大鼠肝类固醇激素和异质素受体基因Nr1i2过表达载体的构建和表达

摘要

目的 构建类固醇激素和异质素受体(SXR)基因Nr1i2过表达质粒,并通过HEK-293细胞系验证其表达.方法 大鼠肝组织中的总RNA逆转录后将含相应酶切位点的引物扩增到Nr1 i2编码框,并将Nr1i2片段亚克隆至pcDNA3.1(+),同时对其进行酶切和测序鉴定,挑选pcDNA3.1(+)-Nr1i2转染至293细胞系中,48 h收集细胞进行荧光定量聚合酶链反应(FQ-PCR),72 h后Western blot法检测.结果 成功扩增Nr1i2编码区,并将其克隆至载体pcDNA3.1中.HEK-293细胞系、pcDNA-3.1(+)、pcDNA3.1(+)-Nr1i2中SXR的FQ-PCR结果分别为1.00±0.09、4.88±0.94、473 274.04±28 784.24,后者分别与前两者比较,差异有统计学意义(P＜0.01).结论 成功构建Nr1i2过表达载体,并在HEK-293细胞中成功表达.%Objective To construct the overexpression plasmid of steroid xenobiotic receptor (SXR) gene Nr1i2,and verify its expression by HEK-293 cells.Methods The inverse transcription of total RNA in rat liver was amplified to Nr1i2 coding frame with enzyme loci primer,and the Nr1i2 was subcloned into the pcDNA3.1,which was subjected to enzyme digestion and verified by sequencing.pcDNA3.1 (+)-Nr1i2 was transfected into 293 cells,and 48 h later,the cells were harvested for fluorescent quantitative polymerase chain reaction (FQ-PCR),and 72 h later for Western blotting.Results The Nr1i2 coding region was successfully amplified,and cloned into the vector pcDNA3.1.The expression of SXR gene Nr1i2 in HEK-293 cells and pcDNA-3.1 (+) transfected group was significantly lower than in pcDNA3.1 (+)-Nr1i2 transfected group (both P ＜ 0.01).Conclusion We have successfully constructedn Nr1i2 eukaryotic overexpression vector,which was successfully expressed in 293 cells.