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福氏志贺菌2a多药耐药基因marA的克隆
及其原核表达
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王松泰1，2,张继瑜1*,魏小娟1，曹小安3，邱昌庆3
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（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，甘肃
兰州730050；2.甘肃农业大学 动物医学院，甘肃 兰州730070；3.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）
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摘要：
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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。 |
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关键词：
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福氏志贺菌2a；marA基因；克隆；原核表达 |
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中图分类号：S 852.612：Q 786 文献标识码：
A 文章编号：1673-4696(2008)06-0500-04 |
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Cloning and prokaryotic expression of multidrug resistance gene marA
of Shigella flexneri 2a |
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WANG Song-tai1,2,ZHANG Ji-yu1,WEI
Xiao-juan1,CAO Xiao-an3,QIU Chang-qing3 |
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（1.Key and Open Laboratory for New Animal Drug Engineering of Chinese Academy of Agricultural Sciences/Lanzhou Institute
of Animal Sciences and Veterinary Pharmaceutics,Chinese Academy of Agricultural Sciences,Lanzhou 730050,China;2.College of
Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070，China;3.State Key Laboratory of Veterinary Etiological
Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China） |
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Abstract: A pair of specific primers with two unique restriction sites of BamHⅠand SalⅠ 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 DH5α.When the fragment was inserted into pET-30a plasmid by double digestion with BamHⅠ+SalⅠ 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. |
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Key words: Shigella flexneri 2a;marA gene;cloning;prokaryotic expression |
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