Construction And Biological Characteristics Of △rpoE Mutant Strain Of Pasteurella Multocida

Rabbit pasteurellois, an acute septic infectious disease also known as rabbit haemorrhagic septicemia, is caused by pasteurella multocida and it has caused vital economic losses around the world. Our laboratory has constructed hfq mutant strain recently and the transcriptome sequencing results showed that the deletion of hfq induced the up regulation of rpoE. The rpoE gene, known as a kind of sigma factor, plays the role of controling protein transcription and translation and it has vital function in sustaining the growth of bacteria and the stable of internal environment. Thus, it was selected as my objective gene. A precisely defined △rpoE deletion mutant of P. multocida C51-17 was successfully constructed by positive screening homologous recombinant technology, identified by PCR, Western blot and sequencing. We also analyzed the growth rate, genetic stability, stress resistance, adhesion, pathogenicity level and regulatory function of it. We further evaluated the role of rpoE in P. multocida and to illustrate that whether it is a virulence gene or not in pasteurella multocida. Futhermore, the pull down experiment was performed to primarily explore the interaction between Hfq and RpoE in protein level. It will lay the foundation of the research in attenuated vaccine and provide the basis of futher research in pathogenic mechanism of P. multocida.The C51-17 strain genome was used as template to amplify the up and down homologous arms of rpoE. The PCR products and KanR cassette of pUC-4K were cloned into pBC-SK to construct the recombinant plasmid pBC-SK-△rpoE. Then the recombinant plasmid was transferred into P.multocida C51-17 competent cell by electrotransformation. The resistance screening method was used to screen the positive rpoE mutant strains that resistant to Kan and sensitive to Cm, and PCR was used to identify the screened strains. The result of PCR demonstrated that the △rpoE mutant strain was constructed successfully. Then, the recombinant RpoE was purified and used to immune the mice and the anti-RpoE serum was collected as primary antibody to perform the western blot assay to identify the mutant strain and standard strain. The results indicated that the △rpoE mutant had no expression of RpoE. The biological characteristics of △rpoE mutant strain, including growth rate, genetic stability, stress resistance, adhesion, pathogenicity level and regulatory function, were tested.The results indicated that the mutant strain was constructed successfully and had a good stability. The growth rate was similar with the wild type strain after monitoring the optical density of OD600 during 10 hours, but it was interesting that the mutant strain has a much slower growth speed compared with the wild type in high permeability and low temperature environment in the same method. Its adhension activity to RK-13 and DF-1 cells was approximately the same with the patent strain, but it had an obviously difference in two kinds of cells in adhension. The LD50 of △rpoE mutant was determined by the challenge with a lethal dose of 1×102 CFU、1×104 CFU and 1×106 CFU to mice. The mortality rate was 20%, 60% and 100%, respectively, and the LD50 of △rpoE mutant was calculated as 1.89×103 CFU. However, all mice died whthin 2 days after the challenge with a dose of 1×102 CFU of C51-17 strain, which indicated that the rpoE was an important virulence gene in P.multocida. The pull down assay was performed to verify the interaction of RpoE and Hfq in vitro, and the result was negative which indicated that the interaction was indirect or in a protein-gene level in P.multocida. The regulative way in P.multocida needs further investigate.In this study, we obtained the △rpoE mutant strain successfully using positive screening technology and rpoE was preliminarily identified as a virulence gene in P.multocida after the analysis of its pathogenicity. Furthermore, it was demonstrated that RpoE and Hfq interacted indirectly. This study provided reference to the study of Hfq regulating RpoE grid channel in P.multocida and laid the foundation for the P.multocida mutant live vaccine research.