Construction And Biological Characteristics Of△prfA Strain Of Pasteurella Multocida

Pasteurella multocida is encapsulated gram-negative bacteria, it can cause fowl cholera in poultry, hemorrhagic septicemia in cattle, pneumonia and atrophic rhinitis in swine, snuffles in rabbits, et al. It caused great damage to the animal breeding industry. Pasteurella multocida also can cause human diseases, but the mortality rate is very low. Early, we using selective capture of transcribed sequences(SCOTS) technique screened PrfA which participates the Pasteurella multocida infections, PrfA may be an important virulence factor.Homologous arms of prfA gene was amplified by PCR and cloned into pBC-SK plasmid to construct recombination plasmid PBC-prfASX. Kanamycin resistance gene expression-box was inserted in homologous arms of prfA gene to construct suicide plasmid pBC-ΔprfA. Then plasmid pBC-ΔprfA was transferred into Pasteurella multocida C51-17competent cells and was screened by kanamycin resistance and verified by PCR and sequencing. The genetic stability of ΔprfA mutant was good and the biochemical characteristics of ΔprfA mutant did not change, which accorded with parent strain. In vitro, growth curves showed growth rate of ΔprfA mutant and the parent strain at37℃were significantly higher than both of42℃culture conditions, at both37℃and42℃,the proliferation of the ΔprfA mutant is faster than the parent strain. Under UV conditions, ΔprfA mutant survival rate was24.20%, compared with the parent strain survival rate of66.67%(P>0.05). At50℃, both of them can grow, growth rate of parent strain was faster than ΔprfA mutant(P>0.05). While treatment with200mM H2O2, both of them were almost killed, ΔprfA mutant survival was0.42%and survival of the parent strain is only0.02%, their difference reach highly significant (P<0.01). The pathogenicity level of prfA deletion strain to mice were13.5-fold lower than C51-17, LD50was350CFU, while C51-17was26CFU. The adhesion efficiency of ΔprfA mutant and parent strain was1.7%and0.97%(P<0.01), the adhesion efficiency of ΔprfA mutant was increased.The result of real-time reverse transcriptase-polymerase chain reaction was show that the transcriptional level of Hpt, PM209and RpoH were up-regulated by1.79-fold,6.31-fold and25.53-fold between ΔprfA mutant and C51-17, Hpt, PM209and RpoH regulated by PrfA. PrfA in the pathogenesis of Pasteurella multocida. Construction and characterization study of ΔprfA mutant verified PrfA in the pathogenesis of Pasteurella multocida and it will provide the basis for further study PrfA regulate other protein mechanism.