前肽缺失体vWF改善FVⅢ基因断裂转移细胞的重链和活性分泌  

Propeptide-deleted von Willebrand Factor Improves Heavy Chain Secretion and Bioactivity by Split FVⅢ Gene Co-transfected Cells

作　　者：朱甫祥[1] 刘泽隆[1] 缪静[1] 屈慧鸽[1] 迟晓艳[1] 

机构地区：[1]鲁东大学生命科学学院,烟台264025

出　　处：《中国生物工程杂志》2012年第7期20-25,共6页China Biotechnology

基　　金：山东省自然科学基金(ZR2010CM061);教育部留学回国人员科研启动基金(20071108)资助项目

摘　　要：通过转von Willebrand因子(vWF)的前肽缺失突变体(vWF-ΔPro)基因,探讨了vWF-ΔPro对双载体转凝血VⅢ因子(FVⅢ)基因的影响。将vWF-ΔPro基因和B-区缺失型FVⅢ(BDD-FVⅢ)的重、轻链基因共转染HEK293细胞48 h后,ELISA检测重链的分泌量为(142±29)ng/ml,明显高于未转vWF-ΔPro基因细胞(87±15)ng/ml;未转vWF-ΔPro基因时单独转重链基因的重链分泌水平很低,vWF-ΔPro存在时重链分泌量明显提高,但不如共转基因时的重链分泌,提示轻链可反式促进重链分泌;单独转轻链或与重链共转基因时轻链分泌水平均较高,且不受vWF-ΔPro影响;Coatest法检测分泌的凝血活性显示,转vWF-ΔPro基因可使细胞分泌的凝血活性明显高于未转vWF-ΔPro基因细胞(0.80±0.15 IU/ml vs 0.41±0.08 IU/ml)。另外,转vWF-ΔPro基因条件下,合并培养转重链和转轻链基因细胞的培养上清中,也检测到FVIII凝血活性(0.23±0.09IU/ml),提示vWF-ΔPro有助于分泌的重、轻链形成功能性异源二聚体。表明转vWF-ΔPro基因可促进双链转FVⅢ基因,为进一步动物体内实验奠定了基础。Dual-vector based FVⅢ gene split delivery has been developped as an altenative strategy to overcome the packaging limitation of adeno-associated virus （AAV） vectors in hemophilia A gene therapy but the efficacy is undesired for the inefficient secretion of heavy chain. A propeptide-deleted mutant of von Willebrand factor（ vWF-△Pro）, which functions as a carrier of FVIII was tested for its effect on dual vector FVⅢ gene delivery. 48 hours post-transfection of HEK293 cell with vWF-△Pro, heavy and light chain genes of a B-domain deleted FVⅢ （BDD-FVIII） , a chain-specific ELISA showed high levels of heavy chain secretion of 142±29ng/ ml in the culture supematant, greater than that of cell without vWF-△Pro co-transfection （87± 15ng/ml）. The heavy chain transfected cells showed a very low levels of heavy chain secretion in absence of vWF-APro althoughimproved in presence of vWF-△Pro, but lower than that of heavy and light chain co-transfected cells indicating a pro-secretion effect of light chain on heavy chain in trans. The light chain showed higher efficient secretion in light chain gene transfection alone or co-transfection with heavy chain, which was not affected by vWF-△Pro. The Coatest assay showed an obviously higher bioactivity （0.80±0.15IU/ml） in supernatant of vWF-△Pro, heavy and light chain genes co-transfected cell, compared to vWF-△Pro-free co-transfection cell （0.41±0.08IU/ml）. The supernatant from combined cells separately transfected with heavy and light chain displayed FVⅢ bioactivity of 0.23 ＋ O. 09IU/ml in presence of vWF-△Pro, suggesting a function of vWF-△Pro in assembly of secreted heavy and light chain into a functional hetero-dimer. The data demonstrated that vWF-△Pro transfection could improve dual-vector mediated FVⅢ gene delivery forming a basis for ongoing in vivo study.

关 键 词：前肽缺失型von WILLEBRAND因子 凝血VⅢ因子 双载体转基因 

分 类 号：Q7[生物学—分子生物学] R55[医药卫生—血液循环系统疾病]