J4 ›› 2014, Vol. 13 ›› Issue (2): 37-42.

Previous Articles     Next Articles

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

  

  1. 1. Pre-clinical College, Dali University, Dali, Yunnan 671000，China; 2. School of Life Sciences, Yunnan University, Kunming 650031，China; 3. School of Life Sciences, Nankai University, Tianjin 300071，China
  • Received:2013-11-27 Online:2014-02-15 Published:2014-02-15

PDF (PC)

231

Abstract:

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 cDNA 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 p3XFLAGCMV™-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 cells. 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.

Key words: nuclear respiratory factor 2, human hepatoma BEL-7402 cells, eukaryotic expression vector

CLC Number: