vWF促进intein剪接的BDD-FVⅢ的分泌和活性  

vWF improves secretion and activity of intein spliced BDD-FVⅢ

作　　者：朱甫祥[1] 杨树德[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,山东烟台264025

出　　处：《药学学报》2010年第5期595-600,共6页Acta Pharmaceutica Sinica

基　　金：山东省自然科学基金资助项目(Y2005D14);烟台市科技计划项目(2008152);教育部留学回国人员科研启动基金项目(20071108);鲁东大学学科建设经费资助项目

摘　　要：vWF是由巨核细胞和血管内皮细胞合成的糖蛋白,其主要功能之一为凝血VIII因子(FVIII)的载体,防止后者被血浆酶降解。作者最近的工作证明intein可应用于双载体转移B结构域缺失型FVIII(BDD-FVIII)基因,通过翻译后的蛋白质反式剪接形成完整的功能性BDD-FVIII蛋白。为了研究vWF对于intein连接的BDD-FVIII分泌和活性的影响,通过intein融合的BDD-FVIII重、轻链基因和vWF基因共转染培养的293细胞,采用ELISA观察了分泌至培养上清液中的由intein剪接的全长BDD-FVIII抗原量,并用Coatest检测了由其产生的活性。结果显示,vWF基因共转染细胞上清液中,全长BDD-FVIII抗原量为(235±21)ng·mL-1,活性为(1.98±0.2)u·mL-1,明显高于未转染vWF的细胞[(110±18)ng·mL-1和(1.10±0.15)u·mL-1],也明显高于单独转染BDD-FVIII基因的对照细胞[(131±25)ng·mL-1和(1.22±0.18)u·mL-1],表明vWF基因共转染可显著改善双载体转BDD-FVIII基因后intein剪接的BDD-FVIII蛋白的分泌和生物活性。为基于蛋白质剪接的断裂BDD-FVIII基因转移的甲型血友病基因治疗研究中,应用vWF基因共转移以提高治疗的功效提供了依据。As synthesized by vascular endothelial cells and megakaryocytes,the von Willebrand factor （vWF） plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII （FVIII） and preventing its enzymatic degradation.Our recent work demonstrated intein can efficiently ligate BDD-FVIII （B-domaim deleted FVIII） posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system.In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII.We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy-and light-chain genes simultaneously with the vWF gene co-transfected.The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 ± 21 ng·mL^-1 and 1.98 ± 0.2 u·mL^-1,respectively,greater than that of non-vWF co-transfected cell （110 ± 18 ng·mL^-1 and 1.10 ± 0.15 u·mL^-1） or just BDD-FVIII gene transfected control cell （131 ± 25 ng·mL^-1 and 1.22 ± 0.18 u·mL^-1） indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein.It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

关 键 词：von WILLEBRAND因子 凝血VIII因子 共转染 分泌 INTEIN 蛋白质反式剪接作用 

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]