禽多杀性巴氏杆菌C48-3株ompH基因敲除突变株的构建及其生物学功能

Construction and characterization of ompH gene knockout mutant of avian Pasteurella multocida C48-3

摘要:

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。

Abstract:

Abstract:［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 knockout mutant of C48-3ΔompH and parent strain C48-3，as well as the complemented strain C48-3C.［Results］ C48- 3ΔompH was successfully constructed． Electron microscopy examination of C48-3ΔompH 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 C48-3ΔompH adhered to CEF cells was significantly lower than that of C48-3 and C48-3C. C48-3ΔompH was relatively attenuated in mice by intraperitoneal injection.［Conclusion］The construction of C48-3ΔompH would facilitate further study on pathogenesis of avian Pasteurella multocida.