弗氏志贺菌5a型M90T株ClpB蛋白在大肠杆菌中的融合表达和纯化

Fusion Expression of ClpB Protein of Shigella flexneri 5a M90T Strain in E.coli and its Purification

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 clpB gene of Shigella flexneri 5a M90T strain expression plasmid by liga- tion-independent eloning（LIC） method and express it in E.coli and purify it. Methods ＆ Results： The 2574 bp clpB gene fragment and linearized expression vector pET24a were prepared by PCR amplification, and they were li- gated with LIC method to construct pET-ClpB. The resultant expression vector was transformed into E.coli BL21 （DE3） to express recombinant ClpB-T7. The expression condition and purification procedure were then optimized. At 30℃, by induction of 1 mmol/L IPTG lasted for 2 h, soluble expression protein was obtained. The fusion pro- tein was purified with T7 tag antibody agarose to get the high purity fusion protein ClpB-T7 （95 kD）. Conclu- sion： High purity of the ClpB-T7 protein was obtained.