CYP2C19基因多态性真核表达载体的构建及稳定转染细胞系的建立

Construction of human CYP2C19 gene polymorphism eukaryotic expression vector and establishment of stably transfected HepG2 cell line expressing human CYP2C19

目的:构建细胞色素P450CYP2C19*1、*2、*3真核表达载体,并分别转染人肝癌细胞(HepG2),建立高表达目的基因的稳定转染细胞系。方法:应用化学合成法合成CYP2C19*1(野生型)序列,再以此为模板,体外定点突变PCR法构建*2、*3(突变型)。DNA重组技术将其定向插入到真核表达载体pcDNA3.l(-),经菌落PCR、酶切以及测序鉴定后,脂质体转染法转染HepG2细胞,通过G418选择培养,建立稳定转染细胞系,RT-PCR、Western Blot分别检测CYP2C19*1、*2、*3 mRNA以及蛋白的表达。结果:成功构建了pcDNA3.1(-)-CYP2C19*1、*2、*3表达质粒,并建立了相应稳定转染细胞系。进一步的RT-PCR和Western Blot检测结果表明,目的基因均得到了稳定的表达。结论:人CYP2C19*1、*2、*3稳定表达细胞系为研究创新药物或新药先导化合物临床前代谢性质的筛选和预测以及为临床潜在药物相互作用提供了有效实验平台。

To construct the eukaryotic expression vector of CYP2C19＊ 1,＊ 2,＊ 3, re- spectively, then stably transfect HepG2 cells with them. METHODS ： The full-length CYP2C19 wild-type cDNA fragment was ampli- fied by PCR from the human liver cDNA library and mutagenesis build ＊ 2, ＊ 3 （mutant） cDNA in vitro, then those cDNA were inserted into eu- karyotic expression vector pcDNA3. 1 （--）. Af- ter identification of restriction digestion and PCR, the recombinant plasmid was transfected into HepG2 cells by lipofectamine. After screen- ing culture by G418, a stably-transfected cell line was established, and the transcription and ex- pression of the CYP2C19 gene SNPs were identi- fied by RT-PCR, Western Blot assay. RE-SULTS：The eukaryotic expression vector spcD- NA3.1-CYP2C19＊ 1,＊2, ＊3 were successfully constructed and transfected stably into HepG2 cells, respectively. Stably-transfected cell lines were established and the CYP2C19 gene SNPs was expressed successfully. CONCLUSION： The establishment of the stably-transfected cell line and the expression of the target gene provide a solid experimental foundation for screening and prediction the metabolic of new drugs on the function of theCYP2C19 gene.