摘要
【目的】探讨ompH基因在禽多杀性巴氏杆菌致病过程中的作用。【方法】利用同源重组原理构建中间为四环素抗性基因,两侧为ompH基因上下游同源序列同源的敲除载体pWSK29ΔompH,将敲除载体电击转入C48-3株感受态细胞中,通过四环素抗性和菌落PCR筛选ompH基因的敲除突变株,并通过组合PCR、逆转录PCR和DNA测序对突变株进行验证。用生物学功能实验比较野生株、互补株和突变株在生长速率、荚膜结构、粘着能力和致病性等方面的差异。【结果】组合PCR、逆转录PCR和DNA测序结果证实ompH基因的敲除突变株C48-3ΔompH构建成功,电镜观察结果证实ompH基因的缺陷影响细菌的荚膜合成能力,粘附实验结果显示与野生株C48-3和互补株C48-3C相比突变株C48-3ΔompH对CEF细胞的粘附能力显著降低(P<0.01),而小鼠毒力实验结果表明突变株C48-3ΔompH的致病性相对减弱。【结论】本实验构建的突变株C48-3ΔompH,为进一步研究多杀性巴氏杆菌的致病机理奠定基础。
[ Objective] To study the role of the outer membrane protein H （OmpH） in pathogenicity of avian Pasteurella multocida. [ Methods ] The ompH knock-out mutant of avian P. multocida C48-3 was constructed by homologous recombination. The DNA replacement was confirmed by PCR, RT-PCR and Western blot. We compared the differences of biological characteristics such as growth rate, capsular structure, adhesion ability and virulence between the ompH knock- out mutant of C48-3AompH and parent strain C48-3, as well as the complemented strain C48-3C. [ Results] C48- 3AompH was successfully constructed. Electron microscopy examination of C48-3AompH shows the absence of capsular material compared to the parent strain C48-3 and complemented strain C48-3C. The adhesion assay shows that the number of C48-3AompH adhered to CEF cells was significantly lower than that of C48-3 and C48-3C. C48-3AompH was relatively attenuated in mice by intraperitoneal injection. [ Conclusion l The construction of C48-3AompH would facilitate further study on pathogenesis of avian Pasteurella multocida.
出处
《微生物学报》
CAS
CSCD
北大核心
2013年第1期66-73,共8页
Acta Microbiologica Sinica
基金
国家自然科学基金项目(30972206)
湖南省研究生科研创新基金项目(CX2011B397)~~
