【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。更多还原

【Abstract】 Objective To construct the carbonic anhydrase II(ICAⅢ)plasmid expression vectors with and withouttyrosine aminotransferase(TAT),and then express and purify them,in order to verify the transmembrane ability ofTAT-CAⅢ in vitro. Methods The CAIII and TAT-CAⅢ genes obtained from PCR were cloned into plasmid pET-28aand expressed in E. coli. BL21(DE3). The transformants were induced by IPTG and the fusion proteins with His-tagwere purified with a Ni-NTA-agarose column. The purified proteins were verified by means of SDS-PAGE,Westernblot and phosphatase activity staining were applied subsequently. The C2C12 cells were treated with serum-free medi-um containing 1μM TAT-CAⅢ or 1μM CAIII respectively for 1 hour and the intracellular distributions of fusion pro-teins were observed by indirect immunofluorescence. Results and Conclusion The TAT-CAⅢ and CAⅢ plasmid ex-pression vectors were successfully constructed. The fusion protein TAT-CAⅢ and CAIII were expressed and purifiedefficiently,and their relative molecular mass weighted about 35 000 and 32 000. TAT-CAⅢ in incubation group cellsrevealed strong green fluorescence,while no green fluorescence was seen in CAⅢ group,indicating that TAT couldmediate CAIII transferring into cells efficiently.更多还原