【摘要】 本文通过全细胞PCR克隆了宋内志贺氏菌侵袭性I相大质粒上的virG基因的部分片段 (virG ) ,并用天冬氨酸半醛脱氢酶基因 (asd)取代了virG 的中间部分 ,随之插入自杀性诱动质粒pXL2 75中 ,获得重组质粒pKNVA1。通过细菌间交配和体内同源重组 ,pKNVA1整合至I相大质粒中 ,在另一温度敏感的结合转移质粒pTH10的帮助下 ,共整合质粒被诱动转移至福氏志贺氏菌FWL0 1和大肠杆菌X6 0 97中 ,并通过氯霉素富集和蔗糖反选择使自杀质粒pKNVA1从大质粒上脱落下来 ,玻片凝集试验和大质粒电泳图谱证实了宋内I相大质粒的成功诱动转移 ,表明这是一种定向转移大质粒行之有效的方法。更多还原

【Abstract】 In the present research a fragment of virG gene(virG *) was cloned from a invasive form I large plasmid of Shigella sonnei by whole cell PCR The central section of virG * was replaced by aspartic semialdehyde dehydrogenase gene(asd) The fragment of virG * asd virG * was then inserted into a mobilizable suicide plasmid pXL275, making a recombinant plasmid pKNAV1 By bacterial mating and homologous recombination in vivo , the pKNVA1 was integrated into form I plasmid of Shigell sonnei With the help of a temperature sensitive mobilizable plasmid pTH10, the co integrated plasmid was mobilized into Shigella flexneri FWL01 and E coli X6097 Hybrid strains that had undergone a second homologous recombination were then selected Slide agglutination assay and agarose gel electrophoretic profiles of large plasmid further indicated that Shigella sonnei form I large plasmid had been transfered into FWL01 and X6097 These data suggested that this methods could be an effective way to transfer large plasmids更多还原