金黄色葡萄球菌铁调节表面决定子（IsdA）缺失突变株的构建

Objective: In order to investigate the function of iron-regulated surface determinant (IsdA) in prokaryote, the IsdA deletion mutant of S. aureus strain was constructed by homologous recom bination. Methods: The upstream and downstream sequences of IsdA were generated by PCR using chromosomal DNA of S. aureus Newman as template. The homologous recombination vector pMAD?IsdA was constructed based on shuttle vector pMAD (carrying the screening markers, the tem perature-sensitive replication origin, erythromycin resistance gene (erm), and β-lactase gene (bgaB)). S. aureus strain RN4220, a restriction deficient strain, was used as the initial recipient for transformation and the modified vector was then electroporated into S. aureus Newman. The strain was incubated alternately at 30℃ and 42℃and the IsdA deletion mutants were selected by antibiotic resistance and β-lactase activity. Results: Results from genome PCR, real-time quantitative PCR and direct sequencing indicates that the IsdA gene of the screened mutants was deleted from chromosomal DNA of S. aureus Newman. Conclusions: The successfully constructed IsdA deletion mutant of S. aureus offered a useful tool to investigating the function of IsdA gene in S. aureus.