Fengjin Hao, Yueqin Feng and Yifu Guan* Pages 37 - 43 ( 7 )
Objective: Botulinum toxin has many applications in the treatment of central diseases, as biological macromolecules, it is difficult to pass through the blood-brain barrier which greatly limits their application. In this paper, we verified whether the botulinum toxin heavy chain HCS has a specific neural guidance function.
Methods: We have constructed a fusion protein with botulinum toxin heavy chain and a membrane penetrating peptide TAT (TAT-EGFP-HCS). Recombinant plasmid of botulinum toxin light chain (LC) and TAT were also constructed. The biological activity of HCS, LC, TAT-EGFP-HCS and TAT-EGFP-LC were measured by its ability to cleave protein SNAP-25. The intracellular expression efficiency was evaluated by detecting the fluorescence intensity of EGFP in the cells by fluorescence microscopy and FACS. In addition, we also determined the effect of the above plasmid expression on the apoptosis of PC12 cells. Finally, the tissue specificity of TAT-EGFP-HCS in vivo experiments was also examined.
Results: In the present study, we have constructed a fusion protein with botulinum toxin heavy chain and a membrane penetrating peptide TAT which can lead the entire molecule through the blood-brain barrier and reach the central nervous system. Moreover, we also examined the biological activities of this recombinant biological macromolecule and its physiological effects on nerve cells in vitro and in vivo.
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.
Botulinum toxin, Heavy chain, TAT, transmembrane, CNS delivery, central disease.
Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning, 110122, Department of Ultrasound, the First Affiliated Hospital of China Medical University, Shenyang, Liaoning, 110001, Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning, 110122