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培养细胞实验表明,亮氨酸拉链通过改善内含肽(intein)的蛋白质剪接效率,提高双载体转B区缺失型凝血因子Ⅷ(BDD-FⅧ)基因细胞剪接FⅧ蛋白的分泌量和活性.本文从C57BL/6小鼠门静脉注射含亮氨酸拉链和Ssp DnaB内含肽融合的BDD-FⅧ的重链和轻链基因双表达载体,48 h后,检测到血浆的重链分泌量和FⅧ活性分别为(298±67)μg/L和(1.15±0.29)U/mL,明显高于不含亮氨酸拉链的双载体转BDD-FⅧ基因对照小鼠((179±59)μg/L和(0.58±0.19)U/mL).结果表明,亮氨酸拉链通过改善蛋白质反式剪接,提高基于蛋白质剪接的双载体转BDD-FⅧ基因小鼠血浆的凝血活性,为进一步双腺相关病毒(AAV)载体转BDD-FⅧ基因的甲型血友病基因治疗研究提供了依据.
Cell culture experiments show that leucine zipper improves the secretion and activity of splicing FⅧ protein of the double-carrier translocation factor B (BDD-FⅧ) gene by improving the protein splicing efficiency of intein. The heavy and light chain gene double-expression vectors of BDD-FⅧ fused with leucine zipper and Ssp DnaB intein were injected into the portal vein of C57BL / 6 mice. After 48 h, the heavy chain secretion and FⅧ activity of plasma were detected (298 ± 67) μg / L and (1.15 ± 0.29) U / mL, respectively, which were significantly higher than that of the BDD-FⅧ transgenic control mice ((179 ± 59) μg / L) without leucine zipper (0.58 ± 0.19) U / mL) .The results showed that the leucine zipper could improve the blood clotting activity of BDD-FⅧ transgenic mice by improving protein trans-splicing, AAV) vector BDD-F VIII gene provides a basis for the study of hemophilia gene therapy.