大肠杆菌多重耐药调控基因evgS的克隆及其原核表达

Cloning and prokaryotic expression of multi-drug resistant regulator evgS gene of Escherichia coli

以大肠杆菌ATCC25922的基因组为模板,根据GenBank中大肠杆菌的evgS基因序列设计引物,PCR扩增出约894 bp的基因片段,将所得片段与pMD18-T simple vector连接,转化至大肠杆菌JM109中,筛选阳性克隆,其质粒中插入序列测序结果与GenBank中报道一致。提取阳性克隆质粒,经EcoRⅠ和XhoⅠ双酶切,回收目的片段,定向克隆到pET-28a原核表达载体中,提取阳性质粒,转化至大肠杆菌BL21(DE3)中,获得阳性克隆。经IPTG诱导阳性菌,收集表达产物,通过SDS-PAGE分析证实,pET-28a-evgS可成功地在大肠杆菌中表达EvgS蛋白。Western-blot检测证明,表达产物具有良好的反应原性。

A construction pMD18-T-evgS was generated by inserting the sequence of 894 bp obtained by PCR into pMD18-T simple vector and selecting the sense clones.The result showed that the cloned sequence coincides with the designed sequence by sequencing.This construction was digested with the same enzyme（EcoRⅠand XhoⅠ） and ligated the pET-28a vector.Then the plasmid pET-28a-evgS was transformed to the competent cell of BL21（DE3）. The sense clone was induced with IPTG.SDS-PAGE analysis showed that the target gene fragment was expressed successfully.Western-blot analysis indicated that the expressed protein possesses satisfactory reactivity with E.coli EvgS.