食品中潜在环境内分泌干扰物的受体通道研究
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摘要
本文旨在通过运用分子对接技术初步筛选可能与受体hERα、hERRγ、hVDR结合的食品中广泛使用和存在的潜在EDCs,再构建hERα、hERRγ、hVDR三种重组质粒,原核表达hERα、hERRγ、hVDR这三种受体蛋白,建立探索食品中潜在EDCs与重组受体蛋白结合活力的磷酸对硝基苯酚法,探索可能通过hERα、hERRγ、hVDR受体通道而对人体产生危害的物质。
通过从H295R细胞克隆hERRγ基因,从Caco-2细胞克隆hVDR基因,分别构建重组质粒pGEX-4T -1-hERRγ、pGEX-4T-1-hVDR。重组质粒转化感受态大肠杆菌DH5α,经过蓝白斑筛选、酶切验证、测序等证明hERRα、hVDR已成功克隆和转化。原核表达并纯化了GST-hERRγ、GST-hVDR融合蛋白,同时表达并纯化了实验室已构建的重组菌pGEX-4T -1-hERα、pET28-TIF2-BAP的GST-hERα、TIF2-BAP融合蛋白。建立了hERα结合活性的磷酸对硝基苯酚法、hERRγ结合活性的磷酸对硝基苯酚法、hVDR结合活性的磷酸对硝基苯酚法。
运用分子对接技术,初步筛选出可能具有hERα、hERRγ、hVDR结合活性的六大类食品中广泛存在的潜在EDCs,分别为:对羟基苯甲酸甲酯类、水杨酸酯类、邻苯二甲酸酯类、双酚A及其类似物、异黄酮类、重金属类。
采用重组受体蛋白结合活性的磷酸对硝基苯酚法,测定了对羟基苯甲酸酯类、水杨酸酯类、邻苯二甲酸酯类、双酚A及其类似物、异黄酮类、重金属类这六大类物质与受体hERα、hERRγ、hVDR的结合活性,并进行了比较分析。结果表明,对羟基苯甲酸酯类物质与hERα、hERRγ均有结合活性,水杨酸酯类物质除水杨酸苯酯与hERRγ几乎没有结合活性外,其他水杨酸酯类物质都与hERα、hERRγ有结合活性。邻苯二甲酸类物质与hERα均有结合活力,但是仅邻苯二甲酸二甲酯和邻苯二甲酸二乙酯与hERRγ有微弱活性,EC50分别为2.77×10-4mol/L、3.07×10-3mol/L。双酚A及其类似物中,4,4-联苯二酚与hERα具有很强的结合活性,EC50达到6.80×10-6mol/L,四溴双酚A仅与hERRα具有结合活性,EC50达到3.11×10-5mol/L。异黄酮类、重金属类物质与hERα、hERRγ均有一定的活性。hVDR受体仅与重金属具有活性。
运用分子对接模拟技术,对采用重组受体蛋白结合活性的磷酸对硝基苯酚法测定出的典型EDCs进行进一步打分验证,结果表明,分子对接模拟结果与基于重组受体蛋白结合活性的磷酸对硝基苯酚法测定出的结果一致。
The purpose of this study is utilizing molecular docking techniques to screen the potential EDCs which may combined with hERα, hERRγand hVDR, to build hERα, hERRγand hVDR three kinds of recombinant plasmid and to prokaryotic expression hERα, hERRγ, hVDR proteins. And it will explore the potential EDCs which contains in food by using the method of p-nitrophenylphosphate-alkaline phosphatase.
With gene cloned method of hERαgene from H295R cells and hERRγfrom Caco-2 cells, we recombinant plasmid pGEX-4T-1-hERRγand pGEX-4T-1-hVDR.Recombinant plasmid was transformed into Escherichia coli DH5α, through blue-white screening, enzyme restriction, and sequencing, which proved hERRαand hVDR been cloned and transformed successfully.
