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基于蛋白质反式剪接的双载体转凝血第八因子(FⅧ)基因受到剪接效率低的不利影响,本研究旨在增强蛋白质剪接元件蛋白内含子(intein)之间的相互作用,通过改善蛋白质反式剪接效率提高小鼠体内双载体转FⅧ基因后血浆剪接FⅧ蛋白的分泌量和凝血活性.近期培养细胞水平证明,FⅧ重链和轻链分别进行Cys点突变(Met226Cys和Asp1828Cys)后,可形成链间二硫键,并提高蛋白质剪接效率产生更多的FⅧ蛋白.在此基础上,将蛋白内含子融合到含Cys点突变的人FⅧ重链和轻链基因,构建一对表达载体,门静脉注射C57BL/6小鼠进行肝脏靶向转基因.48h后分别检测小鼠血浆中分泌的人FⅧ蛋白量和由其所产生的凝血活性,结果显示FⅧ重链抗原分泌量为(442±151)ng/mL,凝血活性为(1.46±0.37)IU/mL,接近于单载体转FⅧ基因产生的血浆凝血活性((1.79±0.59)IU/mL),明显高于双载体转FⅧ基因对照小鼠血浆的重链分泌量((305±103)ng/mL)和血浆凝血活性((0.85±0.23)IU/mL).结果表明,链间二硫键交联可通过改善蛋白质反式剪接效率提高双载体转FⅧ基因的功效.
Based on the protein trans-splicing, the factor VIII (FⅧ) gene of double-carrier coagulation is adversely affected by the low splicing efficiency. The purpose of this study is to enhance the interaction between protein inteins of protein splicing elements, The efficiency of splicing improves the secretion and clotting activity of plasma spliced FⅧ protein after transfection with double-vector FⅧ gene in mice.Recently, the level of CⅧ point mutation (Met226Cys and Asp1828Cys) can be formed after FⅧ heavy chain and light chain respectively Interchain disulfide bonds and improve the protein splicing efficiency to produce more FⅧ protein.On the basis of this, the intein was fused to the human FⅧ heavy chain and light chain gene containing Cys point mutation to construct a pair of expression vector, The C57BL / 6 mice were injected with C57BL / 6 mice via portal vein for liver transfection, and the amount of human FVIII secreted in the plasma of mice and the coagulation activity were detected respectively 48 hours later. The results showed that the secretion of FⅧ heavy chain antigen was (442 ± 151) (1.46 ± 0.37) IU / mL, which was close to that of the single vector transfection with FⅧ gene (1.79 ± 0.59 IU / mL), which was significantly higher than that of the control Plasma weight (305 ± 103) ng / mL and plasma coagulation activity (0.85 ± 0.23) IU / mL, respectively.The results showed that interchain disulfide cross-linking could improve the efficiency of protein trans-splicing Gene efficacy.