Construction and identification of recombinant adenovirus vector pAdeasy-1/pAdtrack-CMV-GFP-β-NGF / 中华神经医学杂志

ABSTRACT

Objective To construct the recombinant adenovirus vector carrying mouse β-nerve growth factor (β-NGF) labeled with green fluorescent protein (GFP). Methods Adult healthy male Kunming mice, weighting (60-70) g, were chosen in our study. The full-length β-NGF cDNA was amplified from the total mRNA of mouse submandibular gland by RT-PCR, and then, this fragment was cloned into the adenovirus shuttle plasmid pAdtrack-CMV (marked with GFP); and then, the pAdeasy-1/pAdtrack-CMV-β-NGF was constructed with defective adenovirus genome pAdeasy-1 by homologous recombination in bacteria; at last, restriction analysis was performed on this adenovirus vector pAdeasy-l/pAdtrack-CMV-GFP-β-NGF.Results Amplified full-length mouse β-NGF fragment was obtained by RT-PCR; the pAdTrack-CMV-β-NGF contained the target gene; double digestion indicated that the recombinant plasmid pAdTrack-CMV-β-NGF was successfully constructed;restriction enzyme digestion analysis indicated that pAdeasy-l/pAdtrack-CMV-βNGF adenovirus vector by homologous recombination was constructed. Conclusion The recombinant adenovirus vector pAdeasy- 1/pAdtrack-CMV-GFP-β-NGF is successfully constructed.