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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。
Objective To construct the prokaryotic expression vector of survivin, and to optimize the expression conditions in E. coli. The survivin / His fusion protein was purified and the activity of antigen was identified. Methods Specific primers targeting survivin gene were designed and cloned into prokaryotic expression vector pET28a (+) by polymerase chain reaction (PCR) to amplify the full-length human survivin gene (538 bp) to construct recombinant expression vector pET28a-survivin The vector was transformed into E. coli BL21 (DE3), the survivin / His fusion protein was induced by IPTG induction, and the recombinant protein was purified by Ni affinity chromatography gel. The purified recombinant protein was identified by Western blot and ELISA. Results The recombinant expression vector was identified by BamHⅠ and Hind Ⅲ. After induced by IPTG, SDS-PAGE analysis showed that recombinant protein with molecular weight (M r) of 24 000 was obtained. The purified protein reached 90% purity. Western blot and ELISA confirmed the purified survivin protein reacted with specific antibody, indicating that it has good antigen activity. Conclusion The prokaryotic expression vector pET28a-survivin was successfully constructed. The fusion protein was expressed and purified by E.coli expression system. The purified survivin protein has high antigenic activity.