摘要
目的 构建表皮葡萄球菌agr阴性突变株 ,以期得到除agr基因以外相同遗传背景的突变株与野生株。方法 首先构建同源重组质粒pBT2 △agr,后电转入金黄色葡萄球菌RN4 2 2 0 ,再转入表皮葡萄球菌 32 98。通过pBT2载体对温度敏感的特点 ,含重组质粒的表皮葡萄球菌 32 98在 4 0℃多次传代 ,最终筛选出agr阴性的突变株。结果 同源重组质粒pBT2 △agr通过酶切鉴定证明构建成功 ,表皮葡萄球菌 32 98接受来自金黄色葡萄球菌RN4 2 2 0的重组质粒 ,经酶切鉴定正确。 4 0℃多次传代后 ,经抗生素抗性筛选 ,并经斑点杂交及序列分析 ,证明获得表皮葡萄球菌 32 98 agr阴性突变株。结论 用同源重组的方法完成表皮葡萄球菌 32 98 agr阴性突变株的构建 ,使agr基因被大部分剔除掉。
Objective Construction of S.epidermidis ag r deletion mutant and getting the agr negative mutant and wild-type strain with t he same gene background except agr gene. Methods Plasmid pBT2-△agr was constructed for homologous recombination of the agr system of S.epidermidis by the insertion of erythromycin resistance gene and two PCR-am plified agr-flanking regions into plasmid pBT2. The homologous recombination ve ctor was firstly transformed to S.aureus RN4220 by electroporation and then transformed to S.epidermidis 3298. S.epidermidis 3298 with recombina tion vector was incubated at 40℃. S.epidermidis agr deletion mutant was sel ected. Results Restriction endonucleases results indicate d that the homologous recombination vector was correct. S.epidermidis 3298 a ccepted the recombination vector. The agr deletion mutant was proved by antibiot ic-resistance, Southern blot and direct sequencing of the chromosomal DNA at th e borders of the PCR-derived regions. Conclusion S.epi dermidis agr deletion mutant was successfully constructed, most of sequences o f S.epidermidis 3298-agr were replaced by erythromycin resistance gene. It could be of value in investigating the relationship between agr gene and the imp ortant functional genes related to biofilm-forming and the virulence related pr oteins. [
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第9期728-732,共5页
Chinese Journal of Microbiology and Immunology
基金
十五"2 11"工程重点学科建设项目
