摘要
目的 :用Red系统快速敲除痢疾杆菌asd基因。方法 :本研究首先合成一对引物 (每一条的 5′端与asd基因同源 ,3′端与氯霉素抗性基因同源 ) ,并用来获得氯霉素抗性基因的PCR产物 ,然后电击转化至大肠杆菌DH5α和痢疾杆菌福氏 2a 2 4 5 7T中 ,在λRed重组系统的帮助下 ,通过氯霉素抗性基因两侧的asd基因序列在体内与asd基因发生同源重组 ,置换了DH5α和 2 4 5 7T基因组中的asd基因 ,最后又利用氯霉素抗性基因两端的FRT位点 ,通过FTP位点专一性重组将氯霉素抗性基因剔除。结果与结论 :获得了asd基因被完全敲除且不带氯霉素抗性基因的大肠杆菌和痢疾杆菌 ,使Red系统在大肠杆菌以外微生物中的应用获得成功。
Objective:To knock_out Shigella flexneri asd gene quickly with Red system.Methods:A pair of primers, which are homologous to asd gene and chloramphenicol_resistant gene, were synthesized. The chloramphenicol_resistant gene was then amplified by PCR with the primers. The PCR products were electro_transferred into Escherichia coli strain DH5α and S.flexneri 2a strain 2457T. With the help of Red recombinant system, asd genes of DH5α and 2457T were in vivo replaced by chloramphenicol gene. Due to FRT sites flanking chloramphenicol gene, the latter was eliminated by FLP_promoted recombination events. Results and Conclusions:The mutants of E.coli and S.flexneri ,from which asd gene was completely deleted, were constructed and were chloramphenicol sensitive. This work indicates that Red system can be successfully applied to other bacteria besides E.coli .
出处
《军事医学科学院院刊》
CSCD
北大核心
2002年第3期172-175,共4页
Bulletin of the Academy of Military Medical Sciences
基金
国家重大基础研究规划 (973 )(G19990 5 410 3 )
首都二四八重大创新工程 (H0 10 2 10 3 60 119)资助项目
关键词
杆菌性痢疾
RED重组系统
基因敲除
聚合酶链反应
dysentery,bacillary
Red recombinant system
gene knock_out
polymerase chain reaction
