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本文旨在通过B区缺失型凝血因子8(BDD-FVⅢ)重、轻链间二硫键形成,改善蛋白质反式剪接效率,提高双载体转BDD-FVⅢ基因功效.将BDD-FVⅢ重链A2区的Met662和轻链A3区的Asp1828突变为Cys,用蛋白内含子融合的重链和轻链基因共转染HEK293细胞,Western印迹检测到细胞内BDD-FVⅢ剪接量的提高以及重、轻链间二硫键的形成,用ELISA和Coatest测得细胞分泌至培养上清的剪接BDD-FVⅢ的量(119±14 ng/mL)和活性(1.06±0.08 IU/mL),明显高于野生型BDD-FVⅢ重链和轻链基因共转染细胞的量(81±12 ng/mL)和活性(0.70±0.15 IU/mL);混合培养的转突变重链和轻链基因细胞培养基中剪接BDD-FVⅢ的量(17±5 ng/mL)和活性(0.15±0.03 IU/mL),与混合培养的转野生型重链和轻链基因细胞(分别为21±9 ng/mL和0.18±0.05IU/mL)相近,反映不依赖细胞机制的蛋白质反式剪接.结果表明,重、轻链间二硫键形成通过增强蛋白质反式剪接提高双载体转BDD-FVⅢ基因的功效.为进一步运用双AAV载体动物体内转BDD-FVⅢ基因提供了实验依据.
The aim of this study is to improve the trans-splicing efficiency and improve the efficiency of BDD-FVIII gene transfer by deleting the heavy and light chain disulfide bonds in region B of BDD-FVIII.The BDD-FVIII heavy chain A2 Met662 in the region and Asp1828 in the A3 region in the light chain were mutated to Cys. HEK293 cells were co-transfected with the heavy chain and light chain genes fused with the intein, and the increase of intracellular BDD-FVIII splicing was detected by Western blotting, as well as heavy and light The formation of interchain disulfide bonds was measured by ELISA and Coatest. The amount of spliced BDD-FVIII (119 ± 14 ng / mL) and activity (1.06 ± 0.08 IU / mL) secreted to the culture supernatant by the ELISA were significantly higher than those of the wild (81 ± 12 ng / mL) and activity (0.70 ± 0.15 IU / mL) of cotransfected BDD-FVIII heavy and light chain genes; The amount of spliced BDD-FVIII (17 ± 5 ng / mL) and activity (0.15 ± 0.03 IU / mL) were compared to the mixed-culture wild-type heavy and light chain gene cells (21 ± 9 ng / mL and 0.18 ± 0.05 IU / mL), reflecting the cell-independent trans-splicing of proteins.The results showed that the formation of disulfide bonds between heavy and light chains increased double translocations by enhancing protein trans-splicing Efficacy BDD-FVⅢ transfected gene Providing further experimental evidence for the use of bis AAV vector in vivo gene transfer BDD-FVⅢ.