金黄色葡萄球菌基因敲除质粒的构建及应用

摘要

BACKGROUND:Methicil in-resistant Staphylococcus aureus has been a primary pathogen of nosocomial infections worldwide. To construct a quick and easy knockout method is an important technique of studying virulence and resistance of methicil in-resistant Staphylococcus aureus. OBJECTIVE:To construct the Staphylococcus aureus gene knockout plasmid for understanding the antibiotic resistance and virulence of Staphylococcus aureus. METHODS:pUC19 was considered as a basic skeleton of construction. pLE194Ts temperature-sensitive replicon and tetracycline resistance gene fragment pHY300PLK plasmid in pCL52.1 were bound to EcoR I site in pUC19 by high assurance amplification. Al multiple clone sites in pUC19 were reserved. The Escherichia coli-Staphylococcus aureus shuttle plasmid was obtained. The N315 dapB gene knockout plasmid was obtained through gene knockout technology. This strain was eventual y identified by multiplex-PCR. RESULTS AND CONCLUSION:The Escherichia coli-Staphylococcus aureus shuttle plasmid, pYZ1 and pYZ8, was successful y constructed, and had been used in Staphylococcus aureus gene knockout. Homologous recombinant plasmid pYZ-ΔdapB was constructed by restriction enzyme digestion and overlap technique. After genetical y modification in RN4220, the constructed gene knockout plasmid pYZ-ΔdapB was introduced to N315 to be screened in the low culture temperature. The deletion strain was successful y obtained after being identified by multiplex-PCR. Above data suggested that pYZ1 and pYZ8 can be successful y used for Staphylococcus aureus gene detection, which provides a tool to study resistance and virulence of clinical Staphylococcus aureus strains.%背景：耐甲氧西林金黄色葡萄球菌已经成为全球院内感染的首要致病菌，建立快速简单的基因敲除方法是研究耐甲氧西林金黄色葡萄球菌毒力、耐药性的重要技术。　　目的：构建适用于金黄色葡萄球菌基因敲除的质粒，用于金葡菌耐药性及毒力的研究。　　方法：以pUC19为构建的基本骨架，通过overlap PCR的技术手段，把pCL52.1中pLE194Ts温敏复制子及质粒pHY300PLK中四环素抗性基因片段，通过高保证扩增后，连接入pUC19的EcoRⅠ位点，保留pUC19中所有的多克隆位点，构建大肠杆菌-金黄色葡萄球菌穿梭质粒，并对金黄色葡萄球菌N315 dapB基因进行敲除，通过多重PCR对基因敲除菌株进行鉴定。　　结果与结论：成功构建了大肠杆菌-金黄色葡萄球菌穿梭质粒pYZ1和pYZ8，并用于金葡菌基因的敲除，通过酶切及overlap技术构建了同源重组质粒pYZ-ΔdapB，电转入RN4220进行修饰后，再电转入N315，通过温度筛选，获得了dapB基因突变菌株，成功的敲除了dapB基因。提示pYZ1和pYZ8能够成功用于金黄色葡萄球菌耐药菌株基因的敲除，为进一步研究临床金黄色葡萄球菌分离株耐药性及毒力提供了工具。