| Estrogens like drug residues in animal foods not only cause the damage t o human health, but also the effect to the environment and the sustained and healthy development of animal husbandry. And it is very concerned in the wor Id wide. Thought we have the detection methods for estrogenic drug residues s uch as gas chromatography (GC), high performance liquid chromatography (HP LC), enzyme immunoassay (ELISA) and etc., these methods have their some li mitations. These methods have high demand for operating people, sophisticated detection equipment and sample purity and also are limited by the detection s ite. Therefore they are not applicable the regular routine supervision for animal food safety. So it is particularly important to establish the simple, fast, effecti ve detection method for estrogenic drug residues in the animal body.This paper was to conducted to constructe the recombinant human estroge n receptor (hER) gene yeast cell of W303-1A/hER-ERE-Lac Z and W303-1A/h ER-ERE-yEGFP respectively. W303-1A/hER-ERE-Lac Z is a kind of yeast cell sensor, whose Lac Z reporter gene regulated by hER. When estrogen like dru gs combined with hER, this make the Lac Z gene expression product, (3-galac tosidase, increased. So we can monitor estrogenic drugs in animal urine by me asuring the activity levels of β- galactosidase. This is especially suitable for ba sic units to carry out routine monitoring. For another the recombinant human e strogen receptor (hER) gene yeast cell of W303-1A/hER-ERE-yEGFP is a kin d of fluorescent yeast cells, by measuring the fluorescenct density of which the estrogenic drugs in urine can be determined. This cytosensor is no need to ad d any reagent with fast and convenient features.Materials and methods:(1) pMP206/ERE and pLZ-yEGFP, which were regulated by the estrogen response element, were transformed in the recombinant human estrogen receptor (hER) gene yeast cell using the methods of LiAC respectively and the positive transformants were selected by the SD/-trp,-ura selective culture plate. The positive clones were identified by PCR. After that, the recombinant Lac Z-gene yeast cells and yEGFP-fluorescent yeast cells regulated by estrogen were constructed successfully and were named W303-1A/hER-ERE-Lac Z and W303-1A/hER-ERE-yEGFP respectively. Then these cells were inoculated on SD/-trp,-ura selective culture plate by streaking and put into the incubator culture for 3-4d at 30℃.(2) A single clone of the recombinant gene yeast was picked out from the yeast culture plates and incubated into the yeast culture liquid with oscillation at 30℃ to be activated. The activated yeast culture was diluted 3 times with yeast cell culture.(3) The urine samples of 22 from calf and pregnant cow calves were collected from large-scale farms respectively, and the urine samples of 78 from piglet and sow from another large-scale farms. The samples were treated by processes of filtration, hydrolysis, C18 column adsorption, chloroform elution and using dimethyl sulphoxide (DMSO) to a constant volume. W303-1A/hER-ERE-Lac Z cell and W303-1A/hER-ERE-yEGFP were treated with 0.010ml of the above DMSO for 6 hours with oscillation at the temperature of 30℃, and the β galactosidase activity or the fluorencent density of the cell were measured. Estradiol and distilled water were used as positive control and as the reagent blank control.Results:The results of all spiked calf urine simples (estradiol, diethylstilbestrol and ethinylestradiol) by two kinds of recombinant gene yeast cells were positive. For the results from W303-1A/hER-ERE-Lac Z detection, the detection limit of estradiol was 10-10mol/L. And it was found that estrogen like compounds in urine from different period of pregnant cows, calf, piglets and sows are no significant difference compared with the blank control (P>0.05). And also estrogen compounds in urine from the different periods of piglets and sows have no statistical significant difference (P>0.05). As far as W303-1A/hER-ERE-yEGFP research was concerned, though estrogenic compounds in urine samples from adult cow were higher than that of calf, but the statistical analysis showed that there were no significant differences (P>0.05). For piglets and sows the estrogenic compound concentrations in the urine were very lower even below the detection limit levels of W303-lA/hER-ERE-yEGFP cells(< 10-9 mol/L estradiol equivalent).Conclusion:(1) This methods can detect the compounds or drugs with estrogenic activities and have the characteristics of convenient, fast and so on; it is especially suitable for a large sample of primary screening at the basic units.(2) The urine sample is the detective object for this method and compared with that of the other samples it has the characteristics of the convenient collection and can be realized in vivo. And also the cost of animal products quality and safety monitoring can be decreased and carry out the timely and effective early warning for animal products quality and safety.(3) The large-scale aquaculture enterprises investigated have standard feeding and no illegal use of the fattening drugs such as estrogens. |
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