福氏志贺菌2a多药耐药基因marA的克隆及其原核表达  

Cloning and prokaryotic expression of multidrug resistance gene marA of Shigella flexneri 2a

作　　者：王松泰[1] 张继瑜[1] 魏小娟[1] 曹小安[2] 邱昌庆[2] 

机构地区：[1]中国农业科学院兰州畜牧与兽药研究所中国农业科学院新兽药工程重点开放实验室 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2008年第6期500-503,共4页Chinese Veterinary Science

基　　金：国家自然科学基金项目(30671582)

摘　　要：根据福氏志贺菌2a多药耐药基因marA的序列,设计并合成了1对特异性引物,在引物的5′和3′端分别加入含有BamHⅠ和SalⅠ限制性酶切位点的序列,以福氏志贺菌2a菌株基因组DNA为模板扩增了marA基因序列。将PCR产物与pMD18-T载体连接,转化到大肠杆菌DH5α。鉴定成功获得目的片段后,经BamHⅠ+SalⅠ双酶切并与载体pET-30a连接,构建原核表达质粒pET-30a-marA,并将其转化宿主菌BL21(DE3)pLys,用IPTG诱导其表达。SDS-PAGE分析表明,重组载体pET-30a-marA可成功地在大肠杆菌中表达MarA蛋白。Western-blotting检测证明,该蛋白能与志贺菌阳性血清发生特异性反应。A pair of specific primers with two unique restriction sites of BamH Ⅰ and Sal Ⅰ were designed according to the marA gene sequence published previously. The full-length marA gene was amplified from genomic DNA of Shigella flexneri 2a by PCR with the specific primers. The plasmid of positive clone was identified after the PCR product was cloned into pMD18-T vector and transformed into the Escherichia coli strain DHSα. When the fragment was inserted into pET-30a plasmid by double digestion with BamHⅠ ＋Sal Ⅰ and the prokaryotic expression vector pET-30a-marA was constructed,and the recombinant plasmid was transformed into the host strain bacteria BL21（DE3）pLys and induced by IPTG. SDS-PAGE analysis showed that the target gene fragment was expressed successfully. Western-blot analysis indicated that the fusion protein could react specifically with positive sera against Shigella.

关 键 词：福氏志贺菌2a marA基因 克隆 原核表达 

分 类 号：S852.612[农业科学—基础兽医学] Q786[农业科学—兽医学]