关键词: 凝血因子VIII, 免疫球蛋白重链结合蛋白, 蛋白质剪接, 双链转基因, 免疫印迹法, 酶联免疫吸附测定, 发色分析法

Abstract: Objective To investigate the effect of RNA Interfering-mediated down -regulation ER chaperon protein, immunoglobulin heavy-chain binding protein (Bip) on secretion of spliced FVIII and bioactivity from protein splicing based two-chain co-transgenic HEK293 cell. Methods After treatment with Bip-siRNA, the HEK293 cells were co-transfected with intein-contained B-domain-deleted FVIII(BDD-FVIII) heavy and light chain genes. The expression of Bip was observed by Western blotting with the cell growth atatus assayed by MTT. The quantitative secretion of spliced BDD-FVIII protein and bioactivity were measured by ELISA and coatest chromogenic method respectively. Results Bip was obviously down-regulated by RNA interference technonogy but with no effect on the cell proliferation. It showed a great higher level of spliced BDD-FVIII and heavy chain in terms of secretion by Bip-downregulated co-transgenic cell ［(142±33)μg/L and (197±43)μg/L］ compared to control［(89±23)μg/L and (120±27)μg/L］. The FVIII bioactivity in cell culture supernatant showed an elevated levels in Bip-downregulated co-transgenic cell ［(1.05±0.16)IU/mL］, greater than that of control ［(0.64±0.17)IU/mL］. Co

Key words: Coagulation factor VIII, Bip, Protein splicing, Two-chain gene transfer, Western blotting, Enzyme-linked immunosorbent assay, Cotest chromogenic method