Expression, purification of tetanus toxin C fragment/cardiotrophin-1 recombinant fusion protein, target delivery to CNS and neurotrophy biology ability / 中华外科杂志

ABSTRACT

<p><b>OBJECTIVE</b>Expression, purification of tetanus toxin C fragment/cardiotrophin-1 recombinant fusion protein (CT-1/TTC) in BL21 (DE3) E. coli, examined whether tetanus toxin C fragment mediate the cardiotrophin-1 target delivery to the central nervous system and the cardiotrophin-1 has the neurotrophic ability.</p><p><b>METHODS</b>Induction by IPTG, the fusion protein was expressed and then purified by GST affinity agarose. The interest protein was viewed by SDS-PAGE, further characterized by Western Blot Rat sciatic nerve transected model was selected. Using drug by nerve-regeneration-chamber and intramuscular injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained with immunohistochemistry method. And select the new born SD rat sciatic nerve transected model, using CT-1/TTC fusion protein by muscle injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained by Nissl's staining.</p><p><b>RESULTS</b>After induction, the fusion protein was about 15% of the total protein and the soluble part was predominant. Purified by GST fusion protein column, the interest protein's concentration is 2.7 g/L. The CT-1/TTC fusion protein was found in lumbar intumescentia by immunohistochemistry method. And after sciatic nerve transected, the numbers of cornu anterius medullae spinalis motoneurons in L4-L6 segments, compared to CT-1/TTC protein grope, have a lower survival rate.</p><p><b>CONCLUSIONS</b>The recombinant CT-1/TTC protein can be expressed and purified in BL21 (DE3) E. coli. This fusion protein has two biological activities of targeting delivery to central nervous system and protecting the cornu anterius medullae spinalis motoneurons.</p>