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目的:构建金黄色葡萄球菌一氧化氮合酶基因(nos)缺失突变株。方法从金黄色葡萄球菌RN6390的基因组DNA中扩增了nos基因的上、下游片段;以大肠杆菌和金黄色葡萄球菌穿梭质粒pMAD(含有温度敏感性的复制起点,红霉素抗性基因(erm)和β-半乳糖苷酶基因(bgaB)为筛选标记)为骨架,构建基于nos基因位点的同源重组载体pMAD△nos,该载体经金黄色葡萄球菌RN4220修饰后再转入金黄色葡萄球菌RN6390。经过在30℃和42℃交替培养,通过抗生素抗性和β-半乳糖苷酶活性筛选nos基因缺失突变株。结果筛选得到的突变菌株,经基因组PCR、定量PCR及序列分析表明,金黄色葡萄球菌RN6390基因组中的nos基因被成功地敲除。结论利用同源重组的方法构建了金黄色葡萄球菌RN6390nos缺失突变株,为金黄色葡萄球菌nos基因功能的研究奠定了基础。
Objective: To construct Staphylococcus aureus nitric oxide synthase gene (nos) deletion mutant. Methods The upstream and downstream fragments of nos gene were amplified from the genomic DNA of Staphylococcus aureus RN6390. The E. coli and Staphylococcus aureus shuttle plasmid pMAD (containing temperature-sensitive origin of replication, erythromycin resistance gene (erm ) And β-galactosidase gene (bgaB) as scaffolds, a homologous recombination vector pMADΔnos based on the nos gene site was constructed. After being modified by Staphylococcus aureus RN4220, the vector was transformed into Staphylococcus aureus RN6390. The nos gene-deleted mutants were selected by antibiotic resistance and β-galactosidase activity after being alternately cultured at 30 ° C and 42 ° C. Results The mutant strains were screened by genomic PCR, quantitative PCR and sequence analysis. The result showed that the nos gene in S. aureus RN6390 genome was successfully knocked out. Conclusion The method of homologous recombination was used to construct the mutant of Staphylococcus aureus RN6390nos, which lays the foundation for the study of the function of nos gene in Staphylococcus aureus.