大肠杆菌多重耐药调控基因marR的克隆及其原核表达

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

为获得MarR多克隆抗体，试验以大肠杆菌ATCC25922基因组为模板，根据GenBank中大肠杆菌的marR基因序列设计引物，PCR扩增出长约435bp的marR基因片段，将所得片段与pMD18-T载体连接，转化至JM109大肠杆菌中，筛选阳性克隆，其质粒中插入序列的测序结果与GenBank中报道一致，从阳性克隆中提取质粒，经BamHI和XhoI酶切回收435bp的目的片段，定向克隆到pET～28a（＋）表达载体中，提取阳性质粒转化到大肠杆菌BL21（DE3）中获得阳性克隆。结果表明：经IPTG诱导阳性菌收集表达产物，通过SDS—PAGE分析证实marR基因得到表达；经Western—blot检测该蛋白具有良好的反应原性。

To acquire MarR polyclonal antibody,this experiment introduced the method that a construction pMD18 -T- marR was generated by inserting the sequence of 435 bp obtained by PCR into pMD18 -T simple vector and selecting the positive clones. The cloned sequence coincided with the designed sequence by sequence was showed. This construction was digested with the same enzyme （ BamH I and Xho I ） and ligated the pET - 28a（ ＋ ） vector. Then the plasmid pET - 28a - marR was transformed to the competent cell of BL21 （ DE3 ）. The consequence that the positive clone was induced with IPTG. SDS - PAGE analysis showed that the target gene fragment was expressed successfully. Western - blot analysis indicated that the protein had good reactogenicity.