title: [Construction and identification of Nogo extra cellular peptide residues 1-40 gene lentiviral vector].
abstract: OBJECTIVE
  To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40 (NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells.
  
  
  METHODS
  The DNA fragment of NEP1-40 coding sequence was amplified by PCR with designed primer from the cDNA library including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40, and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene delivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot.
  
  
  RESULTS
  The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation, and NEP1-40 protein expression was detected in 293T cells by Western blot.
  
  
  CONCLUSIONS
  The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP 1-40.
authors.affiliation: Department of Orthopedics, West China Hospital, Sichuan University, Chengdu Sichuan, PR China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Yuan
authors.forename: Yueming
authors.initials: Y
authors.lastname: Song
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.forename: Chunguang
authors.initials: C
authors.lastname: Zhou
authors.forename: Qingquan
authors.initials: Q
authors.lastname: Kong
authors.forename: Liming
authors.initials: L
authors.lastname: Liu
authors.forename: Quan
authors.initials: Q
authors.lastname: Gong
mesh_terms: D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D009185:Myelin Proteins / Q000235:genetics*; D009416:Nerve Regeneration; D010446:Peptide Fragments / Q000235:genetics*; D010957:Plasmids
chemical_list: D009185:Myelin Proteins; C509624:NEP1-40 protein, human; D010446:Peptide Fragments