Construction Of OmpR Gene Deletion Strain Of Bovine Serotype A Pasteurella Multocida And Study On Its Biological Characteristics

Pasteurella multocida（Pm）can be divided into five serotypes A,B,D,E and F according to the different capsular antigens.In China,the main isolates are serotypes A and serotypes B pasteurella multocida from cattle,of which serotypes A infection can cause lung injury and serious respiratory disease,bringing huge economic losses to cattle industry.In addition,the unclear pathogenesis of pasteurella multocida and the lack of effective commercial vaccines pose great challenges to the prevention and treatment of pasteurella multocida,which also seriously restricts the development of animal husbandry.The virulence of pathogens is related to a variety of regulatory systems and genes.The Env Z/OmpR two-component regulatory system commonly found in Gram-negative bacteria can directly or indirectly regulate the growth of bacteria metabolism,biological membrane synthesis,flagellar movement,resistance and virulence and other biological characteristics,but the regulation and control system in pasteurella,there is no related research.Previously,our laboratory found that OmpR was significantly upregulated in vivo and in vitro in mice infected with bovine serotypes A Pasteurella multocida（Pm CQ2）,suggesting that OmpR gene may have a certain effect on some biological or other functional characteristics of Pm CQ2.In this study,we constructed an OmpR gene deletion strain（Pm CQ2-ΔOmpR）to verify the effects of OmpR gene on the biological characteristics,infection process and virulence of Pm CQ2,and evaluated the antibody titer and cross-immune protection rate of Pm CQ2-ΔOmpR inactivated vaccine.On the other hand,this gene was cloned and expressed,and the recombinant protein r PMOmpR was purified by Ni affinity chromatography to explore the role of this protein in the release of inflammatory factors in host infection with Pm CQ2.In terms of application,the antibody titer and immune protection rate of the subunit vaccine prepared by r PMOmpR recombinant protein were evaluated.In order to provide partial theoretical support for the pathogenesis and prevention and control measures of Pasteurella multocida through this experiment,the main research contents and results are as follows.1.Construction of OmpR gene deletion strain（Pm CQ2-ΔOmpR）and its biological characteristicsIn order to explore the role of OmpR gene in Pm CQ2,we constructed OmpR gene deletion strain（Pm CQ2-ΔOmpR）by homologous recombination method.The gene and transcription level of Pm CQ2-ΔOmpR were detected by ordinary PCR and RT-PCR,respectively.The results showed that Pm CQ2-ΔOmpR deletion strain was successfully constructed.By comparing the biological characteristics of Pm CQ2 wild strains and Pm CQ2-ΔOmpR deficient strains,it was found that there were no significant differences in colony size,growth curve and adaptability to hypertonic stress between the two strains.However,Pm CQ2-ΔOmpR deficient strains increased biofilm content,decreased pod content,and had stronger adaptability to acid and alkali stress.2.Pathogenicity of Pm CQ2-ΔOmpRIn order to investigate the effect of OmpR on the virulence of Pm CQ2,mice were infected with wild Pm CQ2 and Pm CQ2-ΔOmpR-deficient strains at the same dose（2.2×10⁵ CFU）through abdominal cavity.The results showed that the death time of mice infected with Pm CQ2-ΔOmpR-deficient strains was significantly delayed,and the survival rate was 20%after 24 h of infection.The LD₅₀ of Pm CQ2-ΔOmpR muscle infection was 2.36×10⁴ CFU,which was significantly higher than that of Pm CQ2（3.43×10³ CFU）.Further studies showed that Pm CQ2-ΔOmpR’s adhesion and anti-phagocytic ability were decreased,and its colonization ability was also decreased in the lungs of mice.The contents of TNF-α,IL-1βand IL-6 were significantly down-regulated after infection.These results suggest that the infectivity of Pm CQ2-ΔOmpR is lower than that of wild Pm CQ2.3.Cross immune protective evaluation of Pm CQ2-ΔOmpR inactivated vaccineFemale KM mice were immunized with Pm CQ2-ΔOmpR inactivated vaccine twice,and were infected with 2×10⁷ CFU,1×10⁷ CFU and 2×10⁸ CFU doses of pasteurella multocida from cattle,B and F,respectively.The results showed that Pm CQ2-ΔOmpR inactivated vaccine could provide 100%immune protection against Pm CQ2 of serotypes A,but only 20%against serotypes B and F.4.Effects of recombinant protein r PMOmpR on secretion of macrophage inflammatory factors and immunoprotective evaluation of subunit vaccineIn order to investigate the causes of down-regulation of TNF-α,IL-1βand IL-6 after Pm CQ2-ΔOmpR infection in mice,the some function of r PMOmpR protein was studied.Host bacteria BL21（DE3）were induced to express the r PMOmpR recombinant protein.After purification,salt removal and endotoxin removal were performed according to the steps.After the detection reached the standard,co-incubation test of r PMOmpR recombinant protein and mouse peritoneal macrophages was conducted.The results showed that r PMOmpR could induce macrophages to secrete pro-inflammatory cytokines TNF-α,IL-1βand IL-6.At the same time,r PMOmpR recombinant protein was used to make subunit vaccine to immunize mice twice,and Pm CQ2 was challenged with 2×10⁷CFU.The results showed that the antibody titer of this vaccine could reach 1∶102400,but the immune protection rate against Pm CQ2 was only 10%.