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

CHENG Jia; WANG Yong-ji

doi:10.13982/j.mfst.1673-9078.2017.8.007

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.