大肠杆菌耐药质粒的头孢哌酮抗性基因克隆和定位

Cloning and Orientation of Cefoperazone Resistance Gene on Plasmid pFC in E.coli HX88108

用鸟枪法从耐药质粒ｐＦＣ上克隆到头孢哌酮抗性基因。快速小规模制备质粒ＤＮＡ进行限制性酶切分析鉴定含重组质粒的克隆株，经多次传代，克隆株抗性表达稳定。多种限制性内切酶谱分析并构建重组质粒的物理图谱与质粒ｐＦＣ物理图谱比较，定位出头孢哌酮抗性基因在ｐＦＣ物理图谱上３２ｋｂ至４８ｋｂ区间内，其分子量约１６ｋｂ。该基因包含有ＥｃｏＲⅠ、ＳｍａⅠ、及ＰｖｕⅡ位点。

Cefoperazone resistance gene (CPZ r) has been cloned from plasmid pFC of E.coli HX88108 using the vector pMB9 (TC r, 5.3kb). The plasmid pFC DNA was partially digested with Sau3AⅠ, and its 1 2kb fragments were ligated into BamHⅠ site of vector plasmid pMB9. The recombinant DNA was then transformed into E.coli DH5 prepared using calcium chloride. CPZ resistant bacterial colonies were selected on the agar SOB plates containing CPZ (40μg/ml). The resistance to CPZ could be stably reserved in generation after generation. The recombinant plasmids which encoded CPZ resistance were designated pFL11, pFL25, pFL33, pFL82, pFL86 and pFL102. Rapid small scale preparation of plasmid and DNA restrication enzyme analysis were used for identification of bacterial colonies. Five plasmids DNA physical maps have been established. Comparison of recombinant plasmids maps with pFC map confirmed that the CPZ r gene was oriented between nucleotide no. 3200bp and no. 4800+40bp of plasmid pFC total sequence. Its molecular weight was about 1.6kb. There were EcoR Ⅰ, Sma Ⅰ and Pvu Ⅱ sites within CPZ r gene.