RNAi介导的Bip表达下调促进基于蛋白质剪接的双链共转基因细胞分泌FⅤⅢ凝血活性  

RNAi-mediated Bip knockdown improves secretion of FⅤⅢ activity by protein splicing-based two-chain gene transduced 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,山东烟台264025

出　　处：《解剖学报》2012年第5期624-628,共5页Acta Anatomica Sinica

基　　金：山东省自然科学基金资助项目(ZR2010CM061);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)

摘　　要：目的探讨用RNA干扰技术下调内质网蛋白伴侣免疫球蛋白重链结合蛋白(Bip)的表达,对基于蛋白质剪接的双链共转凝血因子ⅤⅢ(FⅤⅢ)基因HEK293细胞分泌剪接FⅤⅢ蛋白的量和活性的影响。方法以培养的HEK293细胞,用含蛋白内含子的B区缺失型FⅤⅢ(BDD-FⅤⅢ)的重链和轻链基因表达载体共转染经Bip-siRNA处理的细胞,免疫印迹法检测Bip的表达,四甲基偶氮唑盐(MTT)法检测细胞生长,用酶联免疫吸附(ELISA)和发色分析法分析转基因细胞分泌的剪接BDD-FⅤⅢ蛋白量和活性。结果 RNA干扰下调Bip表达作用明显,细胞生长不受影响;Bip下调的共转基因细胞分泌的剪接BDD-FⅤⅢ和重链量分别为(142±33)μg/L和(197±43)μg/L,明显高于对照细胞[分别为(89±23)μg/L和(120±27)μg/L];Bip下调的共转基因细胞分泌的FⅤⅢ凝血活性为(1 050±160)IU/L,明显高于对照细胞[640±170(IU/L)]。结论 Bip表达下调通过促进剪接BDD-FⅤⅢ的分泌可有效提高基于蛋白质剪接的双载体转BDD-FⅤⅢ基因功效,为进一步动物体内实验提供了依据。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 . Conclusion Bip downregulation may improve the efficacy of protein-splicing based dual-vector delivery of BDD-FVIII gene by enhancing secretion of spliced BDD-FVIII providing evidence for ongoing study in vivo. [ Key words Coagulation factor VIII; Bip; Protein splicing; Two-chain gene transfer; Western blotting; Enzyme- linked immunosorbent assay; Cotest chromogenic method

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 

分 类 号：Q51[生物学—生物化学] R96[医药卫生—药理学]