Expression and characterization of biologically active recombinant human nerve growth factor

摘要

DNA fragment encoding proNGF was amplified by PCR from genomic DNA isolated from ' term placental tissue and was cloned into pcDNA3. 1. DNA sequencing indicated that the cloned DNA fragment was identical as previously reported. CHO cells were then tranfected with the recombinant plasmid. After cultured in the selection medium containing G418 for 30 days, the expressed product in the culture supernatant was tested. The culture supernatant of the cells harboring recombinant plasmid showed strong ability to promote the outgrowth of nerve fiber from the chicken dorsal root ganglion. The results of ELISA showed that the concentration of the recombinant human NGF in the culture supernatant was about 0.5 mg/L. The specific activitiy was about 4 × 106 units per milligram. In conclusion, the proNGF gene was successfully cloned and the protein was correctly expressed and processed in the CHO cells. The expressed rhNGF has a high specific bioactivity.