【摘要】 目的构建表皮葡萄球菌icaC阴性突变株,以期获得仅icaC基因不同而其他遗传背景与表皮葡萄球菌1457株相同的突变株,对icaC基因产物在细菌形成生物被膜中的作用进行研究。方法构建同源重组质粒pBT2-△icaC,经金黄色葡萄球菌RN4220株修饰后电转化入表皮葡萄球菌1457株。将含重组质粒的表皮葡萄球菌1457株在40℃多次传代,筛选出icaC敲除突变株(△icaC株)。检测△icaC株生长曲线,采用微量板半定量法检测细菌生物被膜的形成能力,并采用斑点免疫印迹法检测细菌多糖黏附因子的合成。结果使用同源重组法敲除表皮葡萄球菌1457株基因组中的icaC基因,△icaC株的生长曲线与表皮葡萄球菌1457株相似,但生物被膜形成能力及细胞外多糖黏附因子含量显著降低。结论表皮葡萄球菌1457株中icaC基因的缺失对细菌生长无明显影响,但显著降低细胞外多糖黏附因子含量及生物被膜形成能力,为进一步研究表皮葡萄球菌icaC基因在生物被膜形成中的作用奠定基础。更多还原

【Abstract】 Objective To construct Staphylococcus epidermidis (Se) icaC deletion mutant (△icaC strain) for acquiring the mutant and wild-type strain with the same genetic background except icaC gene, and thus to find the function of icaC gene in biofilm formation process of bacteria. Methods Plasmid pBT2-△icaC was constructed for homologous recombination in Se by inserting erythromycin resistance gene (ermB) and two icaC-flanking fragments into vector pBT2. Se1457 with recombination plasmid was continuously subcultured at 40℃un-till the△icaC strain was selected. The growth curve, biofilm formation and polysaccharide intercellular adhesion synthesizing were compared between Se1457 wild and A△icaC strain. Results The icaC gene was deleted by homologous recombination in Se1457 strain. Compared with wild strain, the growth of the△icaC strain showed no change, while the biofilm formation and the extracellular polysaccharide intercellular adhesin declined dramatically. Conclusion Se1457△icaC strain is successfully constructed. Though the deletion of the icaC gene has little effect on Se growth, the biofilm formation and the extracellular polysaccharide intercellular adhesion both dramatically decline in the△icaC strain.更多还原