title: [Construction and identification of recombinant adeno-associated virus shuttle vector expressing nerve growth factor beta gene].
abstract: OBJECTIVE
  To construct a recombinant adeno-associated virus (AAV) shuttle vector expressing nerve growth factor beta (NGF-beta) gene.
  
  
  METHODS
  By PCR amplification, the structural element of pAAV-multiple cloning site (MCS) and the functional element of pGenesil-1.1 were obtained and cloned into T-easy vector, respectively; the recombinant T-easy vectors were digested by restriction enzyme, then the target fragments were reclaimed and connected by DNA ligase, so the recombinant AAV shuttle vector pAAV-U6/CMV-enhanced green fluorescent protein (EGFP) containing U6 promoter and CMV promoter was obtained. The vector was transfected into 293 cells. The human Miapaca-2 cell line was cultured, and total RNA was extracted, then human NGF-beta gene was obtained by RT-PCR. T-easy-NGF-beta vector was constructed by cloning human NGF-beta gene into T-easy vector and identified by RT-PCR, digestion, and DNA sequencing. As NGF-beta gene was cloned into pAAV-U6/CMV-EGFP vector, the recombinant AAV shuttle vector expressing NGF-beta gene was obtained and identified by RT-PCR, digestion, and DNA sequencing.
  
  
  RESULTS
  The bands of 800 bp and 4 250 bp were detected when pAAV-U6/CMV-EGFP was digested. The GFP was detected when pAAV-U6/CMV-EGFP was transfected into 293 cells. The bands of 736 bp and 3 015 bp were detected when T-easy-NGF-beta was digested; DNA sequencing result of T-easy-NGF-beta was fully consistent. The bands of 736 bp and 4 250 bp were detected when pAAV-U6/CMV-NGF-beta was digested. DNA sequencing result of pAAV-U6/ CMV-NGF-beta showed that sequences were completely correct.
  
  
  CONCLUSIONS
  The AAV shuttle vector pAAV-U6/CMV-NGF-beta is successfully constructed, providing experimental basis for investigation of the repair of spinal cord injury.
authors.affiliation: No. 1 Department of Orthopedics, Xianyang Center Hospital, Xianyang Shaanxi, 712000, PR China. duguruofei@163.com
authors.forename: Ruofei
authors.initials: R
authors.lastname: Li
authors.forename: Dalong
authors.initials: D
authors.lastname: Gao
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Dang
authors.forename: Kunzheng
authors.initials: K
authors.lastname: Wang
authors.forename: Leigang
authors.initials: L
authors.lastname: Yang
authors.forename: Zhongmin
authors.initials: Z
authors.lastname: Tu
mesh_terms: D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D020932:Nerve Growth Factor / Q000235:genetics*
chemical_list: D020932:Nerve Growth Factor