金黄色葡萄球菌sigB同源重组质粒的构建

发布时间：2018-06-27 07:14

本文选题：金黄色葡萄球菌 + 生物膜　；参考：《第三军医大学》2006年硕士论文

【摘要】： 金黄色葡萄球菌(Staphylococcus aureus)是医院感染的重要病原菌之一,也是生物膜感染常见的病原菌,在ICU导管相关感染败血症的致病原中,金黄色葡萄球菌占15.4%,居第二位。形成生物膜的金黄色葡萄球菌具有高度的耐药性并能逃避免疫系统的攻击,其感染易慢性化并难于控制,因此对金黄色葡萄球菌等临床重要病原菌的生物膜形成机制的研究已成为抗感染研究领域的热点。目前对金黄色葡萄球菌生物膜的形成机制仍不清楚。初步研究显示生物膜的形成首先是由细胞壁相关因子调节的细胞表面的附着,随后细菌主要通过ica操纵子表达产物共同催化合成的多糖胞间粘附素(polysaccharide intercellular adhesin, PIA)介导,细胞增殖并相互聚集成细胞团块,继而形成生物膜。该过程中有多个基因参与调控生物膜的形成。其中革兰氏阳性菌中σ因子编码基因sigB的表达可能是生物膜形成的关键调控因素之一。它除可通过直接调节控制生物膜形成的ica操纵子的表达外,尚可影响ica的调控基因sarA和/或agr的表达。但新近采用对特定的金黄色葡萄球菌实验室菌株敲除sigB基因的研究发现,sigB缺失的菌株在某些条件下仍能形成生物膜。鉴于这些研究多局限于几株特定的实验室菌株,为明确sigB基因在金黄色葡萄球菌形成生物膜中的意义,本研究以第三军医大学第一临床医学院2004年7月-2004年12月分离的野生菌株为研究对象,在明确其耐药及生物膜形成情况的基础上,PCR扩增分析金黄色葡萄球菌中生物膜形成相关调节基因的存在情况;筛选可形成生物膜的菌株,采用融合PCR的方法构建金黄色葡萄球菌sigB同源重组质粒,为建立sigB缺失的野生金黄色葡萄球菌模型奠定基础。研究分三部分进行: 1.临床分离金黄色葡萄球药物菌敏感性和形成生物膜的检测 2.金黄色葡萄球菌生物膜形成相关基因的PCR分析 3.金黄色葡萄球菌sigB同源重组质粒的构建 1.方法 1.1金黄色葡萄球菌敏感性(最小抑菌浓度)用琼脂稀释法测定;生物膜形成的检测用96孔板番红染色法。 1.2 icaAD、icaBC、sar、agr和sigB基因采用PCR法扩增,并对产物进行测序和
[Abstract]:Staphylococcus aureus (Staphylococcus aureus) is one of the important pathogens of nosocomial infection and a common pathogen of biofilm infection. In the pathogeny of ICU catheter related septicemia, Staphylococcus aureus occupies 15.4%, ranking second. Staphylococcus aureus forming biofilm has high resistance and can escape immunity. The infection of the system is easy to be chronicity and difficult to control. Therefore, the study of the mechanism of the biofilm formation of clinical important pathogens such as Staphylococcus aureus has become a hot spot in the field of anti infection research.
The formation mechanism of Staphylococcus aureus biofilm is still unclear. Preliminary study showed that the formation of biofilm was first attached to cell surface regulated by cell wall related factors, and then bacteria mainly catalyzed by ica operon to co catalyze the polysaccharide intercellular adhesin, PIA In this process, there are several genes involved in regulating the formation of biofilms. In gram positive bacteria, the expression of the sigma factor encoding gene sigB may be one of the key regulatory factors of biofilm formation. It can be used to control the ICA of biofilm by direct regulation. The expression of the operon can affect the expression of the regulatory gene sarA and / or agr of the ICA. However, recent studies on the knockout of the sigB gene of a specific Staphylococcus aureus laboratory strain have found that the sigB missing strain can still form a biofilm under certain conditions. The significance of the sigB gene in the formation of Staphylococcus aureus in the biofilm was studied. This study was based on the study of the wild strains isolated from the first clinical medicine hospital of Third Military Medical University in December July 2004. On the basis of its drug resistance and biofilm formation, PCR amplification was used to analyze the regulation of biofilm formation in Staphylococcus aureus. The existence of the gene, screening the strains that can form biofilm, and using the fusion method of PCR to construct the homologous recombinant plasmid of Staphylococcus aureus sigB, lay the foundation for the establishment of the sigB deletion of the Wild Staphylococcus aureus model.
The study is divided into three parts:
1. clinical isolation of Staphylococcus aureus drug sensitivity and biofilm formation detection
PCR analysis of 2. genes related to biofilm formation in Staphylococcus aureus
Construction of 3. homologous recombination plasmid of Staphylococcus aureus sigB
1. method
1.1 the sensitivity (minimum inhibitory concentration) of Staphylococcus aureus was determined by agar dilution method, and the detection of biofilm formation was made by 96 hole plate red staining.
1.2 the icaAD, icaBC, SAR, agr and sigB genes were amplified by PCR and sequenced.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

【引证文献】