大肠杆菌多药耐药基因AcrA的克隆及其原核表达

Chemical Synthesis and Prokaryotic Experssion of Part of AcrA Gene

从大肠杆菌 Acr A的编码序列中设计引物 ,以大肠杆菌基因组为模板 ,扩增出 Acr A基因中约 6 91 bp的 c DNA片段 ,将所得片段与 p MD- 1 8T载体连接 ,转化到 DH5α大肠杆菌中 ,成功地筛选到阳性克隆 ,其质粒测序结果与文献报道一致。从阳性克隆中提取质粒 ,经 Hind 和 Bam H 酶解 ,回收 6 91 bp的目的片段 ,定向克隆到 p ET- 2 8a表达载体中 ,提取质粒 ,再次转化到 BL2 1 (DE3)中 ,成功地筛选出阳性克隆。经 IPTG诱导阳性菌 ,通过 SDS- PAGE检测出Acr

A construction AcrA pMD 18T was generated by inserting the sequence of 691 bp obtained by a PCR into pMD 18T 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 enzymes (HindⅢ and BamHⅠ) and ligated the pET28a vector. Then the plasmid AcrA pET28a was transformed to the competent cell of BL21(DE3).The sense clone was induced with IPTG.The expression of AcrA was observed on SDS PAGE.