The construction and the expression of V5 epitope fused human androgen receptor vector in the yeast cell / 生物医学工程学杂志

When we try to establish the gene recombinant yeast cell to screen the androgenic endocrine disruptors, the key procedure is the androgen receptor (AR) expression in the yeast cell. For this purpose, we obtained the GPD (glyceraldehyde-3-phosphote dehydrogenase) promoter from the yeast genosome of W303-1A using PCR system and inserting it into Swa I and BamH I sites of pYestrp2. The new constructed vector was named pGPD. The V5 epitope tag DNA with a 5'-BamH I and a 3'-EcoR I sticky end was cloned into the corresponding site of the pGPD vector to yield the vector of pGPDV5. The 2 723 bp full length AR ORF amplified by PCR from pcDNA3.1/AR was fused to V5 epitope tag DNA in pGPDV5 to give the AR yeast expression vector of pGPDV5/AR. This fused vector was transformed into the yeast cell (W303-1A). Western blot was used to detect the V5 fused protein of AR, in the protocol of which the primary monoclonal antibody (IgG(2a)) of mouse anti-V5 and the polyclonal secondary antibody of goat anti-mouse (IgG) linked to horseradish peroxidase (HRP) were used to detect the specific protein in the given sample of the transformed yeast extract. The result showed that the fused protein of AR was expressed successfully in the yeast cell.

The effects of TATA-box in CYC1 promoter on the reporter gene regulated by ERE in the recombinant yeast cell / 生物医学工程学杂志

The complementary oligonucleotides, each with two consensus estrogen response element (ERE)-sequences and 5'-Hind III and 3'-Sph I sticky ends were artificially synthesized. A solution with both the complementary DNA sequences was heated to 95'C and cooled down to room temperature to form double strand DNA (dsDNA). The set was cloned into the corresponding sites of CYC1 promoter of the pERE-CYC-yEGFP to yield pERE-CYCalpha-yEGFP vector. The two different reporter vectors, pERE-CYC-yEGFP and pERE-CYCalpha-yEGFP, the 2ERE, were placed in the CYC1 promoter. The former promoter downstream ERE contains alpha and beta-TATA boxes and the latter has only alpha-TATA box. The two different reporter vectors were transformed into the yeast cells that express human estrogen receptor alpha (ERalpha). Incubation of the recombinant yeasts with the six estrogenic compounds for 4 hours showed that the recombinant cell containing pERE-CYCalpha-yEGFP would give very poor dose-response curves, in contrast to the recombinant cell containing pERE-CYC-yEGFP which produced well-shaped dose-response curves. So it is necessary for this bioassay that alpha and beta-TATA boxes in the minimal CYC1 promoter when the promoter is used as a rapid and high throughput system for screening estrogenic chemical products.

Bioassay of recombinant green fluorescent protein gene yeast cell for a high throughput to screen estrogenic compounds / 生物工程学报

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 alpha(hERalpha) was driven by 3-glyceraldehydephosphate 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.

Monitoring of Electrophile Chemical Pollutants Using Transgenic Firefly Luciferase Gene Cells / 环境与健康杂志

Objective To assess quickly the potential environmental pollution by using the luciferase transgenic cells. Methods Many electrophile compounds in the environment can generate oxidative stress,so that the transcription of certain protective genes is induced via specific DNA motifs called electrophile response elements (EPREs). We have made a vector containing a single EPRE fused to the TK minimal promoter and the gene encoding firefly luciferase (EPRE-LUC) by adopting bio-molecular techniques. From this vector the stable LUC expression vector regulated by EPRE had been successfully reconstructed. This reporter construct was transfected into HeLa cells,and the clones resistant to G418 were selected. The resistant cells were treated by the different concentrations of sodium arsenate(NaAsO2),cadmium chloride (CdCl2),mercury chloride(HgCl2) and diethyl mateate (DEM). After that,the expression of luciferase was determined by luciferase assay kit. Results The correct construct frame of LUC reporter vector regulated by EPRE was identified by DNA sequencing; the dose-dependent relationships between the LUC expression and the test substance concentration were found. Among them,the relationship produced by DEM was the most significant. Conclusion The LUC transgenic cells regulated by EPRE have been successfully constructed.

Development of a Rapid Screening System of Environmental Estrogen Based on Recombinant Yeast Cell / 环境与健康杂志

Objective To develop a rapid screening method of environmental estrogen in vitro. Methods Estrogen responsive element (ERE) was inserted into the upstream of Lac Z (?-galactosidase ) reporter gene and the reporter gene was regulated under estrogen receptor(ER) in the recombinant yeast cells by the molecular biological technique. When the yeast cells was affected by environmental estrogen, conformational change of ER lead to expression through binding to ERE. There exist the functional relationships between the expression and the pollution degree of environmental estrogen. Therefore, the pollution degree can be reflected by assaying the activity of ?-galactosidase. Results It was identified by PCR that recombinant yeast cell, whose Lac Z reporter gene regulated under estrogen receptor, was successfully reconstructed. The experimental results showed that the time-dose-effect response had no significant changes when the recombinant yeast cell bad been exposed to ?-estradiol for 4 and 8 hours respectively; among the three methods of yeast disintegration, the ultrasonic vibration method had more advantage compared with that of the other methods and this dose-effect curve exhibited "S" shape; among 3 test chemical products, ?-estradiol was the most estrogenic activity, biosphenol A take second place, estradiol benzoate was the lowest. Conclusion The assay system of yeast cell for environmental estrogen hormone mediated by estrogen receptor is stable and applicable.