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双载体共转B区缺失型凝血因子Ⅷ(BDD-FⅧ)重链和轻链基因是克服腺相关病毒载体(AAV)容量限制的一种策略,但重链分泌的低效性产生链不均衡性,影响转基因的功效.假设重、轻链链间二硫键形成可提高双载体转BDD-FⅧ基因的功效,本文将BDD-FⅧ的重链A2区的Met662和轻链A3区的Asp1828突变为Cys.双载体培养的COS-7细胞共转染突变重链和轻链基因,Western blot显示细胞内二硫键交联的重、轻链蛋白形成,ELISA和Coatest分析细胞培养上清的BDD-FⅧ重链分泌量和生物活性,分别为(109±13)μg/L和(0.65±0.12)U/ml,明显高于共转野生型重链和轻链基因细胞((71±11)μg/L和(0.35±0.05)U/ml).结果表明,链间二硫键形成可提高双载体转BDD-FⅧ基因的功效,为进一步动物体内运用双AAV载体转BDD-FⅧ基因提供了实验依据.
Binary carrier co-transfecting region B deletion factor-VIII (BDD-FVIII) The heavy and light chain genes are a strategy to overcome the limitation of adeno-associated virus vector (AAV) capacity, but the inefficiency of heavy chain secretion leads to a chain imbalance And affect the efficiency of transgene.It is hypothesized that the formation of disulfide bonds between heavy and light chains can increase the efficiency of BDD-FⅧ gene transfection by dual vectors.In this paper, the Met662 of heavy chain A2 of BDD-FⅧ and the Asp1828 of A3 of light chain COS-7 cells transfected with Cys double vector were co-transfected with mutant heavy and light chain genes, Western blot showed intracellular disulfide bridged heavy and light chain protein formation, ELISA and Coatest analysis of cell culture supernatant BDD (109 ± 13) μg / L and (0.65 ± 0.12) U / ml respectively, which were significantly higher than that of the co-transgenic wild-type heavy chain and light chain genes ((71 ± 11) μg / L and (0.35 ± 0.05) U / ml respectively.The results showed that the formation of interchain disulfide bonds could enhance the efficacy of BDD-FⅧ gene transfer by dual vectors and provide further evidence for BDD-FⅧ gene transfer using double AAV vectors in vivo Experimental basis.