【摘要】 中心蛋白是一种在微管组织中心的复制和分离中起着重要作用的蛋白质。为了进一步进行中心蛋白结构和功能的研究 ,我们克隆了单细胞真核生物游仆虫中心蛋白基因并构建了重组表达质粒pGEX 6P EoCen ,其在大肠杆菌BL2 1中经IPTG诱导后获得了大量的可溶性表达 ,融合蛋白表达水平达到了细菌总蛋白的 32 6 %。GST抗体进行Westernblotting检测结果为阳性。经GST亲和层析和superdex 75凝胶层析后得到 90 %以上电泳纯的蛋白。用纯化后的蛋白免疫大鼠产生抗血清 ,经ELISA检测抗体效价达 1∶2 5 6 0更多还原

【Abstract】 Centrin, a component of microtubule organizing centre (MTOC) in eukaryotes, is a 20 kD acidic calcium binding protein and belonging to the F hand superfamily. To study the structure and function of centrin in ciliates, we cloned a centrin gene from Euplotes octocarinatus (GenBank accession number:Y18899). An amino acid sequence alignment of the Euplotes octocarinatus centrin with three centrin proteins from Homo sapiens revealed 60% to 66% identity. The expression plasmid pGEX 6P EoCen was constructed and GST EoCen fusion protein was overexpressed in an E. coli strain Bl21(DE3) with IPTG induction. The expression level of GST EoCen was 32 6% of the total bacterial proteins. The GST EoCen was detected by Western blotting with anti GST antibodies. The protein was purified to above 90% of purity by GST affinity chromatography and gel filtration superdex 75. Mouses were immunolized by the purified EoCen with the injection dosage of 0 4 μg antigen/g body weight. The specificity of polyclonal antibody and the titer of 1∶2 560 were detected by indirect ELISA.更多还原