【摘要】 采用绿色荧光蛋白(GFP)作为报告基因,用酵母细胞构建环境雌激素(EEH)的评价体系,可敏感、快速、方便地筛选出EEH化合物。通过分子生物学技术,将GFP基因插入到pM P 206/ERE启动子CYC 1的下游,用GFP取代L ac Z基因,并转化于酵母细胞。DNA测序发现ERE-CYC 1-GFP框架正确。对转化子进行PCR鉴定,发现有雌激素受体基因(hER cDNA)和GFP基因特异条带,说明hER cDNA和GFP已重组于酵母细胞。荧光显微镜下发现大量的绿色荧光颗粒,表明GFP基因在酵母细胞中成功表达。酵母细胞经不同浓度雌二醇作用后,与GFP表达量有剂量效应关系。与其他酵母评价体系相比,以GFP为报告基因评价EEH不需要破坏细胞壁,也不需要底物,可在96孔板中完成,这为高通量筛选EEH化合物奠定了基础。更多还原

【Abstract】 An estrogen transcription activation assay system using yeast cells was developed,which is sensitive,fast and easy to use in the routine screening of estrogen activity from chemical products according to the reporter gene of green fluorescent protein(GFP).The yeast reporter vector was constructed by inserting GFP into the CYC1 promoter downstream of pMP206/ERE and the lacZ gene in the reporter plasmid was substituted by GFP gene.DNA sequences revealed that CYC1-ERE-GFP frame from the reporter vector was correct.The estrogen receptor gene(hER cDNA) and GFP gene were found by PCR method in the tranformants.This exhibited that hER cDNA and GFP gene had been recombined into the yeast(cell.) A lot of participles that emitted green light were observed under the fluorescent microscope.This illustrated that GFP gene was expressed in the yeast cell.And incubation of yeast with various concentrations of estrogen led to expression of GFP in a dose dependent manner.Compared to other yeast assays,the yeast cell for environmental estrogen bioassay based on GFP reporter gene does not need cell wall disruption nor does it need the addition of a substrate.This GFP assay can be performed completely in 96 well plates.So it creates a cornerstone that can be used as a high throughput system for screening estrogenic chemical products.更多还原