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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pcDNA3.1/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)_2D_3处理转染的细胞,收集总蛋白和m RNA,分别利用免疫印迹(Western blot,WB)和实时定量PCR(q RT-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q RT-PCR结果显示,1 nmol/L浓度的活性1,25(OH)_2D_3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m RNA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。pcDNA3.1/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。
In this paper, a eukaryotic expression vector for vitamin D receptor (VDR) fusion protein was constructed and a cell system stably expressing VDR fusion protein was obtained. A mature cell model was established for the identification of foods and drugs containing active vitamin D. The specific primers were designed and synthesized to amplify VDR gene. The amplified product was cloned into pcDNA3.1 / His eukaryotic expression vector and transfected into HEK293 cells. Immunoprecipitation (IP) was used to identify the expression efficiency of the VDR fusion protein. Based on the successful construction of pcDNA3.1 / VDR-His, the transfected cells were treated with 1,25 (OH) 2D3 and the total protein and m RNA were collected and analyzed by Western blot (WB) and real-time PCR q RT-PCR) was used to detect the expression of VDR fusion protein and its downstream gene CYP24A1. IP results confirm that the VDR fusion protein is transcriptionally active. The results of WB and q RT-PCR showed that 1 nmol / L 1,25 (OH) _2D_3 could effectively activate the expression of VDR protein and enhance the m RNA expression of CYP24A1 (p <0.01). The cell model was used to detect the activity of vitamin D drugs. The results showed that the activity of vitamin D in different drugs was related to the type of drugs. The pcDNA3.1 / VDR-His fusion expression vector was successfully constructed and a cell model for the identification of active vitamin D was constructed.