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凝血VⅢ因子(FVⅢ)尽管与凝血V因子具有相似的结构,但其分泌的低效性不仅限制了重组产品在甲型血友病患者中的广泛应用,也困扰基于转FVⅢ基因的基因治疗。为了提高FVⅢ的分泌效率,在我们以前用内含肽(intein)的蛋白质剪接功能介导的双载体转B区缺失型FVⅢ(BDD-FVⅢ)基因研究的基础上,探讨了重链L303E/F309S双突变对剪接BDD-FVⅢ蛋白分泌的影响。PCR突变法将L303E/F309S双突变引入我们以前构建的融合Ssp DnaB内含肽的野生型重链(HCIntN),得到融合内含肽的重链突变基因(DMHCIntN),与融合内含肽的轻链(IntCLC)基因共转染培养的293细胞,用ELISA检测了培养上清中的重链多肽和剪接的BDD-FVⅢ蛋白浓度,用Coatest法分析了培养上清的凝血生物活性。结果显示,单独转DMHCIntN和DMHCIntN与IntCLC共转染细胞上清中的分泌的重链多肽浓度分别为(35±12)ng/ml和(178±19)ng/ml,高于单独转HCIntN细胞和HCIntN与IntCLC共转细胞[(14±6)ng/ml和(127±23)ng/ml];共转DMHCIntN和IntCLC细胞上清中剪接的BDD-FVⅢ蛋白浓度和活性分别为(128±24)ng/ml和(1.01±0.15)U/ml,明显高于共转HCIntN和IntCLC细胞[(90±12)ng/ml和(0.71±0.14)U/ml];另外,单独转DMHCIntN和IntCLC的细胞合并培养后的上清也检测到剪接的BDD-FVⅢ蛋白[(20±3)ng/ml]和活性[(0.17±0.07)U/ml]。结果表明,双突变可提高重链的分泌效率并明显被轻链顺式提高,伴之以剪接BDD-FVⅢ的分泌增加,表明内含肽对双载体转BDD-FVⅢ基因功效的改善并表现出不依赖细胞机制的BDD-FVⅢ剪接作用。为进一步动物体内实验运用内含肽的双AAV转重链双突变BDD-FVⅢ基因研究奠定了基础。
Coagulation Factor VIII (FVIII) Despite its similar structure to that of coagulation factor V, the ineffectiveness of its secretion not only limits the widespread use of recombinant products in patients with hemophilia A, but also plagues gene therapy based on the FVIII gene. In order to improve the secretion efficiency of FVIII, based on our previous studies on the intein splice-mediated double-carrier to B region deletion type FVIII (BDD-FVIII) gene, we investigated the interaction between heavy chain L303E / F309S Effect of double mutations on the secretion of spliced BDD-FVIII protein. PCR Mutagenesis The L303E / F309S double mutation was introduced into the wild-type heavy chain (HCIntN) of the fused Ssp DnaB intein we previously constructed to obtain the heavy chain mutant gene (DMHCIntN) of the fusion intein, (IntCLC) gene were co-transfected into 293 cells. The concentrations of heavy chain polypeptide and spliced BDD-FVIII protein in the culture supernatant were detected by ELISA, and the coagulation bioactivity of the culture supernatant was analyzed by Coatest method. The results showed that the concentrations of secreted heavy chain polypeptide secreted by DMHCIntN, DMHCIntN and IntCLC co-transfected cells were (35 ± 12) ng / ml and (178 ± 19) ng / ml, respectively, higher than that of HCIntN cells (14 ± 6) ng / ml and (127 ± 23) ng / ml). The concentration and activity of the spliced BDD-FVIII protein in the co-transfection of DMHCIntN and IntCLC cells were (128 ± (90 ± 12) ng / ml and (0.71 ± 0.14) U / ml, respectively). In addition, DMHCIntN and The spliced BDD-FVIII protein [(20 ± 3) ng / ml] and activity [(0.17 ± 0.07) U / ml] were also detected in the supernatants of the cells from IntCLC. The results showed that the double mutation can improve the secretion efficiency of heavy chain and was significantly improved cis-light chain, accompanied by increased secretion of spliced BDD-FVIII, indicating that the intein on the dual carrier BDD-FVIII gene efficacy and improve Not dependent on cellular mechanisms of BDD-FVIII splicing. This study lays the foundation for the further study of BDD-FVⅢ double-AAV to heavy chain double mutation using intein in animal experiments.