Prokaryotic expression and purification of GST-hERRγand GST-hVDR fusion protein were done in this study, and we have expressed and purificated the GST-hERαand TIF2-BAP fusion protein which had been found in recombinant bacteria pGEX-4T-1-hERαand pET28-TIF2-BAP. And we also established a method of p-nitrophenylphosphate-alkaline phosphatase to analyse the effects of chemical on the binding activity between hERα,hVDR ,hERRγand TIF2-BAP.
Using molecular docking techniques, we have screened six kinds of potential EDCs in foods which may have binding activity with hERα, hERRγand hVDR. These EDCs are: parabens, salicylic acid esters, phthalic acid esters, bisphenol A and its analogues, isoflavones, heavy metals.
Using method of p-nitrophenylphosphate-alkaline phosphatase to analyse the effects of chemical on the binding activity between hERα, hERRγ,hVDR and TIF2-BAP, to determinate the binding activity of parabens, salicylic acid esters, phthalate esters, bisphenol A and its analogues, isoflavones, heavy metals with receptors. The results showed that parabens all had some binding activity with hERα, hERRγ.Only phenyl salicylate had no binding activity with hERRγ, other salicylic acid esters all had some binding activity with hERα, hERRγ. Phthalic acid classes all had binding activity with hERα, but only dimethyl phthalate,diethyl phthalate had weak activity with hERRγ, the median effective concentration were 2.77×10-4mol/L, 3.07×10-3mol/L.In bisphenol A and its analogues, there is a strong binding activity between 4,4-dihydroxybiphenyl and ERα,the median effective concentration was 6.80×10-6mol/L. Tetrabromobisphenol A only had binding activity with hERRα, the median effective concentration was 3.11×10-5mol/L. There were some binding activity between Isoflavones, heavy metal substances and ERα,ERRγ. Only heavy metals had some binding activity with hVDR receptor in those six kinds of potential EDCs.
Using molecular docking techniques to test the typical EDCs which been set out through the method of p-nitrophenylphosphate-alkaline phosphatase. the result showed that there had the same conclusion by using the method of Molecular docking techniques and the method of p-nitrophenylphosphate-alkaline phosphatase which can test the binding activity of chemicals and receptors.
通过从H295R细胞克隆hERRγ基因,从Caco-2细胞克隆hVDR基因,分别构建重组质粒pGEX-4T -1-hERRγ、pGEX-4T-1-hVDR。重组质粒转化感受态大肠杆菌DH5α,经过蓝白斑筛选、酶切验证、测序等证明hERRα、hVDR已成功克隆和转化。原核表达并纯化了GST-hERRγ、GST-hVDR融合蛋白,同时表达并纯化了实验室已构建的重组菌pGEX-4T -1-hERα、pET28-TIF2-BAP的GST-hERα、TIF2-BAP融合蛋白。建立了hERα结合活性的磷酸对硝基苯酚法、hERRγ结合活性的磷酸对硝基苯酚法、hVDR结合活性的磷酸对硝基苯酚法。
运用分子对接技术,初步筛选出可能具有hERα、hERRγ、hVDR结合活性的六大类食品中广泛存在的潜在EDCs,分别为:对羟基苯甲酸甲酯类、水杨酸酯类、邻苯二甲酸酯类、双酚A及其类似物、异黄酮类、重金属类。
采用重组受体蛋白结合活性的磷酸对硝基苯酚法,测定了对羟基苯甲酸酯类、水杨酸酯类、邻苯二甲酸酯类、双酚A及其类似物、异黄酮类、重金属类这六大类物质与受体hERα、hERRγ、hVDR的结合活性,并进行了比较分析。结果表明,对羟基苯甲酸酯类物质与hERα、hERRγ均有结合活性,水杨酸酯类物质除水杨酸苯酯与hERRγ几乎没有结合活性外,其他水杨酸酯类物质都与hERα、hERRγ有结合活性。邻苯二甲酸类物质与hERα均有结合活力,但是仅邻苯二甲酸二甲酯和邻苯二甲酸二乙酯与hERRγ有微弱活性,EC50分别为2.77×10-4mol/L、3.07×10-3mol/L。双酚A及其类似物中,4,4-联苯二酚与hERα具有很强的结合活性,EC50达到6.80×10-6mol/L,四溴双酚A仅与hERRα具有结合活性,EC50达到3.11×10-5mol/L。异黄酮类、重金属类物质与hERα、hERRγ均有一定的活性。hVDR受体仅与重金属具有活性。
