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用双载体转运凝血Ⅷ因子基因在甲型血友病基因治疗研究中可克服AAV毒载体容量限制,但存在重链分泌低效和链不均衡性问题。为探索重、轻链间二硫键形成对重链分泌的促进作用,该文用双载体转B结构域大部缺失型FⅧ(BDD-FVⅢ)的重链和轻链基因,将重链的Tyr664和轻链Thr1826突变为Cys,研究了HEK293细胞共转基因后的基因表达、分泌至培养上清的重链量和凝血生物活性。用Western blot检测细胞裂解液结果显示,非还原条件下有明显的二硫键交联的重、轻链蛋白;链特异性ELISA定量检测细胞分泌的重链为(125±29)ng/mL,明显高于共转野生型重链和轻链基因细胞的(75±23)ng/mL;Coatest法显示细胞分泌的凝血活性为(0.78±0.29)U/mL,也明显高于共转野生型重链和轻链基因细胞(0.34±0.12)U/mL。结果表明,重、轻链间的二硫键形成可提高双载体转FⅧ基因的功效,为进一步在动物体内转基因提供了实验依据。
Transfusion of Coagulation Factor VIII Genes by Dual Vectors The AAV vector size limitation can be overcome in hemophilia A gene therapy studies, but there are problems with inefficient heavy chain secretion and strand imbalance. In order to explore the effect of disulfide bond formation between heavy and light chains on the secretion of heavy chain, the heavy and light chain genes of most deleted FⅧ (BDD-FVIII) Tyr664 and the light chain Thr1826 were mutated to Cys, gene expression after HEK293 cell cotransformation, amount of heavy chain secreted to the culture supernatant, and clotting bioactivity were studied. The results of cell lysates detected by Western blot showed that the heavy and light chain proteins were cross-linked by disulfide bonds under non-reducing conditions. The heavy chain secreted by chain-specific ELISA was (125 ± 29) ng / mL, (75 ± 23) ng / mL, which was significantly higher than that of the co-transgenic wild-type heavy chain and light chain genes. The Coatest method showed that the secreting activity of the cells was (0.78 ± 0.29) U / mL, Heavy and light chain genomic cells (0.34 ± 0.12) U / mL. The results showed that the formation of disulfide bonds between heavy and light chains could enhance the efficiency of transfection of FⅧ gene with double vectors and provided experimental evidence for further transgenesis in animals.