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为改善蛋白质反式剪接效率,将B-区缺失型FⅧ(BDD-FⅧ)重链的Tyr664和轻链的Thr1826突变为Cys,双载体共转COS-7细胞,观察了细胞内蛋白质剪接、二硫键形成和细胞培养上清液中分泌的剪接BDD-FVIII量和活性。Western blotting检测结果显示,Cys突变可在细胞内形成链间二硫键,剪接BDD-FVIII蛋白量明显增加;双夹心ELISA检测分泌的剪接BDD-FVIII为(128±24)ng.mL/1,明显高于对照(89±15)ng.mL/1;Coatest法检测结果显示,细胞分泌的凝血活性为(0.94±0.08)u.mL/1,也明显高于对照(0.62±0.15)u.mL/1。结果表明,链间二硫键形成可显著提高基于蛋白质剪接的双载体转BDD-Ⅷ基因作用,为进一步动物体内实验提供了依据。
In order to improve the trans-splicing efficiency of protein, the Tyr664 of the heavy chain of the deleted region of FⅧ (BDD-FⅧ) and the Thr1826 of the light chain of the deleted region of B-region were mutated to Cys. Sulfate formation and volume and activity of secreted spliced BDD-FVIII in cell culture supernatant. The result of Western blotting showed that the Cys mutation could form interchain disulfide bonds in the cells, and the amount of BDD-FVIII splicing protein increased significantly. The double-sandwich ELISA showed that the spliced BDD-FVIII was (128 ± 24) ng.mL/1, (89 ± 15) ng.mL / 1. The results of Coatest assay showed that the secreting activity of the cells was (0.94 ± 0.08) u.mL/1, which was also significantly higher than that of the control (0.62 ± 0.15) u. mL / 1. The results showed that the formation of interchain disulfide bonds could significantly enhance the BDD-Ⅷ gene transfer based on protein splicing, which provided a basis for further animal experiments.