屎肠球菌ORF5基因缺失株的构建及鉴定

Construction and identification of Enterococcus faecium with ORF5 gene deletion

目的构建屎肠球菌ORF5基因缺失株,证实其可能是屎肠球菌感染中较为重要的致病因子,为屎肠球菌的预防和治疗提供理论依据。方法通过重叠延伸PCR技术将ORF5基因上游同源臂、卡纳霉素基因、下游同源臂连接成一个片段,克隆入自杀质粒PCVD442,将重组质粒电转化入屎肠球菌中,通过卡纳霉素以及菌落PCR筛选得到ORF5基因缺失株,进行PCR以及测序鉴定;比较突变株与野生株体外生存能力以及对膀胱癌细胞的粘附率。结果利用重叠延伸PCR技术将ORF5基因上游同源臂、卡纳霉素基因、下游同源臂连接成一个片段,并连接至PCVD442自杀质粒;重组自杀质粒进入屎肠球菌后出现了同源重组事件,ORF5基因被卡纳霉素基因替代,通过PCR及测序分析证实基因组ORF5基因已敲除;通过细胞培养证实野生株对膀胱癌细胞的粘附率高于突变株。结论成功构建屎肠球菌ORF5基因突变株,突变株黏附膀胱癌细胞能力减弱,初步证实ORF5基因与屎肠球菌的致病力有关。

OBJECTIVE To construct the Enterococcus faecium strains with ORF5 gene deletion and confirm that it may be an important pathogenic factor for E. faecium infection so as to provide theoretical basis for prevention and treatment of the E. faecium infection. METHODS The upstream and downstream flanking DNA fragments of ORF5 gene and kanamycin gene were fused by using spliced overlap extension PCR （SOE-PCR） technique and then cloned into the suicide plasmid PCVD442. The recombinant plasmids were electransformed into the E. faeci- um strains. The strains with ORF5 gene deletion were screened by kanamycin and PCR and identified by PCR and DNA sequencing; the in vitro viability and adhesion rate of bladder carcinoma cells were observed and compared between the mutant strains and the wild strains. RESULTS The upstream fragment, kanamycin gene and down- stream fragment were fused to a recombinant fragment by SOE-PCR and connected to the plasmid PCVD442. The homologous recombination occurred after the recombinant plasmids were electransformed into the E. faecium strains, the ORF5 gene was replaced by the kanamycin gene. The PCR and DNA sequencing proved the deletion of the ORF5 gene. The cell culture confirmed that the adhesion rate of bladder carcinoma ceils of the wild strains was higher than that of the mutant strains. CONCLUSION The E. faecium mutant strain with ORF5 gene deletion is constructed successfully, the adhesion ability of the bladder carcinoma cells of the mutant strains is decreased. It is confirmed that the ORF5 gene is associated with the pathogenicity of the E. faecium strains.