Construction And Biological Characteristics Of△lon Strain Of Pasteurella Multocida

Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is often associated with chronic as well as acute infections that can lead to significant morbidity and mortality,manifested as pasteurellosis,pneumonia, atrophic rhinitis, dermonecrosis, cellulitis, abscesses,meningitis, and/or hemorrhagic septicemia.It is the causative agent of fowl cholera in poultry, hemorrhagic septicemia in cattle,snuffles in rabbits,atropic rhinitis and Pneumonia in swine.The ATP-dependent Lon protease plays the major roles in proteins quality control and the specific control of several regulatory proteins and associates with the virulence of bacteria.The lon gene of P. Multocida that was identified in infected rabbit livers by selective capture of transcribed sequences was verified its enzyme activity and constructed its mutant,this has important significance to reveal the pathogenic mechanism and regulation mechanism of Pasteurella multocida.The lon gene was amplified by PCR from P.multocida C51-17strain and cloned to pET30a(+) vector for expression in Escherichiacoli BL21(DE3) under induction with IPTG. Then the recombinant protein was purified by Ni-NTA His-Bind Resin affinity chromatography. The SDS-PAGE analyses shown the expressed recombinant Lon protein was about97ku and mainly existed in a soluble form. Western blot analysis demonstrated that the recombinant Lon protein was recognized by positive sera of P.multocida. The purified Lon protein possessed the ATPase activity and its enzyme activity was27708.47μmol/mg-min.Homologous arms of Lon gene was amplified by PCR and cloned into pBC-SK plasmid to construct recombinant plasmid PBC-lon. Kanamycin resistance gene cassette was inserted in homologous arms of Lon gene to construct recombinant transfer plasmid pBC-Alon. Then plasmid pBC-Δlon was transferred into P. multocida C51-17competent cells and was screened by kanamycin resistance and chloromycetin sensitivity. The Δlon mutant strain was identified by PCR and sequencing and then tested its biological characteristics. The Δlon mutant strain of P. multocida C51-17was successfully constructed and had good genetic stability, the adhesion to RK13cells of Δlon mutant strain and the wild-type strain was9.10±0.75×104CFU and9.7±0.69×103CFU, its growth rate was similar to the wild-type strain in vitro at37℃,their growth rate was faster at37℃than42℃and growth activity was similar,so37℃was more suitable for the growth of Pasteurella multocida;at the stress tolerance experiment,their stress tolerance were similar and reduced to heat shock and UV radiation,but they lost their stress tolerance to H2O2. The virulence test indicated that the LD50of Δlon mutant strain was63.1CFU,while the LD50of the wild-type strain was26.24CFU.In this study, we expressed and tested the ATPase activities of Lon protease from Pasteurella multocida. We constructed the Δlon mutant strain using positive screening homologous recombinant technology. The Δlon mutant had good genetic stability and growth characteristics, adhesion activity of RK13cells, pathogenicity to mice and good growth characteristics.This study will provide a molecular basis for further study of the pathogenesis and regulations of Lon protease in P. multocida.