Aim: Different eukaryotic expression vectors were selected in this research, and then we respectively cloned and constructed recombinant plasmids contained HBX gene or its different fusion forms with GFP. We are aimed to explore the influence of the HBX protein with different structure on its intracellular localization.
Methods: Flag-HBX gene was amplified from pcDNA3.0-HBX plasmid in our laboratory, cloned into pMD-18T vectors, sequenced.and then subcloned into different vectors. After right sequenced, respective recombinant plasmids of pFlag- HBX-IRES2-EGFP, pEGFP-C3-Flag-HBX and pFlag-HBX-EGFP-N3 were transiently transfected into hepatoma HepG-2 cells. The intra-cellular localizations and distributions of HBX protein were examined by indirect immunofluorescence.
Results: Three different Flag-HBX eukaryotic expression vectors were successfully constructed. After transfection of them into HepG2 cells respectively, indirect immunofluorescence demonstrated that HBX protein fused with GFP in different forms show distint intra-cellular distribution characteristics.
Conclusion: We have provided significant experimental evidences for research of the role of HBX protein in the development of hepatocellular carcinoma, and especially the references for explanation of the outcomes in vitro transfection experiments.