【摘要】 目的构建屎肠球菌类透明质酸酶基因(hyl)突变株,研究 hyl 基因的功能。方法用自杀质粒 pTX4577通过体内同源重组,筛选获得 hyl 基因的突变株,研究体外 hyl 基因缺失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研究体内突变株的毒力情况。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 hyl 基因突变株*hyl,突变株在体外的生长能力低于野生株。小鼠腹膜炎模型中,在相同细菌感染浓度(3.7×10~9 CFU/ml)下,野生株 TX2466组小鼠在100h 内的存活率为0,而*hyl 组小鼠的存活率为50%,差异有统计学意义(P<0.01)。兔心内膜炎模型中,TX2466组主动脉瓣和壁性赘生物的菌落计数分别为(3.04±0.63)×10~6 CFU/g 和(6.87±0.58)×10~6 CFU/g,而*hyl 组则分别为(0.21±0.06)×10~6 CFU/g和(0.66±0.05)×10~6 CFU/g,差异有统计学意义(P<0.01)。结论 hyl 基因突变株构建成功,hyl 基因在屎肠球菌致病中起着重要的作用,可能是屎肠球菌的毒力因子之一。更多还原

【Abstract】 Objective To construct a hyaluronidase (hyl) gene mutant strain of Enterococcus faecium and to study the function of hyl gene.Methods An isogenic hyl-defieient mutant (* hyl) with suicide plasmid pTX4577 was constructed and screened by allelic replacement.The mutant strain and wild type strain TX2466 were cultured to observe the growth curves.Both*hyl and TX2466 strains were cultured in solid with kanamycin (KAN) to detect the tolerance to KAN.The study of virulence in rat peritonitis and rabbit endocarditis models in vivo were made.Results A mutant strain,*hyl,was selected by KAN and identified by PCR,pulsed field gel electrophoresis,and Southern blotting.The growth ability of the * hyl strain was remarkably reduced in comparison with the wide-type strain.The survival rate of the rats within 100 h after the inoculation of Enterococcusfaecium.TX2466 was 0,significantly lower than that of the * hyl group (50%,P<0.01).The numbers of colonies in the aortic valve and ventricular wall vegetations of the TX2466 group rabbits were (3.04±0.63)×10~6 and (6.87±0.58)×10~6 CFU/g respectively,both significantly higher than those of the * hyl group [ (0.21±0.06)×10~6 and (0.66±0.05)×10~6 CFU/g respective,both P<0.01].Conclusion Probably a major virulence factor of Enterococcus faecalis,hyl gene is important in the pathogenesis of this bacterium.更多还原