摘要
目的 证明放线菌噬菌体重组酶Sre在大肠杆菌细胞内介导高效的位点特异性重组。方法 在大肠杆菌内构建分子内重组分析系统。质粒pBZP含有方向相同的attB和attP位点,分别位于lacZ基因的两侧。将pBZP电击导入含有sre基因的大肠杆菌细胞。结果 重组的发生导致lacZ基因的切除,使含有X- Gal成分的培养基上细菌菌落从蓝色变成白色。整合后DNA经酶切、PCR和DNA测序证实。发生10 0 %重组率的attB和attP最小片段分别是5 0bp和4 7bp。结论 在大肠杆菌宿主环境中放线菌噬菌体重组酶Sre高效催化位点特异性重组。本分子内重组分析系统是一个简便而有效的方法。
Objective To demonstrate that actinomyces phag e R4 integrase Sre protein efficiently mediates site-specific recombination in E.coli. Methods An intramolecular recombination assay s ystem in E.coli was constructed. The plasmid pBZP contains attB and at tP sites in direct orientation flanking a lacZ gene. When pBZP was introdu ced into E.coli cells, in which the plasmid pSRE4 containing sre gene wa s resident, Sre protein catalyzed integration of attP into attB site, re sulting in excision of the lacZ gene. Results This in tegration changed bacteria colonies from blue to white on agar plates containing X-Gal, which showed that lacZ was removed. The integrant DNAs were identif ied by enzyme digestion, PCR and DNA sequencing. The minimum sizes of attB a nd attP were 50 bp and 47 bp for 100% recombination efficiency. C onclusion The phage recombinase Sre protein efficiently integrated attP into attB site to create attR and attL in E.coli host e nvironment without Streptomyces specific cofactors. This intramolecular assa y system is a simple and efficient system for Sre-mediated recombination in E .coli.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2005年第5期375-378,共4页
Chinese Journal of Microbiology and Immunology