利用酵母同源重组系统克隆肺炎克雷伯菌基因组岛

Cloning of Genomic Islands in Klebsiella Pneumoniae by Using Yeast-Based Homologous Recombination System

利用酵母同源重组系统克隆耐药性条件致病菌肺炎克雷伯菌的基因组岛.具体方法为:对20株从临床中分离的肺炎克雷伯菌进行体外tRIP PCR扩增以搜索外源基因组岛;利用酵母同源重组系统构建作用于arg6 tRNA基因位点的酵母捕捉载体YCV6并克隆该位点的基因组岛.结果表明:在该20株肺炎克雷伯菌中,arg6 tRNA基因位点的tRIP PCR结果都是1.2 kb,没有筛选到大基因组岛插入;所构建的酵母捕捉载体YCV6携带了2个1.8 kb的靶序列,分别与肺炎克雷伯菌arg6 tRNA基因位点两侧的保守序列同源;将线性化的载体YCV6与肺炎克雷伯菌临床株HS04044的总DNA一起转入酵母细胞,利用酵母同源重组克隆临床菌株HS04044染色体上插入arg6位点的小基因组岛.该位点特异性的捕捉载体YCV6可用于克隆肺炎克雷伯菌临床株染色体插入arg6位点的外源DNA序列.

To clone the genomic islands in Klebsiella pneumoniae clinical strains by yeast-based capture system. The tRIP PCR method was used to identify genomic islands in 20 isolates of Klebsiella pneumoniae. The yeast-based capture system was employed to construct yeast capture vector YCV6 which is used for arg6 tRNA gene site and cloning the identified genomie islands in this site of Klebsiella pneumoniae clinical strains. No large bands was obtatined among the 20 isolates of Klebsiella pneumoniae exept 1.2 kb ones by tRIP PCR method. The site-specific capture vector was constructed, which carries targeting sequence homologous to conserved sequences flanking in arg6 tRNA gene site. Then the genomic DNA of K. pneumoniae HS04044 was transformed with the linearized capture vector into Saccharomyces cerevisiae. An arg6-born islet in HS04044 was cloned by the capture vector.