Construction and high expression of retroviral vector with human clotting factor IX cDNA in vitro

摘要

The construction of the high titer and highly expressed safety retroviral vector carrying human clotting factor DC cDNA is reported. Retroviral vectors LNCIX, LDCSN and LCIXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, wereconstructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electropotation Human clotting factor DC was detected in the cultured cells transduced with LNQX and LDCSN but not in the cells transduced with LCDCSN.The viral titer of PA317/LNCIX was 800 000 CFU per mL. With ELBA detection, it was found that the cells transduced with this vector can express human clotting factor DC at the level of 3.3 ng per 10~6 cells in 24 h in human fibrosarcoma cells HT-1080 and 25mu g per 10~6 cells in 24 h in hemophilia B palienls'skin fibroblasl HSF cells, and more than 80% of them were biologically active. The viral tiler and expression of human FIX were increased and the conslruction of retroviral vector backbone was improved and the safety was guaranteed as compared to those vectors used previously. These vectors may produce a sufficient quantity of factor IX proteins to cause the phenolypic modification for hemophilia B patients.