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Construction and identification of recombinant adenovirus vector pAdeasy-1/pAdtrack-CMV-GFP-β-NGF

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Author:
No author available
Journal Title:
Chinese Journal of Neuromedicine
Issue:
9
DOI:
10.3760/cma.j.issn.1671-8925.2011.09.005
Key Word:
神经生长因子;腺病毒载体;绿色荧光蛋白;基因治疗;Nerve growth factor, Adenovirus vector;, Green fluorescent protein;Gene therapy

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.

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