蛋白反式剪接的人/猪嵌合体BDD-FVIII及其分泌的改善  

Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion

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10期1232-1238,共7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(2007[1108]);鲁东大学科研基金项目(LZ20083305)

摘　　要：本研究采用具有促分泌作用的猪FVIII分子的A1和A3区,替换人FVIII分子的相应结构,构建人/猪嵌合体BDD-FVIII(BDD-hpFVIII),利用Ssp DnaB intein的蛋白质反式剪接功能,双载体培养的COS-7细胞瞬时共转嵌合体BDD-hpFVIII基因。免疫印迹法观察了转基因细胞内的蛋白表达和BDD-hpFVIII的剪接,采用ELISA和Coatest法分别观察了分泌至培养上清液中剪接的嵌合体BDD-hpFVIII蛋白和生物活性。结果显示,基因共转染细胞内可见明显的剪接BDD-hpFVIII蛋白,分泌至上清液的剪接BDD-hpFVIII蛋白量为(340±64)ng·mL-1,活性为(2.52±0.32)u·mL-1,明显高于双载体共转人BDD-FVIII基因细胞[质量浓度为(93±22)ng·mL-1,活性为(0.72±0.13)u·mL-1],提示intein介导的双载体共转BDD-hpFVIII基因可明显促进剪接蛋白的分泌。另外,分别转intein融合BDD-hpFVIII重链和轻链基因的细胞混合培养后上清液中仍可检测到剪接的BDD-hpFVIII蛋白及其活性,分别为(44±9)ng·mL-1和(0.32±0.07)u·mL-1,提示intein可进行不依赖细胞机制的BDD-hpFVIII剪接。本研究为旨在提高分泌的动物体内应用intein的双载体转BDD-hpFVIII基因奠定了基础。This study is to construct a chimeric human/porcine BDD-FVIII （BDD-hpFVIII） containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein＇s protein trans-splicng a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to （340± 64） ng·mL^-1 and （2.52 ± 0.32） u·mL^-1 secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [（93 ± 22） ng·mL^-1, （0.72 ±0.13） u·mL^-1]. Furthermore, a spliced BDD- hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.

关 键 词：嵌合体BDD—FVHI 分泌 内含肽 蛋白质反式剪接 

分 类 号：R963[医药卫生—微生物与生化药学]