Cre/loxP介导的伤寒杆菌染色体上插入基因的切除

Cre/loxP Mediated Excision of Inserted Gene Fragment from Salmonella typhi Genome

来源于P1噬菌体的位点专一性重组系统loxP/Cre,已成为一种新的DNA操作的有用工具 ,在体内外都获得了成功的应用 .为了将四环素诱导表达系统引入减毒伤寒杆菌CVD90 8株中 ,tetR loxP neo串联基因已通过同源重组被定位插入在CVD90 8株的△aroC位点中 .构建一Cre酶表达受启动子PLtetO 1控制的自杀质粒 pJG9/Cre ,以切除CVD90 8株△aroC中同向loxP序列之间的neo基因 .质粒pJG9/Cre电转化入菌中 ,加入去水四环素诱导Cre酶表达 ,通过重组切除neo基因 ,再通过自杀质粒上SacB基因的启动 ,使质粒清除出菌细胞 .抗生素鉴定和PCR扩增都证明 ,CVD90

Site specific DNA recombinant system Cre/loxP from bacteriophage P1 has been developed as a novel tool for DNA manipulation, and used successfully both in vitro and in vivo. Here a protocol for effective excision of neo gene located on chromosome of Salmonella have been developed. In order to establish a tetracycline induced expression system in the attenuated Salmonella typhi CVD908 strain, a fused DNA fragment consisting of genes of tetracycline repressor (tetR) as well as neo gene flaked by two loxP sequences in same orientation has been integrated into a defined Δaro C locus of CVD908 strain via homologous recombination. To excise the neo gene from the locus, the suicide plasmid pJG9/Cre expressing Cre recombinase under the control of tetracycline response promoter PL tetO 1 was constructed and electropolated into the CVD908 strain. The expression of the Cre recombinase induced by anhydrotetracycline successfully excised the neo gene. Since the suicide plasmid contains SacB gene encoding an enzyme that is lethal to G bacteria in the presence of sucrose, growing of the bacteria in a medium containing 10% sucrose cured the pJG9/Cre plasmid. Both antibiotic and PCR identification demonstrated the successful excision.