Stable CHO production and functional validation of a recombinant canine NGF-Fc fusion protein.

Recombinant NGF has important applications in neurotrophic and repair-associated models, but well-defined recombinant canine NGF reagents with efficient production and functional validation remain limited. In this study, mature canine NGF was fused to the Fc region of canine IgG and expressed using a stable CHO cell system. The secreted cNGF-Fc protein was recovered from culture supernatant by Protein G affinity chromatography. SDS-PAGE and Western blotting supported the expected Fc-fusion format, with a predominant non-reducing species of approximately 80 kDa and a reducing species of approximately 40-45 kDa; densitometric analysis estimated the electrophoretic purity to be above 90%. Under the production condition used here, the selected stable clone yielded 180 mg/L purified cNGF-Fc. Purified cNGF-Fc showed saturable binding to the recombinant canine TrkA extracellular domain and stimulated TF-1 cell proliferation, with apparent EC50 values of 0.65 μg/mL and 4.5 μg/mL, respectively. It also induced neurite-like process outgrowth in PC12 cells and increased ERK1/2 phosphorylation. Functionally validated cNGF-Fc provides a species-relevant recombinant NGF format for canine NGF/TrkA research, neurotrophic repair models, and further development of NGF-based biologic strategies in companion-animal medicine.