B区缺失的人凝血因子Ⅷ基因在293T细胞表达

B区缺失的人凝血因子Ⅷ基因在293T细胞达 B区缺失的人凝血因子?基因在293T细胞表达 B区缺失的人凝血因子?基因在293T细胞表达 【摘要】 本研究目的是构建含有人凝血因子?and to investigate its expression in 293T cells. B domain deleted factor ? gene fragment as obtained by enzyme digestion and cloned into lentiviral vector pXZ208 to establish the expression vector pXZ208 BDDhF?. Rebinant viral particles ere prepared by cotransfection ith packaging plasmid ΔNRF and envelope plasmid VSV G using calcium phosphate precipitation method. 293T cells ere transfected by viral supernatant. Coagulant activity of F?， BDDhF?mRNA and genome integration ere assayed by one step method, RT PCR and PCR after transfection. The results shoed that 293T cells could be transfected by rebinant virus. The transfection rate of 293T as 59.57%. After transfection, the cells expressed F? efficiently. Detection confirmed that the activity of F? as 12%,43% and 87% respectively at 24,48 and 72 hours after infection. BDDhF? transcription as detected by RT PCR from the infected cells. The gene integration in the targeted cells as also observed. It is concluded that the successfuly constructed lentiviral vector is able to generate high level expression of human F? in 293T cells, hich may provide a potential application of gene therapy to haemophilia A. Key ords lentiviral vector; coagulant factor ?; gene expression 血友病A是一种X连锁隐性遗传性出血性疾病，目前对该病的治疗 主要以蛋白替代疗法为主，但是有感染人免疫缺陷病毒，RNAgents Total RNA Isolation System试剂盒提取细胞的总RNA，按照 Access RT PCR System试剂盒的操作说明，用逆转录 聚合酶链 反应细胞中BDDhF?mRNA的转录。设计1对BDDhF?cDNA的特异 性引物，上游引物