大肠杆菌耐利福定突变基因的克隆及限制性酶切谱分析  

Cloning and map analysls of rifandine resistant E. coli mutant gene digested with restriction endonuclease

作　　者：段传可 王浴生[1] 强文安[1] 李良宏[1] 

机构地区：[1]华西医科大学基础医学院

出　　处：《中国抗生素杂志》1995年第3期163-168,共6页Chinese Journal of Antibiotics

基　　金：国家自然科学基金

摘　　要：采用利福定（ｒｉｆａｎｄｉｎｅ，ＲＦＤ）浓度梯度平皿筛选得４株耐ＲＦＤ大肠杆菌（ＲＦＤ的ＭＩＣ为５００μｇ／ｍｌ），连续传两代其耐药性保持稳定；对４株耐ＲＦＤ大肠杆菌，采用酚—氯仿法，酚—透析法和蛋白酶Ｋ—　玻棒缠绕法提取其总ＤＮＡ和采用煮沸法、　碱法提取质粒，经琼脂糖凝胶电泳分析，结果均未见质粒ＤＮＡ带。　提示所试菌株的耐药系突变耐药，由染色体介导。对耐ＲＦＤ　大肠杆菌２２８０，用蛋白酶Ｋ—玻棒缠绕法提取其染色体ＤＮＡ，并选Ｂｇ１Ⅱ完全酶切，获许多大小不等的ＤＮＡ片段，然后与碱法提取的ＢａｍＨＩ　酶切的载体质粒ｐＢＲ３２２（ＡＭｐｒ、ＴＣｒ失活，４．ｓｋｂ）用Ｔ４－ＤＮＡ连接酶连接，重组体再向感受态大肠杆菌ＨＢ１０１转化，最后在含ＲＦＤ５０μｇ／ｍｌ，ＡＭＰ６０μｇ／ｍｌ的选择性ＬＢ琼脂平板上筛选获得了携有耐ＲＦＤ突变基因重组＊质粒ｐＢＤ８０２的菌落。检测ＲＦＤ的ＭＩＣ为５１２μｇ／ｍｌ，琼脂糖凝胶电泳显示其耐ＲＦＤＤＮＡ片段的大小约１６ｋｂ。选ＥｃｏＲⅠ、ＢａｍＨⅠ、ＨｐａⅠ、ＥｃｏＲⅤ、ＳａｌⅠ、ＨｉｎｄⅢ、ＢａｌⅡ、ＰｓｔⅠ８种限制性内切酶对ｐＢＤ８０２进行完全酶切，分析了其限制性酶切电泳图谱特征?In this paper,four rifandine(RFD)resistant strains(MIC 500μg/ml)were selected from their corresponding sensitive strains by using gradient agar plate method。TheRFD resistance of these strains was stable and still well retained after two subcultures inthe absence of RFD。The total DNA of the four resistant strains were extracted by threedifferent methods。The agarose gel electrophoresis showed no finding of existance of plasmidDNA。In addition,with two extraction methods especially for plasmid DNA,there was noplasmid DNA either,ResuIts strongly suggested that the RFD resistance may not be relatedto plasmid,but mediated by chromosomal mutation。E. coli 2280 RFD resistant strain was chosen to be further studied.Its chromosomalDNA was isolated by using proteinase K-winding up glass rod method and entirely digestedwith BgI I.The digested DNA fragments were ligated with BamH I-digested pBR322(AMpr,TCr was lost,4.3kb)prepared by alkaline lysis with T4-DNA ligase,Afterrecombinations these were transformed into E. coli HB101,a pBD802 plasmid carrying theRFD resistant gene has been isolated from the colonies on the selective agar plates containingRFD 50μg/ml and AMP 60 μg/ml.RFD MIC of pBD802-transformed strain was 5l2μg/ml。Agarose gel e[ectrophoresis determined that the size of the RFD resistant DNA fragment wasapproximately 16 kb.The sites of different restriction endonucleases cluding EcoR I,BamHI,Hpa I,EcoR V,Sal I,Hind Ⅲ,Bgl Ⅱ and Pst I have been defined.hese certainlycontribute for our further subcloning and determining the detail biological characteristics andDNA seqtience of the RFD resistant mutant gene。

关 键 词：利福定 限制性酶 切谱分析 基因克隆 

分 类 号：R978.3[医药卫生—药品]