微小腺病毒载体介导的人凝血IX因子小基因的离体表达

In Vitro Expression of Human Clotting Factor IX Minigene Mediated by Mini-adenoviral Vector

将带有内源内含子的人凝血IX因子cDNA(hFIX小基因 )构建到不含腺病毒基因而只带有反向末端重复顺序 (ITR)和包装信号 ψ等顺式元件的微小腺病毒 (mini adenovirus)载体GT2 0 73上 ,得到载体GTi’IX。将该载体与带有lacZ报告基因的微小腺病毒载体 pRP10 0 1分别转移入腺病毒包装细胞 2 93Cre4中 ,随后再转染辅助病毒AdLC8,从而包装出微小腺病毒AdGTi’IX和AdRP10 0 1。经超离心纯化后对两种病毒的检测结果表明 ,病毒颗粒浓度分别为 2 4× 10 12 /ml和 2 2× 10 12 /ml,辅助病毒所占比例均小于 0 8% ;用AdRP10 0 1以感染复数 (MOI)为 5 0的比例转染 3T3细胞 ,X - gal染色后发现细胞蓝染率在 90 %以上 ;用AdGTi’IX以MOI =5 0的比例转染 3T3细胞 ,ELISA结果表明 ,其瞬时表达为 1 4μg/10 6细胞·2 4h。此结果显示出该载体系统在血友病B基因治疗方面有很好的应用前景 ,为进一步的动物试验及免疫原性研究等临床前研究奠定了良好的基础。

Based on the mini adenoviral vector GT2073(mini Ad) in which the viral protein coding sequences are completely eliminated, vector GTi' IX containing human clotting factor IX minigene driven by CMV promoter was constructed. Mini Ad packaging cell 293cre4 was first transduced with GTi' IX and mini Ad vector pRP1001 containing the reporter gene lacZ, and then was transfected with helper adenovirus AdLC8, thus two kinds of mini Ad, AdGTi' IX and AdRP1001 were obtatined. The particle concentration of purified viruses after CsCl supercentrifugation were 2.2×10 12 /ml and 2.4×10 12 /ml respectively, in which the ratio of helper adenovirus were less than 0.8%. 3T3 cells were transfected with AdRP1001 at a MOI of 50 per cell and more than 90% of cells were blue stained. 3T3 cells were transfected with AdGTi' IX at a MOI of 50 and ELISA showed that transient expression level was 1.4μg/10 6 cell·24hours. These results indicated that the mini Ad vector system might be a promising tool in gene therapy for hemophilia B and offered a preliminary result for further animal and immune response studies.