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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?
Four strains of RFD-resistant Escherichia coli (RFD MIC 500μg / ml) were screened with a concentration gradient plate of rifandine (RFD) and the drug resistance of the two strains was maintained for two consecutive generations. Four strains of RFD-resistant Escherichia coli Phenol-chloroform method, phenol-dialysis method and proteinase K-glass rod winding method to extract the total DNA and using boiling method, alkaline extraction of plasmids, agarose gel electrophoresis analysis, the results showed no plasmid DNA. It suggested that the drug-resistant strains of the tested strains were resistant to mutations and mediated by chromosomes. To RFD-resistant Escherichia coli 2280, the chromosomal DNA was extracted by the protease K-glass rod winding method and completely digested with BglII. A large number of DNA fragments of different sizes were obtained, and then compared with the BamHI-digested vector pBR322 AMpr, TCr inactivation, 4.skb) were ligated with T4 DNA ligase, and the recombinant was transformed into competent E. coli HB101. Finally, the recombinant LB agar plate was screened on a selective LB agar plate containing RFD 50μg / ml and AMP 60μg / ml. Colonies with the RFD mutant gene recombination * plasmid pBD802. The MIC for detection of RFD was 512 μg / ml, and the size of RFDDNA-resistant fragment was about 16 kb by agarose gel electrophoresis. The restriction endonuclease profile of pBD802 was digested with EcoRⅠ, BamHⅠ, HpaⅠ, EcoRⅤ, SalⅠ, HindⅢ, BalⅡ and PstⅠ restriction enzymes.