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

目的：构建弗氏志贺菌cZp8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2574bp的c加曰基因片段，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1mmol／LIPTG诱导2h；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95×10^3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。

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

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.