摘要
该文旨在探索前肽缺失的von Willebrand因子(vWF-ΔPro)对蛋白质剪接的L303E/F309S突变体凝血第八因子(FVIII)分泌的影响。将vWF-ΔPro基因与蛋白内含子融合的FVIII重链和轻链基因共转HEK293细胞。结果显示,转vWF-ΔPro细胞的剪接蛋白FVIII分泌量和活性分别为(196±27)ng/mL和(1.39±0.31)IU/mL,明显高于对照细胞的(116±24)ng/mL和(0.91±0.18)IU/mL。表明vWF-ΔPro可提高剪接的L303E/F309S突变体FVIII蛋白分泌量和活性。
We recently demonstrated that L303E and F309S mutation in the A1 domain of heavy chain of coagulation factor VⅢ (FVⅢ) could improve secretion of spliced FVIII in protein-splicing based dual-vector delivery of FVⅢ gene. In this study, we further investigated the effect of a propeptide-deleted form of the von Wiilebrand factor (vWF-APro), a functional FVIII carrier co-transfection on secretion of protein spliced FVⅢ with L303E/F309S mutation. By co-transfection of HEK293 cell with both heavy and light chain genes fused to intein, a protein splicing element and vWF-4Pro gene, an ELISA was performed to determine secreted spliced FVⅢand Coatest was used to measure secreted bioactivity. The data demonstrated that vWF-APro co-expressed cell displayed a much higher levels of secretion of spliced FVIII (196±27) ng/mL, compared to control cell (116±24) ng/mL. The secreted bioactivity by vWF-APro co-expressed cell (1.39±0.31) IU/mL was also greater than that of control cell (0.91±0.18) IU/mL. Therefore, vWF-APro may further improve efficacy of dual-vector delivery of FVⅢ gene by enhancing secretion of spliced L303E/F309S mutated FVⅢ encouraging our ongoing in vivo investigation for improvement of two-vector FVIII transgene.
出处
《中国细胞生物学学报》
CAS
CSCD
北大核心
2012年第10期988-991,共4页
Chinese Journal of Cell Biology
基金
山东省自然科学基金(No.ZR2010CM061)资助项目~~
关键词
VWF
凝血第八因子
蛋白质剪接
分泌
von Willebrand factor
coagulation factor VⅢ
protein splicing
secretion