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Phthalic acid ester (PAEs) is a kind of environmental endocrine disruptors. It is widely used in our daily life in recent decades, such as plastics, cosmetic, medical appliance and the food additive. Phthalic acid ester is used as the plasticizers, as time goes on, they will transfer from plastics to external environment, because they combination with polyvinyl chloride mainly by hydrogen bonds and van der Waals force. It becomes a widespread global environmental pollutant, and have been identified in river water, sea water, industrial effluents, fish and food supply, it will harmful to biological system and human beings directly. Some researches have shown that, phthalic acid ester has some toxic effects on human and animal liver, reproductive system and immune system, in all of them the endocrine disruption is the most direct effect, otherwise, it has some long-term toxic effects. At present, the United States, Japan, China and some other countries classify it as an priority monitoring pollutants.These effects are mediated by the estrogen receptors. In this study, we carried on a vitro testing to study interaction between estrogen receptor and PAEs in fish, and to lay the foundation for future research.To dibutyl phthalate as the representative of phthalic acid esters, put it into the water to feed carp, in order to induce to produce more estrogen receptors. We separated high-purity estrogen receptors with high speed centrifugation the first step, then with the method ofsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution. In SDS-PAGE experiment, we made a 5% stacking gel and 12% separation gel, took 80V voltage at the first 20min and 120V the rest of time in Tris-Gly buffer (the whole process in the ice bath). For electroelution, we used Tris-Gly buffer (50mmol/L Tris,50mmol/L Gly,0.1% SDS, pH8.9) as electroelution buffer in a conventional vertical electrophoresis tank at 4℃. Elution was performed at 100V for 3h. We obtained 2.1mg receptor from each 10 plastic gel and purity up to 88.5%. This is a simple and high-efficiency method to purification of the estrogen receptor, it can obtain a high-purity protein with a high recovery. It suitables for the proteins with lower content in cells and other proteins of similar molecular weight.In this paper, molecular self-assembly technique was used to prepare the specific chip. The chain avidin (SA) linked to the thiol monolayer which is prepared by 3-mercaptopropionic acid monolayers.70μL receptor was fixed on that chip on the surface plasmon resonance (SPR), then started the binding studies that interacted between DBP and estrogen receptor. As the result, DBP can bind with estrogen receptor steadily, the amount of combination increase with the increasing concentration of DBP, when the DBP concentration of 1×10-9g/L, the receptor binding site is closer to saturation. In addition, fourier transform infrared spectroscopy (FTIR) is used to study the structure of estrogen receptor change that interact with DBP. The result proved that an obvious effect on the structure of receptor after combination with DBP, the structural changes occurred mainly in the amideⅠband, the amideⅢband, the amideⅤband and the fingerprint area.
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