人核呼吸因子2基因真核表达载体的构建与鉴定

Construction and Identification of Eukaryotic Expression Vector of Human Nuclear Respiratory Factor 2

目的:构建人核呼吸因子2(NRF2-α和NRF2-β)的真核表达载体,并检测其在瞬时转染的人肝癌BEL-7402细胞株的表达水平。方法:根据NCBI数据库中NRF2-α和NRF2-β的基因序列设计引物,通过RT-PCR扩增目的基因并构建带FLAG标签的真核表达载体,瞬时转染BEL-7402细胞后,分别使用半定量RT-PCR技术和Western blot技术检测其mRNA和蛋白质表达水平。结果:通过酶切鉴定和DNA序列分析,证实已成功构建了人NRF2-α和NRF2-β的真核表达载体,并能在瞬时转染的人肝癌BEL-7402细胞中实现基因的过表达。结论:成功构建了人NRF2-α和NRF2-β的真核表达载体,为进一步研究其功能奠定了基础。

Objective： To construct eukaryotic expression vectors with FLAG epitope highly expressed human NRF2-α and NRF2-β gene, and detect the expression levels of NRF2 in transient transfected human BEL-7402 cells. Methods： RT-PCR technique was used to amplify the eDNA of NRF2-α and NRF2-β gene from total RNA isolated from BEL-7402 cells. After nucleotide sequencing, the open reading frames （ORF） of NRF2-α and NRF2-β cDNA was respectively cloned into eukaryotic expression vector p3XFLAG-CMVTM-14 to form the recombinant plasmid named as p3XFLAG- NRF2-α and p3XFLAG- NRF2-β. Lipofectamine 2000 was used to transient transfect eukaryotic expression vectors into BEL-7402 ceils. Finally, semi-quantity RT-PCR and western blot were applied to detect mRNA and protein expression levels of human NRF2-α and NRF2-β in BEL-7402 cells. Results： We successfully constructed the eukaryotic expression vectors of NRF2-α and NRF2-β. The results of restriction enzyme digestion and nucleotide sequencing confirmed that the recombinant plasmid was correct. By western blotting, we found that the human NRF2-α and NRF2-β proteins were expressed in BEL-7402 cells. Conclusion： The recombinant eukaryotic expression vector of human NRF2-α and NRF2-β were successfully established, which laid foundation for further study of NRF2 gene and its function.