摘要
目的:构建布鲁氏菌2308株ery基因启动子缺失株。方法:用PCR方法从亲本株2308上扩增ery基因启动子侧翼序列,将该片段与pMD19-T连接,亚克隆为自杀载体pGEM-7zf-Δery-sacB。将自杀载体电转化布鲁氏菌感受态细胞中经同源重组后,分别用100 mg/L氨苄和7%蔗糖筛选。对获得的基因缺失株进行RT-PCR鉴定和遗传稳定性检测。结果:成功获得ery基因启动子缺失株,2308Δery基因启动子缺失株未扩增出eryA基因。并且该缺失株在10代以内未发生回复突变。结论:成功构建2308Δery基因启动子缺失株,为研究布鲁氏菌的毒力基因及其流产机制奠定基础。
Objective:To construct the ery gene promoter deletion mutant of Brucella abortus 2308 Strain(△ery 2308).Method:The upstream and downstream of the target gene was amplified by PCR from Brucella abortus 2308 DNA.Ligation of the fragments in plasmid 19-T Simple Vector.The insert of this plasmid containing the PCR amplified DNA was subcloned in plasmid pGEM-7zf+ generating the suicide plasmid pGEM-7zf-Δery-sacB.The suicide plasmid pGEM-7zf-Δery-sacB was transformed into Brucella abortus 2308 by1 electroporation.After the homologous recombination,the mutant Δery 2308 was screened by 100 mg /L ampicillin and 7% sugar.The mutant was identified by PCR.Its stability was detected by continuous bacteria culture.Result:The ery promoter deletion mutant of Brucella abortus 2308 strain was successfully constructed.The reverse mutation did not occur within 10 passages.Conclusion:These results indicate that the mutant could be utilized for the further study of the virulent function of ery gene and the abortion mechanism caused by Brucella abortus.
出处
《生物技术》
CAS
CSCD
北大核心
2012年第2期9-13,共5页
Biotechnology
基金
国家"973"计划项目(2010CB530203)
国家自然科学基金项目(30960288
31001046)资助
