维生素D受体核定位信号的鉴定

Identification of nuclear localization signal of vitamin D receptor(VDR)

目的:确定维生素D受体(vitamin D receptor,VDR)的核定位信号并验证其功能。方法:通过软件分析及文献调研预测VDR核定位信号,利用点突变试剂盒构建了VDR一系列点突变体,通过蛋白质免疫印迹检测VDR在胞质和胞核里的分布,并结合免疫荧光观察确定其预测突变点对VDR入核的影响,最后采用实时荧光定量链式聚合酶反应(quantitative real-time PCR,qPCR)检测VDR下游基因的表达,以判断VDR核定位突变体的活性。结果:确定VDR核定位信号序列为49RRSMKRKALFLT61,构建的VDR一系列核定位序列(nuclear localization sequence,NLS)点突变体在细胞内的表达主要分布在胞质里,维生素D(vitamin D,VD)既不能诱导这些点突变体入核,也不能促进VD下游基因表达。结论:VDR核定位确定信号能有效控制VDR进入细胞核发挥转录因子作用,进而影响其下游基因的表达,为探索VD及VDR相关疾病的治疗提供新平台和思路。

Objective:To determine the nuclear localization signal of vitamin D receptor(VDR)and verify its function.Methods:The nuclear localization signal of VDR was predicted by software analysis and literature investigation.A series of point mutants of VDR were constructed by a point mutation kit.The distribution of VDR in the cytoplasm and nucleus was obsened by Western blot and immunofluoresceme to determine the effect of mutation points in the entry of VDR into the nucleus.Finally,quantitatve real-time PCR(q PCR)was used to detect the expression of genes downstream of VDR to determine the activity of VDR nuclear localization mutants.Results:The nuclear localization signal sequence of VDR was determined to be49RRSMKRKALFLT61.The expression of a series of NLS(nuclear localization sequence)point mutants of VDR was mainly distributed in the cytoplasm.Vitamin D could neither induce these point mutants into the nucleus nor promote the expression of downstream genes of vitamin D.Conclusion:VDR nuclear localization signal can effectively control the entry of VDR into the nucleus,play the role of transcription factors,and then affect the expression of downstream genes,which provides a new platform and idea for exploring the treatment of vitamin D and VDR related diseases.