Objective: To observe the localization and translocation of human high mobility group protein B1 (HMGB1) protein in eukaryotic cells.
Methods: The vector that expressed the fusion protein of human HMGB1 and enhanced green fluorescent protein (EGFP) in mammalian cells were constructed. HMGB1 and EGFP were subcloned into hytohemagglutinin (HA) tagged vector pcDNA3-HA by two steps, the construct of which was then transfected into 293A cells and observed with fluorescence microscope.
Results: The recombinant plasmid was verified by enzyme digestion, polymerase chain reaction (PCR) and sequence analysis, and the fusion protein was highly expressed in 293A cells. With fluorescence microscopy the green fluorescence was found to be distributed in the nuclei. Eighteen hours after the stimulation with tumor necrosis factor-alpha (TNF-alpha), it was found to have translocated into the cytoplasm in some of the cells.
Conclusion: The expression vector for HMGB1-EGFP fusion protein is successfully constructed and effectively expressed in mammalian cells, and it can serve as an important tool in the study of the signal pathway of HMGB1 in mammalian cells.