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目的构建粪肠球菌丝氨酸蛋白酶基因(sprE)突变株,并研究sprE基因的功能。方法用自杀质粒pTX4577构建粪肠球菌基因重组自杀质粒pCQ001,通过体内同源重组,筛选获得sprE基因的突变株,体外研究不同温度和氧化条件对突变株生长能力的影响,通过小鼠腹膜炎和兔心内膜炎模型来研究突变株的毒力下降情况。结果经同源重组,利用卡那霉素抗性筛选,PCR、脉冲场电泳和Southern印迹进行鉴定获得sprE基因突变株,命名为*sprE,突变株在40℃的生长能力及在氧化条件下的存活率均明显低于野生株。在小鼠腹膜炎模型中,存活率明显高于野生株,差异有统计学意义(P<0.01),引起的兔心内膜炎也较野生株轻,差异有统计学意义(P<0.01)。结论sprE基因突变株*sprE构建成功,sprE基因在粪肠球菌致病中起重要作用,可能是粪肠球菌的毒力因子之一。
Objective To construct the serine protease gene (sprE) mutant of Enterococcus faecalis and to study the function of sprE gene. Methods The suicide plasmid pTX4577 was used to construct the recombinant suicide plasmid pCQ001 of Enterococcus faecalis. The mutant of sprE gene was screened by homologous recombination in vivo. The effects of different temperature and oxidative conditions on the growth of the mutant were studied in vitro. Endocarditis model to study the mutant virulent decline. The results of homologous recombination, the use of kanamycin resistance screening, PCR, pulsed-field electrophoresis and Southern blot were identified sprE gene mutant was named * sprE, the mutant at 40 ℃ growth capacity and under oxidative conditions Survival rates were significantly lower than wild strains. In mouse model of peritonitis, the survival rate was significantly higher than that of the wild-type strain (P <0.01). The incidence of rabbit endocarditis was also lighter than that of the wild-type strain. The difference was statistically significant (P <0.01). Conclusion The sprE gene mutant sprE was successfully constructed, sprE gene plays an important role in the pathogenesis of Enterococcus faecalis, which may be one of the virulence factors of Enterococcus faecalis.