Construction and application of Staphylococcus aureus gene knockout plasmid / 中国组织工程研究

ABSTRACT

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.