中文摘要：

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32000（CAⅢ）和35000（TAT-CAⅢ的融合蛋白，Western blot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。

英文摘要：

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.