Construction of recombinant plasmid pcDNA3-hNGFb and its expression

Objective To construct the eukaryotic expression recombinant plasmid pcDNA3-hNGFb and investigate its expression in L929 cells.Methods The cDNA of human nerve growth factor beta subunit (NGF-β) was amplified by RT-PCR from human brain tissue. By gene recombination technique, human NGF-β cDNA was inserted into eukaryotic expression vector pcDNA3. The recombinant plasmid was verified with restriction enzyme digestion and DNA sequencing. Transfected the recombinant vector into L929 cells with Lipofectamine 2000 transfection reagent. The expression of NGF-β was analyzed by immunocytochemistery as well as western blot.Results The RT-PCR product is 750bp specific segment. By restriction enzyme digestion, the recombinant plasmid was digested into 750bp and 5.2kb fragments. DNA sequence result showed the 750bp fragment was identical with human NGF-β cDNA in GenBank. Immunocytochemistery and western blot showed the NGF-β was expressed successfully in L929 cells.Conclusions The recombinant plasmid pcDNA3-hNGFb was constructed successfully, which will provide the foundation for further research.