Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal
targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane
biological activity.
Methods:
We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed
and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify
whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25.
Results:
Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence,
and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze
intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition,
the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain
tissue.
Conclusion:
TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins
across the cell membrane without influencing the biological activity.
Biological activity of liposome-encapsulated murine interferon gamma is mediated by a cell membrane receptor.