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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。
In this study, virG of virulent I-phase plasmids from Shigella flexneri was cloned by whole-cell PCR and the middle part of virG was replaced by aspartate semialdehyde dehydrogenase gene (asd) , Followed by insertion suicide-inducing plasmid pXL2 75 to obtain a recombinant plasmid pKNVA1. Through interbreeding and in vivo homologous recombination, pKNVA1 is integrated into the I-phase large plasmid. With the help of another temperature-sensitive binding transfer plasmid pTH10, the cosmid is induced to transfer to Shigella flexneri FWL0 1 and the large intestine Bacillus subtilis X6 097, and the suicide plasmid pKNVA1 was detached from the large plasmid by chloramphenicol enrichment and sucrose reverse selection. The slide agglutination test and the large plasmid electrophoresis confirmed the successful induction of I-phase large plasmids in Song , Suggesting that this is an efficient method for targeted transfer of large plasmids.