同源重组构建表皮葡萄球菌纤维蛋白原结合基因缺失突变菌株  

Construction of fbe deletion mutant of Staphylococcus epidermidis via homologous recombination

作　　者：赵旭[1] 朱德妹[1] 张婴元[1] 郑兆鑫[2] 严维耀[2] 

机构地区：[1]复旦大学附属华山医院抗生素研究所,上海200040 [2]复旦大学遗传学研究所

出　　处：《中国感染与化疗杂志》2006年第3期154-158,共5页Chinese Journal of Infection and Chemotherapy

基　　金：国家自然科学基金资助项目(39970040)

摘　　要：目的构建表皮葡萄球菌(表葡菌)纤维蛋白原结合基因(fbe基因)缺失突变菌株,为fbe基因功能研究提供实验工具。方法采用同源基因重组技术。构建质粒ΔpBT2(含fbe::ermB基因),将其电转化进入金葡菌RN4220,再次抽提后电转化进入fbe基因阳性表葡菌(HB)中。将含有质粒ΔpBT2的表葡菌HB在5mg/L红霉素平板42℃经多次传代,使质粒ΔpBT2裂解,fbe::ermB基因同源重组到表葡菌HB的染色体上,通过红霉素耐药、氯霉素敏感等特性筛选出含有fbe::ermB基因的新表葡菌HB-ermB。结果经酶切鉴定后确认质粒ΔpBT2构建成功以及成功转导入金葡菌RN4220和表葡菌HB中,经PCR方法以及测序确认表葡菌HB的fbe基因缺失突变菌株HB-ermB构建成功。结论采用同源重组的方法完成了表葡菌HB的fbe基因缺失突变菌株的构建。Objective Construction of fbe（gene of fibrinogen binding protein） deletion S. epidermidis mutant and acquiring the fbe positive and negative mutant with the same gene background except fbe gene. Methods Homologous recombination method was used. Plasmid △pBT2 （including fbe： ：ermB, ermB is an erythromycin-resistant gene from plasmid PEC1） was constructed, and used to transform S. aureus RN4220 by electroporation. Plasmid △pBT2 was purified from the transformed RN4220, and transferred back into S. epidermidis HB by electroporation. S. epidermidis HB carrying △pBT2 were grown on LB agar plates containing erythromycin （Erm, 5 mg/L） at 42℃ for several generations. Plamid △pBT2 was cleaved. The fbe： ：ermB replaced fbe and was integrated into chromosome of HB via homologous recombination. One strain that grow only on Erm plates but not on plates with chloramphenicol （Chl, 10 mg/L） was selected. It was designated as S. epidermidis HB-ermB. Results Endonuclease digestion analysis indicated that plasmid △pBT2 was constructed and introduced into S. aureus RN4220 and S. epidermidis HB successfully. The fbe deletion mutant S. epidermidis HB ermB was confirmed by PCR amplication and sequencing. Conclusions S. epidermidis fbe deletion mutant is successfully constructed via homologous recombination.

关 键 词：表皮葡萄球菌 纤维蛋白原结合基因 同源重组 

分 类 号：R378.1[医药卫生—病原生物学] Q78[医药卫生—基础医学]