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双载体转凝血Ⅷ因子基因(FⅧ)可有效克服腺相关病毒(AAV)载体容量限制,但FⅧ重链分泌的低效性导致重、轻链分泌的不均衡。重链分泌的低效性源自其A1区存在与内质网蛋白质分子伴侣结合的位点。本文在我们最近运用蛋白质剪接的双载体共转B区缺失型FⅧ(BDD-FⅧ)重链和轻链基因研究的基础上,将重链的A1区替换为猪FⅧ的A1区,用融合蛋白内含子的重链和轻链转基因实验,定量分析了重链的分泌及其对共转重链和轻链基因细胞分泌剪接BDD-FⅧ蛋白和活性的影响。结果显示,变构体重链单独转基因时其分泌得到明显改善,达到89±12 ng/ml,明显高于人BDD-FⅧ重链的分泌(25±9 ng/ml);该变构体重链与轻链共转基因细胞分泌的剪接变构体BDD-FⅧ和活性分别为219±51 ng/ml和1.47±0.22 U/ml,明显高于剪接的人BDD-FⅧ的分泌量和活性(1 16±32 ng/ml和0.8±0.11 U/ml)。单独变构体重链和轻链转基因细胞合并培养后,其培养上清中检测到剪接的变构体BDD-FⅧ和活性,分别为38±7 ng/ml和0.22±0.05 U/ml,提示为不依赖细胞机制的蛋白质剪接所产生。结果表明,A1区替换后重链分泌的增强,可促进基于蛋白质剪接技术的双载体共转重链和轻链基因细胞分泌的剪接BDD-FⅧ水平和活性,并可缓解链分泌的不均衡性,为动物体内应用双AAV载体共转BDD-FⅧ重链和轻链基因研究奠定了实验基础。
The bV-factor VIII gene (FVIII) can effectively overcome the AAV vector capacity limitation, but the ineffectiveness of FⅧ heavy chain secretion leads to the imbalance of heavy and light chain secretion. The inefficiency of heavy chain secretion stems from the presence of sites in its A1 region that bind to the endoplasmic reticulum protein molecular chaperones. In this study, based on the recent studies on the heavy chain and light chain deletion of FⅧ (BDD-FⅧ) in the double-vector co-transgene B region of protein splicing, the A1 region of the heavy chain was replaced by the A1 region of porcine FⅧ, Intron heavy and light chain transgenic experiments quantitatively analyzed the secretion of heavy chain and its effect on the secretion and splicing of BDD-F Ⅷ protein and the activity of the co-transferred heavy and light chain genes. The results showed that the secretion of the variant heavy chain alone was significantly improved to 89 ± 12 ng / ml, which was significantly higher than that of the human BDD-FⅧ heavy chain (25 ± 9 ng / ml) The light chain cotransformationed cells secreted splicing variants BDD-FⅧ with activities of 219 ± 51 ng / ml and 1.47 ± 0.22 U / ml, respectively, which were significantly higher than that of spliced human BDD-FⅧ 32 ng / ml and 0.8 ± 0.11 U / ml). After co-cultivation of the single variant heavy chain and light chain transgenic cells, the spliced variant BDD-FⅧ was detected in the culture supernatant and its activity was 38 ± 7 ng / ml and 0.22 ± 0.05 U / ml, respectively, suggesting that Not dependent on cellular mechanisms arising from protein splicing. The results showed that the enhancement of heavy chain secretion after the substitution in the A1 region can promote the level and activity of spliced BDD-FⅧ secreted by double-vector co-transfected heavy chain and light chain gene cells based on protein splicing technology and can alleviate the unbalance of chain secretion , Which laid the experimental foundation for the study of the co-transfection of BDD-FⅧ heavy chain and light chain genes in animals using double AAV vectors.