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【目的】构建金黄色葡萄球菌RN6390黄素血红蛋白(flavohaemoglobin,HMP)基因缺失突变株,研究其抗一氧化氮(Nitric Oxide,NO)能力及其在细菌生物被膜形成中的作用。【方法】根据同源重组技术的原理,利用PCR扩增RN6390的hmp基因上下游同源臂,经过抗生素和温度交替培养筛选hmp基因缺失突变株,利用基因组PCR、定量PCR对突变菌株进行鉴定。以硝普钠(SNP)为NO供体,检测了hmp基因缺失菌株的抗NO能力,并初步研究了hmp基因在生物被膜形成中的作用。【结果】成功构建了RN6390的hmp基因缺失突变株,外源NO能够诱导菌株hmp基因的表达,hmp基因缺失菌株抗NO能力明显下降,但其生物被膜形成能力有明显提高。【结论】获得了RN6390的hmp基因缺失突变株,该突变株的获得为进一步研究hmp基因的生物功能,以及细菌内源性NO的作用奠定了良好的技术平台。
【Objective】 To construct the mutant of Staphylococcus aureus RN6390 with flavohaemoglobin (HMP) gene and study its anti-nitric oxide (NO) abilities and its role in bacterial biofilm formation. 【Method】 According to the principle of homologous recombination, PCR was used to amplify the upstream and downstream homology arms of hmp gene of RN6390. The hmp gene deletion mutant was screened by antibiotic and temperature alternation. Genomic PCR and quantitative PCR were used to identify the mutant strains. Using nitroprusside (SNP) as NO donor, the ability of anti-NO of hmp gene deletion strain was tested, and the role of hmp gene in the formation of biofilm was also studied. 【Result】 The hmp gene deletion mutant of RN6390 was successfully constructed. Exogenous NO induced the expression of hmp gene. The ability of anti-NO of hmp gene deletion strain was significantly decreased, but the ability of biofilm formation was significantly improved. 【Conclusion】 The hmp gene deletion mutant of RN6390 was obtained. The obtained mutant strain has laid a good technical platform for further study of the biological function of hmp gene and the role of bacterial endogenous NO.