金黄色葡萄球菌agr基因敲除及其对膜泡毒力影响.docVIP

金黄色葡萄球菌agr基因敲除及其对膜泡毒力影响 袁吉振1，杨杰1，袁文常1，胡珍1，尚伟龙1，张霞1，朱军民1，胡启文1，周人杰2， 饶贤才1 （400038 重庆，第三军医大学微生物教研室，重庆市微生物工程实验室(1； 40037 重庆，第三军医大学新桥医院急诊部2） 摘要：【目的】利用同源重组技术构建金黄色葡萄球菌RN4220附属基因调节系统（agr）的敲除株，探讨agr基因缺失对金黄色葡萄球菌分泌膜泡毒力的影响。【方法】构建同源重组质粒pYT3::Δagr，电转化至金黄色葡萄球菌RN4220，利用pYT3质粒对温度敏感的特点筛选agr缺失的突变株。分别制备野生菌RN4220和突变株RN4220::Δagr的膜泡，小鼠毒力实验观察agr基因缺失后对金黄色葡萄球菌膜泡毒力的影响。【结果】同源重组质粒pYT3::Δagr通过酶切鉴定证明构建成功；经PCR及DNA测序证实获得金黄色葡萄球菌RN4220 agr缺失突变株；aGene knockout of Staphylococcus aureus agr and its effect on the virulence of bacterial membrane vesicles Yuan Jizhen 1，Yang Jie1，Yuan Wenchang1，Hu Zhen1，Shang Weilong1，Zhang Xia1，Zhu Junmin1，Hu Qiwen1，Zhou Renjie2，Rao Xiancai1（1Department of Microbiology，Laboratory of Microbiological Engineering，College of Basic Medical Science，Third Military Medical University，Chongqing，400038；2Department of Emergency，Xinqiao Hospital，Third Military Medical University，400037，China） Abstract:[Objectives] To construct the agr gene knockout mutant of Staphylococcus aureus and evaluate the effect of agr on the virulence of secreting membrane vesicles. [Methods] A plasmid pYT3::Δagr was constructed for homologous recombination of the S. aureus and transformed into the S. aureus strain RN4220. S. aureus RN4220 with the recombination vector was incubated at 42℃ and 25℃ alternatively to select the agr deletion mutant. The membrane vesicles were prepared from both RN4220::Δagr mutant and the wild type strain, and the virulence of the membrane vesicles was evaluated after challenging Balb/c mice. [Results] Restriction endonucleases analysis indicated that the homologous recombination vector was correctly constructed and the agr deletion mutant was characterized by PCR and direct sequencing of the chromosomal DNA. The virulence of membrane vesicles prepared from the RN4220::Δagr mutant was significantly decreased comparing to that from the wild type RN4220. [Conclusions] The agr gene of S. aureus has great effect on the virulence of membrane vesicles. The construction of the RN42