Construction and production of recombinant lentiviral vector carrying protein kinase B gene markerd by red flurescent protein

Abstract

Objective To constructe and identify the lentiviral vector carrying protein kinase B (akt1) gene markerd by the Discosoma red fluorescent protein (DsRed). Methods Mouse cDNAs encoding 1488 kb aktl were amplified by reverse transcription-polymerase chain reaction (RT-PCR). By using TA clone technology, aktl PCR product was cloned into T vectors, the BamH Ⅰ, Ecor Ⅰ double restriction enzymatic digesting TA clone parts were norientatial ligated with IRES-dsred, pXZ208 vector by T4 ligase.The correct recombination plasmids named pXZ208-akt1 -IRES-dsred were identified by enzymatic digestion and sequcence. The 293FT cells were co-transfected with the recombination pXZ208-akt1-IRES-dsred and the packaging mixture △NRF, VSVG by using calcium phosphate DNA precipation. The viral liter was observed by fluorescence microscope and fluorescence-activated cell sorter ( FACS) . Western blotting was used to detect AKT1 protein expression. DsRed marked lentivirus and green fluorescent protein ( GFP) marked lentivirus were co-infected into eukaryotic cells, and under the fluorescent microscopy expression efficiency of two genes was observed. Results Recombinant plasmid pXZ208-akt1-IRES-dsred was correct identified by digestion and sequcence. DsRed was observed in 293FT cells by fluorescent microscope, and the viral titer analyzed by FACS was (1. 245 ±0. 250) ×107/ml. Western blotting revealed AKT1 protein expression in infected group was higher than in control group ( P ＜ 0. 05 ). Under the fluorescent microscopy, both dsred and gfp gene expression was observed in 293 FT cells co-transfected with two lentiviruses.Conclusion The three plasmids system of lentiviral vector containing aktl gene marked by DsRed is successfully constructed. The eukaryotic cells co-infected with lentivirus marked by DsRed and GFP express DsRed and GFP. Key words: Lentiviral vector; Protein kinase B; Red fluorescent protein