摘要
目的对福氏志贺菌外排基因acrA进行克隆和原核表达,为进一步研究其在志贺菌外排机制中的作用奠定基础。方法参考福氏志贺菌2aacrA基因序列设计一对特异引物,在引物的5′和3′端分别加入含有BamHⅠ和SalⅠ限制性酶切位点序列。以福氏志贺菌2a菌株基因组DNA为模板,通过PCR扩增acrA基因并与pMD18-T载体连接,然后转化DH5α。提取重组质粒pMD18-acrA,经BamHI/SalI双酶切并与载体pET30a连接后转化宿主菌BL21(DE3)pLys,通过IPTG诱导表达目的蛋白。结果克隆的acrA基因长度为1122bp,核苷酸序列与GenBank上公布的序列完全相同。原核表达经SDS-PAGE及WesternBlotting检测和鉴定,结果表明重组载体pET-30a-acrA可成功地在大肠杆菌中表达AcrA蛋白。结论成功构建了福氏志贺菌acrA基因的原核表达质粒,并在大肠杆菌中得到有效表达,该研究为了解AcrA蛋白的特性、功能以及对福氏志贺菌多药耐药机理的深入研究奠定了基础。
The active efflux gene acrA of Shigella flexneri 2a was cloned and expressed by means of a pair of specific primers designed according to the sequence of acrA gene sequence published by NCB1, in which BarnH Ⅰ and Sal Ⅰ restriction sites were added to the 5′ and 3′ termini of primers. The genomic DNA of S. flexneri 2a was used as tenplate and the acrA gene was amplified by PCR, linked to vector pMD18-T and then transformed to E. coli DH5a. Meanwhile, the recombinant plasmid pMD18-acrA was extracted and transformed to host bacteria BL21（DE3）pLys after BamH Ⅰ/Sal Ⅰ double digestion and linkage with vector pET-30a. The target protein was expressed after induction with IPTG, and the expressed product was analyzed by SDS-PAGE and Western blotting. Experimental results revealed that the cloned acrA gene was 1122 bps in length and its nucleotide sequence was completely identical to that published in GenBank. As analyzed with SDS_PAGE and Western blotting, the recombinant vector pET-30a-acrA could successively express AcrA protein in E. coli and this protein could react with positive sera against shigella organism. These result would provide basis for the further studies on the characterization and function of AcrA protein and the study on the mechanism of multi-drug resistance in S. flexneri 2a.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2008年第10期933-936,共4页
Chinese Journal of Zoonoses
基金
国家自然科学基金项目(No.30671582)
关键词
福氏志贺菌
acrA基因
克隆
原核表达
Shigella flexneri 2a
acrA gene
cloning
prokaryotic expression
