Construction And Identification Of Recombimant Aden-ovirus Vector PAdeasy-1/pAdrack-CMV-GFP-β NGF

BackgrounBackground:Traumatic brain injury treatment now increasingly in the direction of gene therapy, in whichthe virus as a vector of about 85%, the recombinant viral vectors in gene therapy to become thekey.Objective:To Construct recombinant adenovirus vector. Carrying mouse nerve growth factor (nervegrowth factor,βNGF) labeled green fluorescent protein (green flurrescent protein, GFP) .Material:①Animals: adult male healthy Kunming mice2 (weight 60-70g), purchased from theGeneral Hospital of Guangzhou Military Command Center animal experiments, animalexperiment animal ethics meet the disposal standards.②plasmid, strain: adenovirus shuttleplasmid pAdTrack-CMV(Marked green fluorescent protein), backbone plasmid pAdeasy-1, E.coli BJ5183, and DH5αwere purchased from stratagene.③The main enzymes and reagents:Restriction enzymes PacⅠ, PmeⅠ(NEB); reverse transcription kit, plasmid extraction kit, gelextraction kit, T4 DNA ligase, TaqDNA polymerase, restriction enzymes Kpn Iand Xho I(Takara); Trizol (Invitrogen); cDNA primer base by the British Wei Jie (Shanghai) Trading Co.,Ltd. synthesis; sequenced by the Biotechnology Co, Ltd, Guangzhou won the Benson completed.Methods:The lengthβNGF cDNA was amplified from the mouse submandibular gland total mRNA byRT-PCR,the fragment was cloned into the adenovirus shuttle plasmid pAdtrack-CMV(Markedgreen fluorescent protein),then contructed pAdeasy-1/pAdtrack-CMV-βNGF with defectiveadenovirus genome pAdeasy-1 by homologous recombination in bacteria.At last ,to finish therestriction analysis of adenovirus vector pAdeasy-1/pAdtrack-CMV-GFP-βNGF.Results:RT-PCR, sequencing and restriction enzyme digestion analysis, proved successfulpAdeasy-1/pAdtrack-CMV-βNGF adenovirus vector construct. ConclusionConclusion:The recombinant adenovirus vector pAdeasy-1/pAdtrack-CMV-GFP-βNGF was successfullyconstructed.At the same time the success of this experiment laid the experimental foundation forβNGF gene function and the treatment of traumatic brain injury .