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目的:构建弗氏志贺菌clpB基因的原核表达质粒,在大肠杆菌中表达后,纯化带T7标签的ClpB蛋白。方法与结果:PCR扩增得到线性化表达载体pET24a与2574 bp的clpB基因片段,利用不依赖连接反应的克隆法(LIC)进行克隆,得到重组质粒pET-ClpB,转入大肠杆菌BL21(DE3)中进行诱导表达,通过一系列条件优化,确定可溶性表达条件为在30℃下、用1 mmol/L IPTG诱导2 h;利用抗T7单克隆抗体琼脂糖珠进行亲和纯化,得到了纯度很高的相对分子质量为95×103的ClpB-T7融合蛋白。结论:实现了ClpB-T7在大肠杆菌中的可溶性表达,并纯化获得了高纯度的融合蛋白。
OBJECTIVE: To construct a prokaryotic expression plasmid for clpB gene of Shigella flexneri and purify ClpB protein with T7 tag after expressed in E. coli. Methods and Results: The linearized expression vector pET24a and the 2574 bp clpB gene fragment were cloned by PCR and cloned using a Lectin - independent cloning (LIC) method. The recombinant plasmid pET - ClpB was transformed into E. coli BL21 (DE3) , And the expression of soluble protein was determined by a series of optimization conditions. The conditions of soluble expression were that induced by 1 mmol / L IPTG at 30 ℃ for 2 h, affinity purified with anti-T7 monoclonal antibody agarose beads, and the high purity The relative molecular mass of 95 × 103 ClpB-T7 fusion protein. Conclusion: The soluble expression of ClpB-T7 in Escherichia coli was achieved and the fusion protein of high purity was obtained.