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Construction of recombinant prokaryotic expression plasmid of human high mobility group box1 and its expression and purification
Author(s): 
Pages: 40-44
Year: Issue:  4
Journal: Acta Academiae Medicinae Shandong

Keyword:  High mobility group proteinsEscherichia coliProkaryotic cellsPurification;
Abstract: 目的构建人高迁移率族蛋白B1(HMGB1)基因的原核表达载体,观察表达水平并纯化重组蛋白。方法根据GenBank中人HMGB1基因序列,用OptimumGeneTM行密码子优化并合成目的基因,经PCR扩增,NdeⅠ和XhoⅠ双酶切,插入原核表达载体pQE-T7-2的相应位点,构建原核表达质粒pQE-T7-2/HMGB1。经菌落PCR、酶谱分析及序列分析鉴定重组质粒,转化大肠杆菌BL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,采用SDS-PAGE和Westernblot印迹法鉴定重组蛋白,Ni-NTA树脂亲和纯化目的蛋白。结果成功构建人HMGB1克隆载体和表达载体;表达的目的蛋白约占菌体总蛋白的20%以上,Westernblot法显示,重组蛋白能与抗HMGB1抗体和抗His抗体特异结合,亲和层析纯化后纯度达90%以上。结论成功构建高效稳定的重组人HMGB1原核表达载体,获得纯化人HMGB1蛋白,为进一步研究HMGB1的功能奠定基础。
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