[Construction of eukaryotic expression vector for EBF3 and EGFP fusion protein and its expression in HepG2 cells].

Aim: 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.

Methods: 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.

Results: 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.

Conclusion: 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.