A novel system for producing lentiviral vectors / 生物工程学报

ABSTRACT

The aim was to develop a cell culture system capable of producing high titer lentiviral vector stocks with recombinant vaccinia viruses as helpers. BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL and the envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamine2000. After 4 days incubation, the culture supernatant of lentiviral vectors was collected and judged by RT-PCR and the Western blot, the results showed that lentiviral vectors were found in the culture supernatant; approximately (11.71 +/- 0.80) x 10(11) copies of lentiviral vector RNA were present per mL of cell culture supernatant, as detected by Real-time PCR; the vector stocks with titers was up to (1.3 +/- 0.18) x 10(8) tu/mL, as detected by flow cytometry , which is one order of magnitude higher than the output of classical manufacture system. These results suggest that the new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established. It provides the basis for the future development of industrial application.