猪组成型雄烷受体激动剂高通量药物筛选模型的建立

Establishment of a cell-based high-throughput screening model for pig CAR agonist

构建猪组成型雄烷受体(pgCAR)激动剂体外高通量筛选模型。利用RT-PCR技术从猪肝脏总RNA中扩增出pgCAR基因序列,将测序后正确的pgCAR片段连接到pcDNA 3.1 vector上构建表达载体pcDNA 3.1-CAR;将合成的5个拷贝NR1(Nuclear receptor sites)插入PGL3-TK-promoter构成报告质粒(NR1)5-TK-luc,与PRL-TK报告质粒构成双荧光报告系统。用PEI转染技术将表达载体与报告质粒共转染HepG 2细胞株中,通过检测荧光素酶基因表达状况评价具有生物活性配体化合物对pgCAR激动活性。阳性药苯妥英钠明显提高荧光素酶的表达,最大上调倍增数可达约2.53倍,并且在一定浓度下阳性药与相对荧光酶的活性表达有较好量效关系。

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 pGI3-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.