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体外细胞培养实验证明,双载体共转重链A2结构域Met662和轻链A3结构域Asp1828突变为Cys的B区缺失型凝血Ⅷ因子(BDD-FⅧ)基因可使共表达的重、轻链形成链间二硫键,重链的分泌水平得到改善.本文进一步观察小鼠体内双载体转此突变体BDD-FⅧ基因后血浆中重链的分泌量和凝血活性.构建一对含Cys662突变重链和Cys1828突变轻链真核表达载体,经门静脉注射双表达载体至C57BL/6小鼠体内,用ELISA法和Coatest法分别检测小鼠血浆中重链的分泌量和凝血活性为(239±56)ng/mL和(1.09±0.25)IU/mL,明显高于双载体共转野生型BDD-FⅧ基因对照小鼠((84±25)ng/mL和(0.36±0.12)IU/mL).结果表明,链间二硫键形成可有效增强转基因小鼠的血浆重链分泌,提高血浆凝血活性,改善双载体转BDD-FⅧ基因功效,为后续应用双AAV载体的血友病基因治疗研究提供实验依据.
In vitro cell culture experiments showed that the double-carrier co-transfer heavy chain A2 domain Met662 and the light chain A3 domain Asp1828 mutation Cys B region deletion coagulation factor VIII (BDD-F VIII) gene co-expression of heavy and light chain formation Chain disulfide bond, the secretion of heavy chain was improved.In this paper, we further observed the secretion of heavy chain and the activity of coagulation in plasma after transfection of BDD-FⅧ gene with double vectors in mice.We constructed a pair of heavy chain containing Cys662 mutant heavy chain And Cys1828 mutant light chain eukaryotic expression vector. The double expression vector was injected into C57BL / 6 mice via portal vein. The secretion and coagulation activity of heavy chain in mice plasma were measured by ELISA and Coatest method respectively (239 ± 56) ng / mL and (1.09 ± 0.25) IU / mL, respectively, which were significantly higher than that of the binary vector-co-transferred wild-type BDD-FⅧgene control mice (84 ± 25 ng / mL and 0.36 ± 0.12 IU / mL) The results showed that the formation of interchain disulfide bonds could effectively enhance the secretion of heavy chain in plasma of transgenic mice, increase the activity of coagulation and improve the efficacy of BDD-FⅧ transfected by bimodal vector, and provide an experimental study of gene therapy for hemophilia with double AAV vectors in accordance with.