运用分子对接模拟技术,对采用重组受体蛋白结合活性的磷酸对硝基苯酚法测定出的典型EDCs进行进一步打分验证,结果表明,分子对接模拟结果与基于重组受体蛋白结合活性的磷酸对硝基苯酚法测定出的结果一致。
The purpose of this study is utilizing molecular docking techniques to screen the potential EDCs which may combined with hERα, hERRγand hVDR, to build hERα, hERRγand hVDR three kinds of recombinant plasmid and to prokaryotic expression hERα, hERRγ, hVDR proteins. And it will explore the potential EDCs which contains in food by using the method of p-nitrophenylphosphate-alkaline phosphatase.
With gene cloned method of hERαgene from H295R cells and hERRγfrom Caco-2 cells, we recombinant plasmid pGEX-4T-1-hERRγand pGEX-4T-1-hVDR.Recombinant plasmid was transformed into Escherichia coli DH5α, through blue-white screening, enzyme restriction, and sequencing, which proved hERRαand hVDR been cloned and transformed successfully.
Prokaryotic expression and purification of GST-hERRγand GST-hVDR fusion protein were done in this study, and we have expressed and purificated the GST-hERαand TIF2-BAP fusion protein which had been found in recombinant bacteria pGEX-4T-1-hERαand pET28-TIF2-BAP. And we also established a method of p-nitrophenylphosphate-alkaline phosphatase to analyse the effects of chemical on the binding activity between hERα,hVDR ,hERRγand TIF2-BAP.
Using molecular docking techniques, we have screened six kinds of potential EDCs in foods which may have binding activity with hERα, hERRγand hVDR. These EDCs are: parabens, salicylic acid esters, phthalic acid esters, bisphenol A and its analogues, isoflavones, heavy metals.
Using method of p-nitrophenylphosphate-alkaline phosphatase to analyse the effects of chemical on the binding activity between hERα, hERRγ,hVDR and TIF2-BAP, to determinate the binding activity of parabens, salicylic acid esters, phthalate esters, bisphenol A and its analogues, isoflavones, heavy metals with receptors. The results showed that parabens all had some binding activity with hERα, hERRγ.Only phenyl salicylate had no binding activity with hERRγ, other salicylic acid esters all had some binding activity with hERα, hERRγ. Phthalic acid classes all had binding activity with hERα, but only dimethyl phthalate,diethyl phthalate had weak activity with hERRγ, the median effective concentration were 2.77×10-4mol/L, 3.07×10-3mol/L.In bisphenol A and its analogues, there is a strong binding activity between 4,4-dihydroxybiphenyl and ERα,the median effective concentration was 6.80×10-6mol/L. Tetrabromobisphenol A only had binding activity with hERRα, the median effective concentration was 3.11×10-5mol/L. There were some binding activity between Isoflavones, heavy metal substances and ERα,ERRγ. Only heavy metals had some binding activity with hVDR receptor in those six kinds of potential EDCs.
Using molecular docking techniques to test the typical EDCs which been set out through the method of p-nitrophenylphosphate-alkaline phosphatase. the result showed that there had the same conclusion by using the method of Molecular docking techniques and the method of p-nitrophenylphosphate-alkaline phosphatase which can test the binding activity of chemicals and receptors.
引文
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