人survivin蛋白的原核表达、纯化及抗原活性鉴定

发布时间：2018-04-02 06:32

  本文选题：Survivin　切入点：原核表达　出处：《细胞与分子免疫学杂志》2013年08期

【摘要】：目的构建人肿瘤抗原survivin的原核表达载体,优化在大肠杆菌中的表达条件,并对survivin/His融合蛋白进行纯化和抗原活性鉴定。方法设计针对survivin基因序列的特异引物,通过聚合酶链式反应(PCR)扩增人survivin全长基因序列(538 bp)克隆至原核表达载体pET28a(+),构建重组表达载体pET28a-survivin,并将该载体转化大肠杆菌BL21(DE3),经IPTG诱导表达survivin/His融合蛋白,并采用Ni亲和层析凝胶纯化重组蛋白。纯化后的重组蛋白经Western blot法、ELISA鉴定其抗原活性。结果重组表达载体经BamHⅠ和HindⅢ鉴定正确;IPTG诱导后经SDS-PAGE分析表明获得了相对分子质量(M r)24 000大小的重组蛋白;纯化后的蛋白纯度达到90%。Western blot法和ELISA检测证实纯化的survivin蛋白能够与特异性抗体发生反应,表明其具有良好的抗原活性。结论成功构建了原核表达载体pET28a-survivin,利用大肠杆菌表达系统实现了融合蛋白的可溶性表达并进行纯化,纯化后survivin蛋白经鉴定具备较高的抗原活性。
[Abstract]:Objective to construct the prokaryotic expression vector of human tumor antigen survivin, optimize the expression conditions in Escherichia coli, and purify and identify the antigen activity of survivin/His fusion protein.Methods specific primers for survivin gene sequence were designed.The full-length human survivin gene was amplified by polymerase chain reaction (PCR) and cloned into prokaryotic expression vector pET28a (pET28a). The recombinant expression vector pET28a-survivin was transformed into Escherichia coli BL21DE3, and the survivin/His fusion protein was induced by IPTG.The recombinant protein was purified by Ni affinity chromatography gel.The purified recombinant protein was identified by Western blot Elisa.Results the recombinant expression vector was identified by BamH 鈪，

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