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首页> 外文期刊>African Journal of Biotechnology >Construction of a novel lentiviral vector carrying human B-domain-deleted factor VIII gene
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Construction of a novel lentiviral vector carrying human B-domain-deleted factor VIII gene

机译：携带人B结构域缺失因子VIII基因的新型慢病毒载体的构建

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Vectors derived from human immunodeficiency virus-1 (HIV-1) are highly efficient vehicles for gene delivery. The present study aimed to establish a potent expression system of human factor VIII (FVIII) with lentiviral vectors. FVIII3BD gene was obtained by enzyme digestion and inserted into lentiviral vector pXZ208 driven by cytomegalovirus (CMV) promoter/enhancer. Recombinant viral particles were prepared by cotransfection of 293T cells with packaging plasmids?3NRF and VSV-G through calcium phosphate precipitation. A variety of cell lines including 293T, HLF, NIH3T3, BMEC, Chang-Liver cells and MSCs were infected with recombinant virus containing FVIII3BD. The expression of FVIII3BD mRNA, FVIII procoagulant activity and genomic DNA integration were detected. All the above cell lines were successfully transfected by recombinant lentiviruses. The transfection efficiencies in 293T, HLF, NIH3T3, BMEC, Chang-Liver cells and MSCs were 59.57 ± 5.24, 74.52 ± 7.57, 41.33 ± 5.82, 42.34 ± 5.84, 14.38 ± 2.73% and 27.24 ± 6.53, respectively. All the cell lines expressed FVIII after infection to different extents and the activity of FVIII in 293T, HLF, NIH3T3, mBMEC, Chang-Liver cells and MSCs was 43.2 ± 3.2, 54.1 ± 5.6, 14.2 ± 2.8, 8.7 ± 1.3, 22.5 ± 2.9 and 12.5 ± 2.7%, respectively. In addition, FVIII3BD mRNA and genomic DNA integration were detected in all cell lines after transfection. A novel lentiviral vector carrying human FVIII3BD was constructed, which was able to transfect different mammalian cell types accompanied by high-level activity. This lentiviral vector may provide a theoretical basis for the gene therapy of patients with hemophilia A.
机译：源自人类免疫缺陷病毒1（HIV-1）的载体是高效的基因传递载体。本研究旨在建立慢病毒载体与人凝血因子八（FVIII）的有效表达系统。通过酶消化获得FVIII3BD基因，并将其插入由巨细胞病毒（CMV）启动子/增强子驱动的慢病毒载体pXZ208中。通过用磷酸钙沉淀法将293T细胞与包装质粒？3NRF和VSV-G共转染来制备重组病毒颗粒。用含有FVIII3BD的重组病毒感染包括293T，HLF，NIH3T3，BMEC，长肝细胞和MSC在内的多种细胞系。检测FVIII3BD mRNA的表达，FVIII促凝活性和基因组DNA整合。重组慢病毒成功转染了上述所有细胞系。 293T，HLF，NIH3T3，BMEC，Chang-Liver细胞和MSCs的转染效率分别为59.57±5.24、74.52±7.57、41.33±5.82、42.34±5.84、14.38±2.73％和27.24±6.53。感染后所有细胞系均表达不同程度的FVIII，在293T，HLF，NIH3T3，mBMEC，Chang-Liver细胞和MSCs中FVIII的活性分别为43.2±3.2、54.1±5.6、14.2±2.8、8.7±1.3、22.5±分别为2.9和12.5±2.7％。此外，转染后在所有细胞系中均检测到FVIII3BD mRNA和基因组DNA整合。构建了携带人FVIII3BD的新型慢病毒载体，该载体能够转染具有高水平活性的不同哺乳动物细胞类型。该慢病毒载体可为A型血友病患者的基因治疗提供理论依据。

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