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[Construction of the eukaryotic expression vector of mouse beta-NGF and its expression in NIH 3T3 fibroblast cell
1Department of plastic surgery of tangdu hospital, Fourth Military Medical University, Xi'an 710038,China.
Aim:
To construct the eukaryotic expression vector of mouse nerve growth factor gene and express it in NIH 3T3 fibroblast cell lines.
Methods:
By gene recombination technique, mouse beta-NGF cDNA was inserted into mammlian expression vector pcDNA3.1+. The recombinant plasmid was verified with restriction enzyme digestion analysis. NIH 3T3 cell grown to log phase was transfected with this vector using FuGENE(TM) 6 transfection reagent. The transfected cells were grown in DMEM medium containing G418 at 72 hours after transfection, and the positive clones were selected in G418 medium until the 20 th day. The expression of NGF and its biological activity were analyzed by Western blot and the neurite outgrowth of PC12 cells stimulated by culture supernatant of positive clones respectively.
Results:
The beta-NGF gene was expressed successfully in NIH 3T3 cells. The culture supernatant could stimulate the neurite outgrowth of PC12 cells.
Conclusion:
The eukaryocyte expression vector was constructed successfully. The NGF gene was expressed successfully in the transfected NIH 3T3 cells with good biological activity, which laid the foundation for gene therapy of nervous system diseases.
