Establishment of a cell-base high-throughout screening model for pig CAR agonist

• 1、Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030

Abstract：To establish a new high-throughput screening model for the agonist of pgCAR (pig constitutive androstane receptor), pgCAR gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and cloned to pMD-18T Vector for sequencing, then the pgCAR fragment was excised by restriction enzymes, and inserted into pcDNA3.1 Vector to construct expression vector pcDNA3.1-CAR. Insert five copies of NR1 into pGl3-TK promoter vector to construct expression vector (NR1)5-TK-luc. The vector pcDNA3.1-CAR was transiently cotransfected by liposome technology with (NR1)5-TK-luc and pRL-TK Vector into HepG2 cell lines to assay the expression levels of luciferase and evaluate the exciting activity of agonist phenytoin. Finally, phenytoin a positive drug significantly improved the expression level of luciferase, the maximum multiple is up to about 2.53 fold, and it had good dose-effect relationship between positive drug and the relative luciferase expression in a certain concentration of positive drug.

Keywords： pgCAR agonist cotransfection high-throughput screening