[Construction of eukaryotic expression vector for EBF3 and EGFP fusion protein and its expression in HepG2 cells].
ABSTRACT To construct the eukaryotic expression vector for early human B-cell factor 3(EBF3) fused with the enhanced green fluorescent protein (EGFP) and express the fusion protein EBF3-EGFP in HepG2 cells.
The intact EBF3 gene was amplified from RNA isolated from the placental tissue by RT-PCR. Then it was inserted into the pEGFP-N1 vector to construct the recombinant eukaryotic expression vector pEGFP/EBF3. The fusion protein EBF3-EGFP was expressed in HepG2 cells by the transfection of pEGFP/EBF3 into the cells.
The pEGFP/EBF3 recombinant expression vector was constructed and confirmed by DNA sequencing, enzymatic digestion and PCR identification. 24 h after the fusion protein EBF3-EGFP was transfected wiht pEGFP/EBF3, it was observed mainly in the cellular nucleus under the inverted fluorescence microscope. Both pEGFP/EBF3 and pEGFP-N1 were transfected into human HepG2 cells. 24 h after the transfection, the proportion of transfection was about 52% and 59%, respectively. Western blot analysis confirmed that the EBF3-EGFP fusion proteins of M(r) 87 000 were detected in the cytoplasmic and nuclear protein of HepG2 transfected with pEGFP/EBF3 for 24 h or 48 h. The cell proportion in S phase increased in HepG2 cells transfected with pEGFP/EBF3 in comparison with HepG2 cells transfected with pEGFP-N1 or untransfected. These findings suggested that the transfection of EBF3 gene into HepG2 induced the cell proliferation from G1 phase to G2 phase by increasing the number of cells.
The construction of pEGFP/EBF3 eukaryotic expression vector and the expression of EBF3-EGFP fusion protein in HepG2 cells lay a foundation for further study of the relationship between EBF3 and its growth and the proliferation of tumor cells.