采用蛋白质工程技术将αB-晶状体蛋白导入心肌细胞的初步研究  

Cardiomyocytic Import of αB-Crystallin Engineered with Membrane-translocating Sequence

作　　者：蒋磊[1] 刘双[1] 袁灿[1] 肖卫民[1] 王慷慨[1] 尢家騄[1] 肖献忠[1] 

机构地区：[1]中南大学湘雅医学院病理生理学教研室,长沙410078

出　　处：《生物化学与生物物理进展》2002年第2期283-287,共5页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金 ( 39770 30 7);教育部骨干教师资助计划资助~~

摘　　要：为将αB 晶状体蛋白 (αB crystallin ,αB C)导入心肌细胞 ,采用蛋白质工程技术将人αB 晶状体蛋白全长cDNA基因克隆至具有细胞膜通透能力的膜移位序列 (membrane translocatingsequence,MTS)的碱基顺序的下游 ,在大肠杆菌中表达谷胱甘肽 5 转移酶 (GST) MTS αB C融合蛋白 .用谷胱甘肽 Sepharose4B亲和层析分离表达产物 ,用Xa因子将其中GST切除 ,并通过阴离子交换层析纯化MTS αB C .纯化的MTS αB C在SDS 聚丙烯酰胺凝胶电泳 (PAGE)上呈单一条带 ,分子质量 2 3ku ;免疫印迹显示GST MTS αB C与MTS αB C均能为人αB C抗体识别而在相应位置出现清晰条带 .GST MTS αB C与MTS αB C均有分子伴侣活性 .用异硫氰荧光素 (FITC)标记的MTS αB C与心肌细胞共培养后 ,在荧光显微镜下观察到其进入了心肌细胞 .In order to deliver αB crystallin (αB C) into cardiomyocytes,the full length cDNA fragment encoding the human αB crystallin was cloned into the bacterial expression vector pGEX MTS containing membrane translocating sequence(MTS) which could mediate intracellular delivery of peptides and expressed as a fusion protein coupled to glutathione S transferase(GST).After glutathione affinity chromatography and cleaved from GST by factor Xa,the recombinant MTS αB C was separated from GST and factor Xa by anion exchange chromatography.Recombinant MTS αB C was characterized by SDS PAGE and Western immunoblot analysis.The purified MTS αB C migrated on SDS PAGE as a single band to an apparent molecular mass ( 23 ku ) that corresponded to total native αB C and MTS,and was recognized on Western immunoblot by anti human αB crystallin antibody. Both MTS αB C and GST MTS αB C displayed chaperone like function by disaggregating the denatured and aggregated actin induced by H 2O 2 treatment in an ATP containing buffer at 37℃.It was observed under fluorescence microscope that FITC labeled MTS αB C had gone into neonatal rat cardiomyocytes by MTS mediation after the cells were incubated with the MTS αB C for 8 hours.

关 键 词：αB-晶状体蛋白 蛋白质工程 膜移位序列 心肌细胞 心肌损伤 分子机制 

分 类 号：R542.2[医药卫生—心血管疾病] R362[医药卫生—内科学]