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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 CAⅢ plasmid expression vector containing and not containing TAT, and to induce expression and purification of TAT-CA III in vitro. Methods: The DNA sequence encoding the full length of CA Ⅲ and TAT-CA Ⅲ was amplified by PCR and recombined into pET28a plasmid. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) After inducing the expression, the fusion protein was purified by Ni-NTA affinity chromatography. The purified product was analyzed by SDS-PAGE, Western blot and phosphatase activity. Then the C2C12 and CA125 were respectively incubated with 1μmol / L CAⅢ and TAT-CAⅢ Myoblast 1h, indirect immunofluorescence assay both in the cell distribution. RESULTS AND CONCLUSION: CAⅢ plasmid vector with and without TAT was constructed successfully. After transformation of E. coli BL21 (DE3), about 32 000 (CA Ⅲ) and 35 000 (TAT-CA Ⅲ) Western blotting and enzyme activity staining showed that two kinds of fusion proteins were successfully obtained. Indirect immunofluorescence staining showed that there was a strong green fluorescence in the cells of TAT-CAⅢgroup, but no fluorescence in CAⅢgroup , Indicating that TAT can mediate the translocation of CAIII into the cell by extracellular transmembrane.