同源重组构建临床分离金黄色葡萄球菌rbf基因阴性突变株

Detection of rbfvia homologous recombination in a clinical isolate of Staphylococcus aureus

目的利用同源重组技术敲除金黄色葡萄球菌rbf基因,构建金黄色葡萄球菌临床分离株金黄色葡萄球菌20(SAU20)rbf基因的阴性突变株。方法构建pBT2-Δrbf质粒后将之电转入金黄色葡萄球菌RN 4220中,再次将pBT2-Δrbf质粒转入金黄色葡萄球菌临床分离株SAU20;利用pBT2载体对温度敏感的特点,使含重组质粒的SAU20在40℃多次传代,最终筛选出金黄色葡萄球菌rbf基因的阴性突变株。结果成功构建pBT2-Δrbf质粒,pBT2-Δrbf质粒被转入SAU20后,筛选到可疑突变株,经PCR及DNA测序证实SAU20 rbf基因被ermB基因置换。结论利用同源重组技术敲除了金黄色葡萄球菌rbf基因,完成了金黄色葡萄球菌临床分离株SAU20 rbf基因的阴性突变株的构建,为今后研究金黄色葡萄球菌生物膜形成过程中rbf基因的作用及其机制提供了实验载体。

OBJECTIVE To delete rbf in a clinical isolate of Staphylococcus aureus homologous recombination and to get a S.aureus strain 20（SAU20）of the rbf negative mutant.METHODS A plasmid pBT2-Δrbf was constructed with inserting erythromycin resistance gene and two PCR-amplified rbf-flanking regions for homologous recombination of S.aureus.The recombination vector was transformed to S.aureus RN4220 by electroporation firstly and then transformed to S.aureus 20.SAU20 with recombination vector was incubated at 40℃ until the rbf delectation strain was selected.RESULTS pBT2-Δrbf has been successfully constructed,the suspected mutant strains were screened out after the pBT2-Δrbf was transferred to the SAU20,the restriction endonucleases results indicated that the homologous recombination vector was correct and SAU20 accepted the recombination vector.The rbf of SAU20 was replaced by ermB,which was proved by PCR and DNA sequencing.CONCLUSION S.aureus rbf deletiou mutation has been constructed successfully.The sequence of agr gene is replaced by ermB gene in SAU20,which offers the experimental vectors for the study on the rbf and its mechanism in the formation of the S.aureus biofilm.