Construction Of PurF Gene Delete Mutant Strains Of Leporid Pasteurella Multocida And Research On Its Biological Characteristic

Rabbit pasteurellois which caused by Pasteurella multocida is an acute septic infectious disease, andalso known as rabbit haemorrhagic septicemia. Rabbit pasteurellosis was a worldwide distribution, andcaused huge economic losses in the rabbit breeding industry. It was one of major infectious diseases onprevention and control of rabbit infections. The purF gene is bacterial nutrition metabolism related gene andencodes phosphoribosylpyrophosphate amidotransferase, which catalyses the first committed step in purinebiosynthesis. we constructed the ΔpurF mutant strains using positive screening homologous recombinanttechnology and analysised the growth characteristics, genetic stability, adhesion and invasion activity and thepathogenicity to mice. We verified the pathogenicity of purF gene and cleared whether is virulence gene of P.Multocida. The ΔpurF mutant provides a molecular basis to clarify the pathogenic mechanism of PurFprotein in P. multocida.Homologous arms of purF gene was amplified by PCR and cloned into pBC-SK plasmid to constructrecombinant plasmid PBC-purF. Kanamycin resistance gene cassette was inserted in homologous arms ofpurF gene to construct recombinant transfer plasmid pBC-ΔpurF. Then plasmid pBC-ΔpurF was transferredinto P. multocida C51-17competent cells and was screened by kanamycin resistance and chloromycetinsensitivity. The ΔpurF mutant strain was identified by PCR, Western blot and sequencing and then tested itsbiological characteristics. The ΔpurF mutant strain of P. multocida C51-17was successfully constructed andhad good genetic stability, its adhesion activity of Vero cells was enhancer and invasion was similar to theparent strain, its growth rate was similar to the wild-type strain within the first3hour and then faster in vitro.The virulence test indicated that ΔpurF mutant strain was significant attenuated and no death was found inmice even inoculated at a dosage of1.0×106CFU, the wild-type strain was completely lethal at a dosage of1.0×102CFU. The virulence of ΔpurF mutant strain of P. multocida was highly attenuated in mice.In this study, we constructed the ΔpurF mutant strain using positive screening homologous recombinanttechnology and analysised the pathogenicity to mice. The ΔpurF mutant preliminary clarify that the purFgene is virulence gene of P. multocida, and provide a molecular basis for further study to clarify thepathogenic mechanism of PurF protein in P. multocida.