[Construction and expression of recombinant lentiviral vectors of AKT2,PDK1 and BAD].

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 293Tcells.Methods Total RNA was extracted from lung cancer tissues.The full-length coding regions of human ATK2,BADand PDK1cDNA 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 293Tcells using the calcium phosphate method.The over expression of AKT2,BAD and PDK1were detected by Western blot.Results AKT2,PDK1and BAD were subcloned into pCDF1-MCS2-EF1-copGFP,with an efficiency of transfection of 100%,95%,and 90%respectively.The virus titers were 6.7×106 PFU/mL in the supernatant.After infection, the proteins of AKT2,PDK1and BAD were detected by Western blot.Conclusion The lentivial vector pCDF1-MCS2-EF1-copGFPcontaining AKT2,BADand PDK1were successfully constructed and expressed in 293Tcells.