表皮葡萄球菌PhoU同源物(SERP0316)的抗体制备及免疫鉴定  

Expression of a PhoU homologue (SERP0316) in Staphylococcus epidermidis

作　　者：韩海燕[1] 王小飞[1] 武有聪[1] 许涛[1] 刘华勇[1] 蔡霞[1] 瞿涤[1] 

机构地区：[1]复旦大学基础医学院教育部/卫生部医学分子病毒学重点实验室,上海200032

出　　处：《微生物与感染》2014年第3期174-181,共8页Journal of Microbes and Infections

基　　金：国家自然科学基金(81271791;81271594);"十二五"国家科技重大专项(2012ZX09301002-001-005;2012ZX10004401)

摘　　要：表皮葡萄球菌可在植入性医疗材料表面形成生物膜,对多种抗生素耐受,而持留菌的形成是导致细菌生物膜耐药的原因之一。研究发现,大肠埃希菌中PhoU可影响持留菌的形成,与细菌对多种抗生素及压力条件的耐受相关,但表皮葡萄球菌中PhoU的功能仍不清楚。生物信息学分析显示,在表皮葡萄球菌中存在2个phoU同源基因,其中serp0956位于pst操纵子中,命名为phoU1;另一个编码功能未知蛋白的phoU基因同源物(serp0316)命名为phoU2。前期研究显示,敲除phoU1并不影响表皮葡萄球菌生长、生物膜形成等生物学特性,为此本研究对PhoU2进行了初步探讨。利用实时荧光定量反转录-聚合酶链反应(RT-PCR)检测phoU2基因在表皮葡萄球菌SE1457不同生长时期的转录水平,发现其在细菌生长不同时间点持续表达,其中对数期(6h)表达量相对较高。为进一步研究表皮葡萄球菌中PhoU2的表达情况,对PhoU2蛋白进行原核表达和纯化,并用纯化的重组PhoU2蛋白免疫小鼠制备PhoU2多克隆抗体。在此基础上,利用蛋白免疫印迹法检测PhoU2在SE1457菌株不同生长时间点的表达水平,结果显示PhoU2在细菌生长对数期和平台期均有表达。另外,在不同的表皮葡萄球菌临床分离株中均检测到PhoU2蛋白表达,表明PhoU2具有一定的保守性。以上研究提示,PhoU2可能作为PhoU的功能同源物发挥一定的作用,为进一步研究表皮葡萄球菌PhoU2的生物学功能及其在生物膜和持留菌形成中的作用奠定了基础。The pathogenesis of Staphylococcus epidermidis （S. epidermidis ） in persistent infections is primarily attributed to its ability to form biofilms on the surface of indwelling medical devices. The antibiotic tolerance of biofilm is partly due to the presence of persister cells; however, the mechanism of bacterial persistence is poorly understood. In Escherichia coli （E. coli）, PhoU was reported as a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses. There are two phoU homologues （serp0956 and serp0316） in S. epidermidis. As a component of pst operon, serp0956 was designated phoU1. The other phoU homologue serp0316 which encoded a hypothetical protein was namedphoU2. Our previous study revealed that deletion of phoU1 in SE1457 had no effects on biofilm formation capacity. In this report a preliminary study on phoU2 was conducted. The relative transcriptional level of phoU2 in logarithmic phase was higher than that in stationary phase detected by a quantitative real-time polymerase chain reaction （PCR）. PhoU2 was expressed in E. coli with a prokaryotic expression recombinant plasmid and purified to immunize BALB/c mice by intraperitoneal injection, and the anti-PhoU2 antisera were collected. Western blotting with anti-PhoU2 polyclonal antibody was conducted to monitor PhoU2 expression in S. epidermidis. Continuous expression of PhoU2 in both exponential and plateau phases was detected. The expression of PhoU2 was also detected in several clinical isolates of S. epidermidis. These results indicate that PhoU2 is conserved in different strains of S. epidermidis and suggest that PhoU2 may function as a homologue of PhoU in E. coli. These data allow us to further characterize the role of PhoU2 in the formation of biofilm and persisters in S. epidermidis.

关 键 词：表皮葡萄球菌 phoU同源基因 原核表达 持留菌 

分 类 号：R392[医药卫生—免疫学]