Met662和Asp1828的Cys突变增强蛋白内含子对共表达的BDD-FVⅢ重链和轻链的剪接  

Cys Mutation of Met662 and Asp1828 Strengthens Splicing of Co-expressed B-domain-deleted FVⅢ Heavy and Light Chains by Intein

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：《中国生物化学与分子生物学报》2012年第3期234-239,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：山东省自然科学基金(No.ZR2010CM061);烟台市科技计划项目(No.2008152);教育部留学回国人员科研启动基金(No.20071108)~~

摘　　要：本文旨在通过B区缺失型凝血因子8(BDD-FVⅢ)重、轻链间二硫键形成,改善蛋白质反式剪接效率,提高双载体转BDD-FVⅢ基因功效.将BDD-FVⅢ重链A2区的Met662和轻链A3区的Asp1828突变为Cys,用蛋白内含子融合的重链和轻链基因共转染HEK293细胞,Western印迹检测到细胞内BDD-FVⅢ剪接量的提高以及重、轻链间二硫键的形成,用ELISA和Coatest测得细胞分泌至培养上清的剪接BDD-FVⅢ的量(119±14 ng/mL)和活性(1.06±0.08 IU/mL),明显高于野生型BDD-FVⅢ重链和轻链基因共转染细胞的量(81±12 ng/mL)和活性(0.70±0.15 IU/mL);混合培养的转突变重链和轻链基因细胞培养基中剪接BDD-FVⅢ的量(17±5 ng/mL)和活性(0.15±0.03 IU/mL),与混合培养的转野生型重链和轻链基因细胞(分别为21±9 ng/mL和0.18±0.05IU/mL)相近,反映不依赖细胞机制的蛋白质反式剪接.结果表明,重、轻链间二硫键形成通过增强蛋白质反式剪接提高双载体转BDD-FVⅢ基因的功效.为进一步运用双AAV载体动物体内转BDD-FVⅢ基因提供了实验依据.In this study,we aimed to increase the efficacy of dual-vector mediated BDD-FVⅢ gene delivery by improving the efficiency of protein trans-splicing via inter-chain disulfide between heavy and light chains.By introducing Cys mutations at the Met662 in A2-domain of heavy chain and Asp1828 in A3-domain of light chain,a pair of intein fused heavy and light chain expression vectors was co-transfected into HEK293 cell.An enhanced intracellular BDD-FVⅢ splicing and inter-chain disulfide linking were observed by Western blotting.The amount of spliced BDD-FVⅢ and activity in the culture supernatant were 119±14 ng/mL and 1.06±0.08 IU/mL analyzed by ELISA and Coatest method,respectively,greater than wild-type BDD-FVⅢ gene co-transfected cell（81±12 ng/mL and 0.70±0.15 IU/mL）.By combining cells separately transfected with Cys mutated heavy and light chain genes,the spliced BDD-FVⅢ and activity in the culture supernatant were 17±5 ng/mL and 0.15±0.03 IU/mL similar to wild-type heavy and light chain genes transfected cells（21±9 ng/mL and 0.18±0.05 IU/mL） indicating a cellular mechanism-independent protein trans-splicing.It suggested that inter-chain disulfide linking could increase the efficacy of a dual-vector delivery of the BDD-FVⅢ gene by improving protein trans-splicing.It provided evidence for ongoing in vivo BDD-FVⅢ gene delivery by a dual-AAV vector.

关 键 词：B区缺失型凝血因子8 共表达 二硫键 蛋白内含子 蛋白质反式剪接 

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]