鉴定活性维生素D细胞模型的构建

Construction of a Cell Model for the Identification of Active Vitamin D

摘要:

本文构建了维生素D受体（Vitamin D receptor，VDR）融合蛋白的真核表达载体，并获得稳定表达VDR融合蛋白的细胞体系，为含有活性维生素D食品及药物的鉴定建立成熟的细胞模型。设计并合成特异性引物扩增VDR基因，将扩增产物克隆至pcDNA 3.1/His真核表达载体上，将其转染HEK293细胞。采用免疫沉淀（IP）技术鉴定VDR融合蛋白的表达效率。在构建pcDNA 3.1/VDR-His成功的基础上，用活性1,25(OH)2D3处理转染的细胞，收集总蛋白和mRNA，分别利用免疫印迹（Western blot，WB）和实时定量PCR（qRT-PCR）技术，检测VDR融合蛋白以及其下游基因CYP24A1基因的表达。IP结果证实VDR融合蛋白具有转录活性。WB和qRT-PCR结果显示，1 nmol/L浓度的活性1,25(OH)2D3处理转染细胞能有效激活VDR蛋白的表达，以及显著增强CYP24A1的mRNA表达（p<0.01）。利用该细胞模型检测维生素D类药物的活性，结果显示不同药物中维生素D的活性与药物种类有关。pcDNA 3.1/VDR-His融合表达载体构建成功，并以此构建了鉴定活性维生素D的细胞模型。

Abstract:

A eukaryotic expression vector containing a vitamin D receptor (VDR) fusion protein was constructed, enabling the creation of a cell system that could stably express VDR fusion protein, and a mature cell model for the identification of food and drugs containing active vitamin D was established. Specific primers were designed and synthesized to amplify the VDR gene, the gene amplification products were cloned into a pcDNA 3.1/His eukaryotic expression vector, and the vector was then transfected into HEK293 cells. Immunoprecipitation (IP) was used to identify the expression efficiency of the fusion protein. Using the successfully constructed pcDNA 3.1/VDR-His vector, the transfected HEK293 cells were treated with an active form of vitamin D, 1,25-dihydroxyvitamin D3, and then total protein and mRNA were quantified. Western blot (WB) analysis and quantitative real-time PCR (qRT-PCR) were used to detect the expression of the VDR fusion protein and its target gene, CYP24A1, respectively. The IP results showed that the VDR fusion protein had transcription activity. WB and qRT-PCR results showed that 1 nmol/L 1,25-dihydroxyvitamin D3 could effectively activate VDR fusion protein expression in the transfected cell system and significantly increase CYP24A1 mRNA expression (p < 0.01). This cell model was used to determine the activity of vitamin D drugs, and the results suggested that the vitamin D activity of different drugs was related to drug type. The pcDNA 3.1/VDR-His fusion-protein expression vector system was successfully constructed, and a cell model to identify active vitamin D in food and drugs was successfully established using the working vector.