| Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowlcholera in poultry, hemorrhagic septicemia in cattle, atropic rhinitis in swine and snuffles in rabbits.Strains of P. multocida are normally designated on the basis of the capsular serogroup and somaticserotype. There are five serogroups (A, B, D, E, and F) based on capsule specificity, and16somaticserotypes (1–16) based on lipopolysaccharide antigens. The bacterial is a worldwide distribution and animportant disease to prevent and control, causing economical losses to the animal breeding industry.The differentially expressed gene profile of P. multocida C51-17strain in infected rabbit livers wasidentified and compared with that from in vitro culture by selective capture of transcribed sequences. Atotal of31genes were identified, of which28encoded enzymes for amino acid biosynthesis andmetabolism, intermediary metabolism, and energy metabolism, or proteins for regulatory adaptiveresponses, general microbial stress response, transport proteins and secreted proteinases. Three wereunknown, novel genes. Five genes representing different categories were chosen randomly and verifiedby real-time reverse transcriptase-polymerase chain reaction analysis. All Genes (purF, lon, dnaB, ftsQ,and glpT) were upregulated by P. multocida in infected rabbit livers, with changes ranging from1.61-fold to13.55-fold when compared with in vitro cultures.The dnaK gene of heat shock protein70(DnaK) of P. multocida strain C51-17was cloned intoprokaryotic expression vector pPro-EXHTb, transformed into BL21(DE3) and induced by IPTG. Theantigenicity and immunogenicity and biologic activity of recombinant DnaK protein were analysised.The results showed that dnaK gene at the nucleotide level had homology with other P. multocida strainsavboe99.2%, and82.3%to97.5%with other species of bacteria. rDnaK was purified by the Ni-NTAaffinity chromatography. The results of SDS-PAGE and western blot showed that DnaK was amolecular weight of70kDa in soluble form and recognized by positive serum of P. multocida with goodantigenicity. The immunogenicity of rDnaK was determined in ICR mice and showed that the micewere immuned with50μg and150μg rDnaK protein and survived1/5and2/5at the lethal dosage of50CFU of P. multocida, respectively. The adhesion and inhibition activity of rDnaK was detected andindicated that protein was adhered on Vero cells and inhibition of adherence of P. multocida to Verocells. The dnaK gene cloned into eukaryotic expression plasmid pEGFP-C1, transformed into Vero cellsand indicated that DnaK was localizated in cytoplasm.Suicide recombinant plasmid of pBC-DnaK-Km and pBC-aroA-Km were constructed with kanamycingene and transformed into P. multocida by electroporation. The ΔdnaK and ΔaroA mutant strains of P.multocida were screened by kanamycin resistance and chloromycetin sensitivity and identified by PCR.Its virulence of ΔdnaK and ΔaroA mutant strain was tested in mice by intraperitoneal injection. TheΔdnaK and ΔaroA mutant strains of P. multocida C51-17were successfully constructed. Growth rateofΔdnaK and ΔaroA mutant strains were similar to the wild-type strain in vitro. The virulence testindicated that ΔdnaK mutant strain was no significant attenuated and the death was found in miceinoculated at a dosage of1.0×10~2CFU, and ΔaroA mutant strain was significant attenuated and no death was found in mice even inoculated at a dosage of1.0×10~6CFU. The virulence of ΔaroA mutant strain ofP. multocida was highly attenuated in mice.The differentially expressed genes of P. multocida C51-17strain were identified by selective captureof transcribed sequences and verified by real-time reverse transcriptase-polymerase chain reactionanalysis. The ΔdnaK and ΔaroA mutant strains of P. multocida C51-17were constructed using positivescreening homologous recombination technology. This study will provide a molecular basis for furtherstudy of the pathogenesis and attenuated vaccine of P. multocida. |
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