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vWF是由巨核细胞和血管内皮细胞合成的糖蛋白,其主要功能之一为凝血VIII因子(FVIII)的载体,防止后者被血浆酶降解。作者最近的工作证明intein可应用于双载体转移B结构域缺失型FVIII(BDD-FVIII)基因,通过翻译后的蛋白质反式剪接形成完整的功能性BDD-FVIII蛋白。为了研究vWF对于intein连接的BDD-FVIII分泌和活性的影响,通过intein融合的BDD-FVIII重、轻链基因和vWF基因共转染培养的293细胞,采用ELISA观察了分泌至培养上清液中的由intein剪接的全长BDD-FVIII抗原量,并用Coatest检测了由其产生的活性。结果显示,vWF基因共转染细胞上清液中,全长BDD-FVIII抗原量为(235±21)ng·mL-1,活性为(1.98±0.2)u·mL-1,明显高于未转染vWF的细胞[(110±18)ng·mL-1和(1.10±0.15)u·mL-1],也明显高于单独转染BDD-FVIII基因的对照细胞[(131±25)ng·mL-1和(1.22±0.18)u·mL-1],表明vWF基因共转染可显著改善双载体转BDD-FVIII基因后intein剪接的BDD-FVIII蛋白的分泌和生物活性。为基于蛋白质剪接的断裂BDD-FVIII基因转移的甲型血友病基因治疗研究中,应用vWF基因共转移以提高治疗的功效提供了依据。
vWF is a glycoprotein synthesized by megakaryocytes and vascular endothelial cells. One of the major functions of vWF is the carrier of coagulation factor VIII (FVIII), which prevents the latter from being degraded by plasma enzymes. Recent work by authors has demonstrated that intein can be applied to the dual vector-transfer domain B deletion FVIII (BDD-FVIII) gene, which transforms the translated protein to form the complete functional BDD-FVIII protein. To investigate the effect of vWF on intein-linked BDD-FVIII secretion and activity, cultured 293 cells were co-transfected with intein fused BDD-FVIII heavy, light chain and vWF genes and secreted into culture supernatants by ELISA Of full-length BDD-FVIII antigen spliced by intein and the resulting activity was assayed using Coatest. The results showed that the total amount of BDD-FVIII antigen in the co-transfected cells was (235 ± 21) ng · mL-1 and the activity was (1.98 ± 0.2) u · mL-1, which was significantly higher than that of the non- The number of cells transfected with vWF [(110 ± 18) ng · mL -1 and (1.10 ± 0.15) u · mL -1] was also significantly higher than that of control cells transfected with BDD-FVIII alone [(131 ± 25) ng · ML-1 and (1.22 ± 0.18) u · mL-1], respectively, indicating that co-transfection of vWF gene can significantly improve the secretion and bioactivity of intein-spliced BDD-FVIII protein after biotransfection of BDD-FVIII gene. In the study of gene therapy for hemophilia A based on protein splicing, the co-transfer of vWF gene provided the basis for improving the efficacy of treatment.