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