摘要
目的:利用基于T载体快速克隆构建布鲁氏菌突变株的方法,构建缺失VirB启动子区域的绵羊附睾布鲁氏菌株,为绵羊附睾种布鲁氏菌IV型分泌系统功能研究奠定基础。方法:运用同源重组的原理,用Kana抗性基因替换编码分泌系统VirB的启动子区域,从而获得virB的突变株。通过RT-PCR对突变株virB基因的转录进行了分析;分析突变株和野生株在体外模拟的胞内环境下的生存能力,观察virB对B.ovis胞内生存环境适应的影响。结果:成功构建了B.ovis的缺失virB启动子区的突变株,突变株中virB基因的表达抑制。与野生株相比,突变株对胞内环境的适应力发生了改变。结论:利用T载体克隆和抗性替换的方法成功构建了B.ovis的virB的突变株,为B.ovis的IV型分泌系统的功能研究奠定了基础。
Objective： To construct a deletion mutant of virB of B. ov/s for functional analysis of Type IV secretion system by using T vector based rapid cloning technique. Methods： The promoter region of virB was deleted by Kanamycin gene through homologous recombination. Transcription of virB genes were verified by RT- PCR. The survival capabilities of the mutant under stress conditions simulating intracellular environments were tested. Results： A deletion mutant of virB was successfully constructed. Expression of the virB genes were inhibited in the mutant. Compared with the wild type strains, the mutant showed altered survival capabilities under stress conditions. Conclusion： A deletion mutant of virB cloning svstem. and resistance gene replacement. This provide basis for was successfully constructed by using T vector further functional analysis of type IV secretion
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2012年第11期86-91,共6页
China Biotechnology
基金
国家自然科学基金(30901071)
国家"十一五"科技重大专项(2008ZX1004-015)
国家"973"计划(2009CB522602)资助项目
