Recombinant Green Fluorescent Protein Gene Yeast Cell for a High Throughput to Screen Estrogenic Compounds

摘要:

以载体双表达的方式构建重组酵母环境雌激素的评价体系, 用于快速筛选雌激素类化合物。在表达载体中, 用3-磷酸甘油醛脱氢酶(GPD)启动子驱动a人雌激素受体基因(hERa)的表达; 在报告载体中, 用雌激素效应元件(ERE)调控的绿色荧光蛋白(yEGFP)作为报告基因。将两者转化于酵母细胞(W303-1A)中, 构建成重组绿色荧光蛋白酵母细胞。该酵母细胞经不同浓度的雌激素类化合物作用后, 发现GFP的表达量与此类受试物具有明显的剂量效应关系。与其他环境雌激素酵母评价体系相比, 该重组酵母评价细胞, 在应用时不需要破坏细胞壁, 也不需要底物和相关试剂, 可直接在96孔板中操作完成, 具有快速、高通量、敏感性高、重现性好及廉价等特点。

Abstract:

We developed the recombinant green fluorescent protein gene yeast cell to screen estrogenic compounds based on two episomal vectors. In the expression vector the expression of human estrogen receptor a(hERa) was driven by 3-glyceraldehyde- phosphate dehydrogenase (GPD) promoter; in the reporter vector the expression of the yeast enhanced green fluorescent protein (yEGFP) gene was under the control of the estrogen response element (ERE). The vectors were transformed into yeast cell (W303-1A) to construct GFP recombinant yeast cell. Incubation of the yeast cell with various concentrations of the estrogenic compounds led to expression of the reporter gene product GFP in a dose dependent manner. Compared to other yeast bioassays, the yeast cell for environmental estrogen bioassay based on yEGFP reporter gene did not need cell wall disruption or the addition of a substrate or reagent. This yEGFP assay was performed completely in 96 well plates. So this test system can be used as a rapid and high throughput system for screening estrogenic chemical products, which has the characteristics of the sensitivity, reproducibility and cheapness.