摘要
为了研究spiC基因对鸡白痢沙门菌致病性的影响,采用同源重组方法构建该基因缺失株。以鸡白痢沙门菌S06004基因组DNA为模板,PCR扩增spiC基因上、下游DNA片段作为等位基因;将上游片段、卡那霉素抗性基因(KmR)及下游片段依次连接并克隆到pGMB151自杀性质粒上,构建重组自杀质粒pGMB151-ΔspiC/KmR,通过大肠杆菌χ7213与鸡白痢沙门菌S06004固相杂交,将质粒转入S06004中,运用反向筛选法获得含KmR基因的spiC基因缺失株(ΔspiC/KmR);再使用编码FLP重组酶的温度敏感型质粒pCP20敲除KmR,获得无抗性ΔspiC。PCR鉴定和测序结果显示,spiC基因被成功缺失。血清型、生化特性、生长特性、耐药性等鉴定表明,S06004ΔspiC未发生理化特性改变。本研究成功构建了spiC基因缺失株S06004ΔspiC,为进一步研究鸡白痢沙门菌致病性变化及spiC基因的功能奠定了重要基础。
The objective of this study was to knock out the spiCgene from the genomic DNA of Sal monellaPullorum S06004by homologous recombination.Two DNA fragments,which represented the upstream and downstream of spiCgene as allelic genes,were amplified and were flanked at the two ends of kanamycin-resistant(KmR)gene and this recombinant fragment was cloned into suicide vector pGMB151. The recombinant suicide plasmid(pGMB151-ΔspiC/KmR)was transformed into S06004strain by conjugation with Escherichia coliχ7213.AspiCgene-deleted mutant strain(ΔspiC/KmR)was screened out and KmRgene was then eliminated using a temperature-sensitive plasmid,pCP20,encoding the FLP recombinase.There was not spiCgene in chromosome of S06004ΔspiCconfirmed by PCR and sequencing analysis. The serotype,biochemical properties,growth property and resistance to antibiotics of theΔspiC mutant strain were identified to show that they were consistent with those of its parent strain S06004.It was concluded that a Salmonella Pullorum ΔspiC mutant strain was successfully constructed.This study would pave a way for further studies of virulence of this mutant and the functions of the spiCgene.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2014年第4期379-386,共8页
Chinese Veterinary Science
基金
"十二五"农村领域国家高技术研究发展计划(863)项目(2011AA10A212)
国家自然科学基金资助项目(31230070)
江苏省重点实验室开放课题项目(0273896034819)
江苏省博士后基金项目(1302067C)
