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报道了带有人凝血因子IX cDNA的高滴度高表达安全性反转录病毒载体的构建。采用LNL6反转录病毒载体为骨架,构建了由人巨细胞病毒启动子(hCMV)驱动的反转录病毒载体LNCIX,由反转录病毒LTR启动子驱动的反转录病毒载体LIXSN,以及由LTR和CMV启动子共同控制转录的反转录病毒载体LCIXSN,分别用电穿孔方法转导PA317辅助细胞株。LNCIX和LIXSN反转录病毒载体能在离体细胞中表达人IX因子蛋白,而LC’IXSN反转录病毒载体转移到离体细胞中没有检测到人IX因子蛋白。PA317/INCIX细胞的产病毒滴度为8×10~5CFU/ml,该重组病毒感染人纤维肉瘤细胞HT1080及血友病B患者皮肤成纤维细胞(HSF),用ELISA方法分别测定这些细胞的IX因子蛋白产量,LNCIX载体在HT-1080细胞中的人IX因子平均表达量为3.3μg/10~6细胞·d~(-1);在HSF细胞中的平均表达量为2.5μg/10~6细胞·d~(-1),其中80％以上的IX因子具有凝血活性,与过去相比,提高了产病毒滴度,增加了人IX因子的平均表达量,病毒载体骨架的设计更完善,降低了产生野生型病毒的概率,提高了安全性,对于进一步开展血友病B的基因治疗具有重要的意义。
The construction of a high titers high expression safety retroviral vector with human factor IX cDNA was reported. The retroviral vector LNCIX driven by the human cytomegalovirus promoter (hCMV), the retroviral vector LIXSN driven by the retrovirus LTR promoter, was constructed using the LNL6 retrovirus vector as the backbone, The LTR and CMV promoters co-control the transcribed retroviral vector LCIXSN and transduce the PA317 helper cell line by electroporation, respectively. The LNCIX and LIXSN retroviral vectors express human factor VIII protein in ex vivo cells, whereas no transfer of human factor IX protein was detected in the ex vivo cells transfected with the LC’IXSN retroviral vector. PA317 / INCIX cells produced virus titer of 8 × 10 ~ 5CFU / ml, the recombinant virus infection of human fibrosarcoma cells HT1080 and hemophilia B patients with skin fibroblasts (HSF) were measured by ELISA method of these cells IX The average expression level of human factor IX in LNCIX vector was 3.3μg / 10 ~ 6 cells · d ~ (-1) in HT-1080 cells and 2.5μg / 10 ~ 6 in HSF cells In addition, more than 80% of factor VIII has clotting activity. Compared with the past, it increased the titer of virus and increased the average expression of human factor IX. The design of virus vector skeleton is more perfect, Reduce the probability of generating wild-type virus and improve the safety, which is of great significance for further gene therapy of hemophilia B.