酸性区3融合重链促进基于蛋白质内含子的双载体B域缺失型凝血因子Ⅷ转基因表达  

Acidic Region-3 Fused Heavy Chain Ameliorates Intein-based Dual-vector Delivery of The B-Domain Deleted FⅧ Gene

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,烟台264025

出　　处：《生物化学与生物物理进展》2010年第11期1225-1231,共7页Progress In Biochemistry and Biophysics

基　　金：山东省自然科学基金(ZR2010CM061);烟台市科技发展计划(2008152);教育部留学回国人员科研启动基金(20071108);鲁东大学科研基金(LZ20083305)资助项目~~

摘　　要：最近的研究表明,蛋白质内含子(intein)介导的B区缺失型凝血因子Ⅷ(BDD-FⅧ)的轻链和重链剪接可顺式促进后者的分泌,而且剪接反应在细胞内、外均可发生.为进一步提高基于蛋白质内含子的双载体转BDD-FⅧ基因的功效,将具有促进重链分泌作用的位于Pro1640～Ser1690的酸性区3(acidic region-3,AR-3)引入重链,检验对蛋白质内含子剪接的BDD-FⅧ蛋白分泌和活性的影响.用融合蛋白内含子的附加ar-3重链(HCAR3IntN)基因和轻链(IntCLC)基因共转染培养的HEK293细胞,分别用ELISA和Coatest法定量分析分泌至培养上清中剪接BDD-FⅧ蛋白量和生物活性,并用免疫印迹观察了细胞内的BDD-FⅧ剪接.结果显示,共转HCAR3IntN和IntCLC基因细胞,分泌至上清的剪接BDD-FⅧ蛋白量和活性分别为(173±26)μg/L和(1.31±0.15)U/ml,明显高于未添加ar-3的蛋白质内含子融合重链(HCIntN)与轻链(IntCLC)基因共转染细胞[(102±12)μg/L和(0.79±0.09)U/ml],提示AR-3对蛋白质内含子剪接的BDD-FⅧ蛋白分泌和活性有明显改善作用.而且,分别转HCAR3IntN和IntCLC基因细胞混合培养后的上清中,亦检测到剪接的BDD-FⅧ蛋白和活性[(35±7)μg/L和(0.28±0.08)U/ml],表明蛋白质内含子可进行不依赖细胞机制的蛋白质剪接.另外,转基因细胞总蛋白呈现明显的可与FⅧ多克隆抗体进行反应的剪接BDD-FⅧ蛋白条带,直观地反映细胞内BDD-FⅧ的剪接.为动物模体内运用蛋白质反式剪接技术的双腺相关病毒载体(AAV)转BDD-FⅧ基因实验提供了依据.It was previously demonstrated that an intein-catalyzed splicing of B-domain deleted coagulation factor Ⅷ（BDD-FⅧ ） heavy and light chains could improve the secretion of heavy chain in cis and the splicing can occur independently of cellular entities exhibiting a splicing activity in and out of the cell.In order to improve the efficacy of intein-based dual-vector delivery of the BDD-FⅧ gene,here an additional acidic region-3（AR-3） between Pro1640 and Ser1690 of FⅧ proven to be helpful for the secretion of heavy chain was incorporated into BDD-FⅧ heavy chain to examine its effect on secretion and bioactivity of an intein-spliced BDD-FⅧ protein.By co-transfection of the cultured HEK293 cells with genes of the ar-3 incorporated heavy chain and light chain with fused intein（HCAR3IntN and IntCLC） ,an ELISA and Coatest assay were performed to determine the amount of spliced BDD-FⅧ protein and coagulation activity secreted in the culture supernatant,and the intracellular BDD-FⅧ splicing was observed by Western blotting.The data showed that the amount of spliced BDD-FⅧ protein（173±26） μg/L and coagulation activity（1.31±0.15） U/ml of supernatant from gene co-transfected cells were greater than supernatant from intein-fused ar-3-free heavy and light chain genes（HCIntN and IntCLC） co-transfected cells（102±12） μg/L and（0.79±0.09） U/ml indicating the additional AR-3 in the heavy chain could dramatically improve the secretion and activity of intein spliced BDD-FⅧ .A spliced BDD-FⅧ protein（（35±7） μg/L） and coagulation activity（（0.28±0.08） U/ml） was also detected in the culture supernatant of combined cells separately transfected with the HCAR3IntN and IntCLC genes implying the cellular entities independent BDD-FⅧ splicing of the intein.The total protein from gene co-transfected cells displayed an obvious protein band of the spliced BDD-FⅧ detected by a FⅧ polyclone antibody indicating the intracellular BDD-FⅧ splicing.It paved a way for ongoin

关 键 词：凝血因子Ⅷ 酸性区3 分泌 蛋白质内含子 蛋白质反式剪接 

分 类 号：Q7[生物学—分子生物学]