TAT-CAⅢ融合蛋白的表达、纯化及其跨膜转运功能鉴定  

Expression and Purification of TAT-CAⅢ Fusion Protein and Characterization of Its Transmembrane Ability

作　　者：尚西亮[1] 陈世益[1] 任惠民[2] 李云霞[1] 陈疾忤[1] 华英汇[1] 

机构地区：[1]复旦大学附属华山医院运动医学科,上海200040 [2]复旦大学神经病学研究所

出　　处：《中国运动医学杂志》2009年第5期534-538,共5页Chinese Journal of Sports Medicine

基　　金：国家自然科学基金(30771044)资助项目

摘　　要：目的:构建含有及不含有TAT的CAⅢ质粒表达载体,并进行诱导表达、纯化,在体外初步鉴定TAT-CAⅢ的跨膜转运功能。方法:采用PCR的方法扩增编码CAⅢ和TAT-CAⅢ全长的DNA序列,分别重组入pET28a质粒表达载体中,测序鉴定后转化大肠埃希菌BL21(DE3),构建重组体的表达菌株;IPTG诱导表达后,用Ni-NTA亲和层析柱分离纯化融合蛋白,纯化产物进行SDS-PAGE分析、Western blot鉴定及磷酸酶活性染色;然后分别以1μmol/L纯化的CAⅢ及TAT-CAⅢ孵育C2C12成肌细胞1h,间接免疫荧光法检测两者在细胞内的分布情况。结果及结论:成功地构建了含有及不含有TAT的CAⅢ质粒表达载体;转化大肠埃希菌BL21(DE3)后表达并纯化出相对分子量分别约32 000(CAⅢ)和35 000(TAT-CAⅢ)的融合蛋白,Westernblot和酶活性染色鉴定表明成功地获得了两种融合蛋白;间接免疫荧光染色显示,TAT-CAⅢ孵育组细胞内可见有较强的绿色荧光,而CAⅢ组细胞内则未见荧光,表明TAT可介导CAⅢ由胞外跨膜转导进入胞内。Objective To construct the carbonic anhydrase Ⅲ（CAⅢ） plasmid expression vectors with and without tyrosine aminotransferase（TAT）, and then express and purify them, in order to verify the transmembrane ability of TAT-CAⅢ in vitro. Methods The CAⅢ and TAT-CAⅢ genes obtained from PCR were cloned into plasmid pET-28a and expressed in E. coll. BL21 （DE3）. The transformants were induced by IPTG and the fusion proteins with His-tag were purified with a Ni-NTA-agarose column. The purified proteins were verified by means of SDS-PAGE, Western blot and phosphatase activity staining were applied subsequently. The C2C12 cells were treated with serum-free medium containing 1 μM TAT-CAⅢ or 1 μM CAⅢ respectively for 1 hour and the intracellular distributions of fusion proteins were observed by indirect immunofluorescence. Results and Conclusion The TAT-CAⅢ and CAⅢ plasmid expression vectors were successfully constructed. The fusion protein TAT-CAⅢ and CAⅢ were expressed and purified efficiently, and their relative molecular mass weighted about 35 000 and 32 000. TAT-CAⅢ in incubation group cells revealed strong green fluorescence, while no green fluorescence was seen in CAⅢ group, indicating that TAT could mediate CAⅢ transferring into cells efficiently.

关 键 词：碳酸酐酶Ⅲ 融合蛋白 纯化 蛋白质转导域 

分 类 号：R87[医药卫生—运动医学]