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目的：构建金黄色葡萄球菌（金葡菌）OatA 基因敲除菌株及其回补菌株。方法：以pBT2 为载体，构建金葡菌OatA基因敲除质粒pBT2-驻OatA，经金葡菌RN4220 修饰后电转入金葡菌USA300，利用pBT2 载体对温度敏感的特点，在42 ℃多次传代，筛选出金葡菌OatA 基因敲除菌株。以pLI50 为载体，构建pLI50-OatA 全基因回补质粒，电转入金葡菌RN4220，再次抽提后电转入敲除菌株驻OatA，获得基因回补菌株pOatA。结果：成功构建基因敲除质粒pBT2-驻OatA，经RN4220修饰电转入USA300，经PCR 及测序鉴定，获得了金葡菌OatA 敲除菌株驻OatA。经PCR 及酶切鉴定，确认pLI50-OatA 回补质粒构建成功，通过金葡菌 RN4220 修饰后电转入敲除菌株驻OatA，获得基因敲除回补菌株pOatA。结论：成功构建金黄色葡萄球菌OatA 基因敲除菌株及其回补菌株，为进一步研究金葡菌OatA 的功能及其作用机制奠定基础。

英文摘要:

Objective:Construction of OatA deletion mutant and complementation of Staphylococcus Aureus （S.Aureus ）Methods:Plasmid pBT2-驻OatA of S.aureus was constructed on the vector of pBT2, and used to introduced into RN4220 by electroporation. Plasmid pBT2-驻OatA of the transformed RN4220, was used to introduced into USA300 by electroporation. Taken the advantage of the characteristics of pBT2 vector that was sensitive to temperature, and then repeatedly passaged at 42 ℃, the deletion mutant 驻OatA was screened. Plasmid pLI50-OatA was constructed on the vector of pLI50, and used to transformed into RN4220 by electroporation. Plasmid pLI50-OatA was purified from the transformed RN4220, and transferred back into 驻OatA. The complementation pOatA was obtained.Results:Endonuclease digestion analysis indicated that plasmid pBT2-驻OatA was constructed and introduced into S.aureus RN4220 and USA300 successfully. The constraction of 驻OatA S. aureus was confirmed by PCR amplification and sequencing. Endonuclease digestion analysis indicated that plasmid pLI50-OatA was constructed and introduced into RN4220 and 驻OatA successfully. The S.aureus pOatA was confirmed by PCR amplification and sequencing.Conclusion:deletion mutant and complementation of were successfully constructed and would lay the foundation for the further work on the study of function and its mechanism.

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