title: [Construction and expression of recombinant lentiviral vectors of AKT2,PDK1 and BAD].
abstract: OBJECTIVE
  To construct human protein kinase B (ATK2), phosphoinositide-dependent kinase 1 (PDK1) and bcl-2-associated death protein (BAD) lentiviral expression vector, and to determine their expressions in 293T cells.
  
  
  METHODS
  Total RNA was extracted from lung cancer tissues. The full-length coding regions of human ATK2, BAD and PDK1 cDNA were amplified via RT-PCR using specific primers, subcloned into PGEM-Teasy and then sequenced for confirmation. The full-length coding sequence was cut out with a specific restriction enzyme digest and subclone into pCDF1-MCS2-EF1-copGFP. The plasmids were transfected into 293T cells using the calcium phosphate method. The over expression of AKT2, BAD and PDK1 were detected by Western blot.
  
  
  RESULTS
  AKT2, PDK1 and BAD were subcloned into pCDF1-MCS2-EF1-copGFP, with an efficiency of transfection of 100%, 95%, and 90% respectively. The virus titers were 6.7 x 10(6) PFU/mL in the supernatant. After infection, the proteins of AKT2, PDK1 and BAD were detected by Western blot.
  
  
  CONCLUSIONS
  The lentivial vector pCDF1-MCS2-EF1-copGFP containing AKT2, BAD and PDK1 were successfully constructed and expressed in 293T cells.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.forename: Bo-Jiang
authors.initials: BJ
authors.lastname: Chen
authors.forename: Na
authors.initials: N
authors.lastname: Huang
authors.forename: Wei-Min
authors.initials: WM
authors.lastname: Li
mesh_terms: D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D014162:Transfection; D051025:bcl-Associated Death Protein / Q000235:genetics*
chemical_list: C494819:BAD protein, human; C000631429:PDK1 protein, human; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D051025:bcl-Associated Death Protein; C494921:AKT2 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt