hVDR原核表达及其与重金属镉、铅的结合活性

Prokaryotic Expression of Human Vitamin D Receptor(hVDR) and Its Binding Activities to Cd and Pb

人类维生素D受体(hVDR)是重金属影响骨骼代谢的潜在受体通道,但目前没有重金属影响hVDR通道的报道.通过构建含有hVDR的pGEX-4T-1表达质粒,建立了原核表达具生物学活性的hVDR的方法;然后根据核受体与其配体结合后可以与核受体转录激活因子2(Transcriptional intermediary factor 2,TIF2)-细菌碱性磷酸酶(bacterial alkaline phosphatase,BAP)融合蛋白(TIF2-BAP)结合,建立了研究化学物质影响hVDR与核受体转录激活因子结合活性的磷酸对硝基苯酚法.通过该方法研究发现,镉(CdCl2)或铅(PbAc2)均能提高hVDR与TIF2-BAP的非配体依赖性结合.当加入100μmol/L和1000μmol/L氯化镉(CdCl2)后,结合活力分别显著上升至对照组的3.95和4.39倍(p<0.05);加入100μmol/L和1000μmol/L醋酸铅(PbAc2)后,结合活力分别显著上升至对照组的2.29和3.52倍(p<0.05).这表明镉和铅可能通过干扰hVDR受体通道的正常功能导致骨代谢异常和骨质疏松症的发生.

Human vitamin D receptor（hVDR） is a potential receptor channel for heavy metals to affect bone metabolism,while to date there is no report about the binding activity between heavy metals and hVDR.This study established a prokaryotic expression of hVDR system,by cloned hVDR-LBD into pGEX-4T-1 vector.Then according to the principle that the nuclear receptor binding with its ligand can be combined with nuclear receptor coactivator 2-bacterial alkaline phosphatase fusion protein（TIF2-BAP）,we established a method of p-nitrophenylphosphate-alkaline phosphatase to analyse the effects of chemical on the binding activity between hVDR and TIF2-BAP.Using this method,we studied the binding activities between hVDR and TIF2-BAP after exposure of cadmium and lead.The results showed that the binding activities significantly increased to 3.95 and 4.39 times that of the control after exposure of 100 μmol/L and 1 000 μmol/L cadmium chloride（CdCl2）,and the binding activities significantly increased to 2.29 and 3.52 times that of the control after exposure of 100 μmol /L and 1 000 μmol /L lead acetate（PbAc2）,respectively.These results indicate that cadmium and lead can mimic the activity of 1,25-（OH）2 D3,disrupt the normal function of hVDR receptor channel,which may be the underlying mechanism of abnormal bone metabolism and osteoporosis caused by cadmium and lead.