Construction And Immunoprotective Evaluation Of Attenuated Mutant Strain Pm?ORF224 Of Pasturella Multocida

Pasteurella multocida(Pm)is one of the common pathogenic microorganism in animal's clinical detection.It causes varieties of diseases such as avian cholera,porcine pulmonary disease and bovine hemorrhagic septicemia,and it makes a high fatality rate in infected animals,which is harm to the development of the global breeding industry.Due to the widespread use of antibiotics in clinical animal,the phenomenon of multiple drug resistance of bacteria is widespread,which leads to the bacterial diseases occurring repeatedly in the breeding industry.Pm has many serotypes,genotypes,and it has a low efficiency of Pm's vaccine,and its hosts are widely.Secondly,the pathogenic mechanism of Pm is poorly known in current reports,and many virulence factors have not been identified.Therefore,it is of great significant to explore new virulence factors and develop safe and efficient candidate vaccine strains through genetic engineering technology to prevent the Pasteurellosis polycide in animals.In this study,we used the technique of Ng Ago homologous recombination to constructed Pm?ORF224 by eliminating the ORF224 gene in the lysogenic phage cluster of the virulent strain Pm C48-1 without trace,then C-Pm ORF224 was constructed.Furthermore,many biological characteristics were detected.The results showed that the mutant strain of Pm?ORF224 had a good genetic stability within 20 generations.The time of mutant strain was reduced in the lag-phase,and it was increased in the logarithmic phase.The reverting mutant strain of C-Pm?ORF224 partially restored the growth performance.The results of pathogenicity test showed that the mortality rate of the mice that infected by Pm?ORF224 at 2×10~7 CFU dose was 20%,and that of the group of parents strain at 100 CUF dose was100%,so we got the conclusion that the virulence of the mutant strain was weakened obviously.The results of dectecting biofilm showed that Pm?ORF224 had a higher biofilm,and C-Pm?ORF224 had partially reduced biofilm.Mice were immunized with Pm ORF224 mutant strain at a dose of 5×10~6 CFU.Then,the results of serum Ig G antibody level that detected by ELISA method showed that the antibody level of both test group and positive control group were significantly increased.The results of complement mediated antibacterial assay and spleen lymphocyte proliferation assay showed that there was no significant difference between the test group and the positive control group,and they could provide same protection to the experimental group's mice and the positive control group's mice for challenging parent strain.These results indicate that ORF224 gene affect the virulence and formatting biofilm of Pm C48-1strain.After mice were immunized with Pm?ORF224 mutant strain,it will induce mice to produce effective humoral and cellular immune responses,and it can help the mice to resist the challenge of the parent strain,which laid a foundation for screening of Pm attenuated vaccine strains.