利用同源重组技术敲除金黄色葡萄球菌临床分离株sarA基因  

Deletion of sarA in a clinical isolates of Staphylococcus aureus via homologous recombination

作　　者：余方友[1] 陈坚[1] 王薇薇[1] 李美兰[1] 黄金伟[2] 陈增强[1] 张雪青[1] 朱涛[3] 瞿涤[3] 

机构地区：[1]温州医学院附属第一医院检验科,浙江温州325000 [2]浙江省丽水市中心医院检验科,浙江丽水323000 [3]复旦大学教育部/卫生部医学分子病毒学重点实验室,上海200032

出　　处：《中国卫生检验杂志》2009年第1期17-20,共4页Chinese Journal of Health Laboratory Technology

基　　金：浙江省自然科学基金资助项目(NO.Y206461)

摘　　要：目的:利用同源重组技术敲除金黄色葡萄球菌临床分离株sarA基因。方法:构建含有Em抗性基因(ermB)和sarA基因上、下游同源DNA片段的pBT2-ΔsarA质粒,将pBT2-ΔsarA质粒电转入金黄色葡萄球菌RN4220中,再把pBT2-ΔsarA质粒转入金黄色葡萄球菌30临床分离株中,把含重组质粒的金黄色葡萄球菌30临床分离株在42℃条件下振摇培养,筛选金黄色葡萄球菌30菌株的sarA基因缺失可疑突变株(SA30-ΔarlS);对重组到SA30染色体基因组的相应序列进行PCR扩增及DNA测序。结果:成功构建pBT2-ΔsarA质粒,pBT2-ΔsarA质粒被转入金黄色葡萄球菌30后,筛选到可疑突变株,经PCR及DNA测序证实金黄色葡萄球菌30sarA基因被ermB基因置换。结论:利用同源重组技术成功敲除金黄色葡萄球菌30临床分离株sarA基因。Objective：To delete sarA in a clinical isolate of Staphylococcus aureus via homologous recombination.Methods：A plasmid pBT2-ΔarlS was constructed with inserting erythromycin resistance gene（ermB） and two PCR-amplified sarA-flanking regions for homologous recombination of sarA of the isolate of Staphylococcus aureus.Recombinant plasmid pBT2-ΔsarA first was transformed to S.aureus RN4220 and then transformed to the isolate of S.aureus 30 by electroporation.S.aureus 30 with recombinant plasmid pBT2-ΔsarA was continuously subcultured at 42℃ with shaking until the sarA deletion strain was selected.Results：Restriction endonucleases results indicated that the plasmid pBT2-ΔsarA was constructed successfully.The recombinant vector was transformed into S.aureus RN4220 and S.aureus 30 successfully by electroporation The sarA deletion S.aureus 30 was screened by antibiotic-resistant profile then proved by PCR and DNA sequence.Conclusion：S.aureus sarA deletion mutant is constructed successfully.The sequence of sarA gene is replaced by ermB gene in S.aureus 30.

关 键 词：葡萄球菌 金黄色 同源重组 sarA基因 

分 类 号：R378.11[医药卫生—病原生物学]