| Pasteurella multocida(P.multocida,Pm)is one of the primary pathogens associated with bovine respiratory disease in cattle industry around the world,causing loss of productivity and high mortality.There are various different types of virulence-associated genes in P.multocida.But the expression patterns and pathogenicmechanism of virulence genes are not clear.There are various different types of virulence-associated genes in P.multocida.In this study,we selected some isolates which were identified as capsular type A.We divided virulent strain and attenuated strain using a series of in vivo experiments and Polymerase Chain Reaction(PCR).Then,transcriptome analyses were carried out on both virulent and attenuated strain,including that with differentially expressed genes.The virulence genes were screened and enriched on KEGG-map to analyze the pathogenic mechanism of Pasteurella multocida.1.Isolation,identification of Pasteurella multocida of bovineP.multocida field strains(n=12)were obtained from bovine with pneumonia,and identified by biochemistic identification and PCR.All 12 clinical isolates were identified as capsular type A by PCR.2.Transcriptomic analysis of virulent strain and attenuated strainIn this study,we divided strains into virulent type and attenuated type.Using in animals experiments and virulence gene amplification.Transcriptome analysis was performed to select 72 differentially expressed genes from the cDNA library.According to the comparison analysis,69 genes were down-regulated while 3 genes were up-regulated in the attenuated strains than those in the virulent strains.Compared with the virulent strains,the expression level of Znua and Rope are significantly lower than that of attenuated strains by transcriptome sequencing.3.Purification and functional verification of DnaJ proteinThe transcriptome sequencing results showed that the expression level of Dnaj was most significantly down-regulated.In this study,pET-28 a was used as expression vector and ligated with DnaJ to construct the pET-28a-DnaJ.pET-28a-DnaJ was transformed into DE3 cells,and we obtained a recombinant protein(37.8kDa)after inducting IPTG.Then this recombinant protein was purified by affinity chromatography(Ni-NAT).Mice were immunized with recombinant DnaJ,and Western blot was used to evaluate immunogenicity. |
