QS系统中LasR和RhlR蛋白对铜绿假单胞菌生物被膜形成的影响以及免疫原性研究

摘要

为探讨铜绿假单胞菌PAO1中lasR和rhlR基因表达产物的分子生物学特性,研究它们对铜绿假单胞菌生物被膜形成的影响以及对小鼠的免疫保护效果,采用聚合酶链式反应(PCR)方法扩增铜绿假单胞菌标准株PAO1中的lasR和rhlR基因,全自动荧光测序仪测序,并用Blast方法检测克隆片段.利用pGEX4T-1载体分别构建lasR/1hlR-pGEX4T-1重组质粒,在大肠杆菌BL21(DE3)中诱导表达,并经过免疫印迹实验验证其生物学活性.用硅胶膜培养法建立生物被膜模型,诱导转入了pGFPuv质粒的铜绿假单胞菌PAG0305形成生物被膜,并测定LasR蛋白和RhlR蛋白对生物被膜形成的影响.同时用纯化的重组蛋白免疫小鼠,菌落计数法检测免疫组和对照组鼠肺对铜绿假单胞菌的清除率.以PAO1染色体DNA为模板的PCR结果显示,lasR的全基因序列为720 bp,rhlR基因序列为726bp,经序列分析和同源性比较分别与GenBank中lasR/rhlR基因(登录号:M59425;AE004768)的同源性为100%.大肠杆菌BL21(DE3)分别转化重组质粒lasR/rhlR-pGEX4T-1后,经IPTG诱导和SDS-聚丙烯酰胺凝胶电泳分析,表达的融合蛋白分子质量均为54 ku左右,与预期蛋白质分子质量相同.荧光显微镜观察和测定结果表明,在硅胶膜上PAG0305能够形成典型的发荧光的生物被膜,LasR或Rh1R蛋白(10 mg/L)存在的情况下,PAG0305生物被膜的形成速度在前三天比对照组平均提高40.77%,而且两蛋白单独存在与同时存在时的作用相同.体内实验中,免疫小鼠肺部对铜绿假单胞菌的清除率显著高于未经免疫的正常组(P＜0.05).上述结果表明:构建的lasR/rhlR-pGEX4T-1重组质粒能够在大肠杆菌BL21(DE3)中成功地表达并具有生物学活性.LasR/RhlR蛋白在体外模型中能够加快铜绿假单胞菌生物被膜的形成速度,是调节铜绿假单胞菌生物被膜形成的重要因素之一.免疫结果表明,重组蛋白对小鼠表现出一定的保护作用,这为进一步开展疫苗研究奠定了基础.%New strategies are needed for prevention of Pseudomonas aeruginosa (P. aeruginosa) infections, a widespread disease caused by P. aeruginosa with strong drug resistance. The immunoprotective capacity of the receptor of autoinducers protein LasR/RhlR was examined in the BALB/c mice. At first, specialized expression plasmids were developed to facilitate expression of LasR/RhlR proteins in Escherichia coli (E. coli). Then, biofilms were grown from clinical isolated P. aeruginosa PA0305 to investigate the relative contributions of cell signaling for biofilm formation. Morphological characters of biofilm were quantified using Image-Pro Plus software. Fluorescence analysis demonstrated that cell signal molecule LasR/RhlR significantly (P ＜ 0.05) influenced development of P. aeruginosa biofilm. Active immunization of mice with LasR/RhlR was found to provide significant protection against homologous challenge with P. aeruginosa in mice lungs. In 10 days after lungs inoculation, the bacterial clearance rate of the immunized mice was clearly higher than that of non-immunized groups on the basis of microbiological and histological assays. The protective effects of immunization with LasR and Rh1R together were the same as the result of LasR or Rh1R immunized mice alone. These data indicate that the manner ofLasR, Rh1R or both is an important determinant of immunoprotection in mice lungs infection.