Identification Of PhoP Gene And Evaluation Of Its Mutant Vaccine In Riemerella Anatipestifer

Riemerella anatipestifer (RA) is one of the most serious diseases pathogens in China, which could cause fibrinous parahepatitis, pericarditis and air sacculitis in ducks. It leads to massive death of ducklings and growth retardation, thus inhibits the development of duck industry in China. More than 21 serotypes of R. anatipestifer have been confirmed and there is little cross-protection among different serotypes. Commercial vaccines were available, but has some defects. For instance,oil-emulsion inactivated vaccine can not provide complete protection (around 70% protection rate) and its absorption is not ideal after intramuscular injections, et al.In the study, we constructed an attenuated RA-CH1 vaccine by genetic engineering techniques, aiming at finding an effective attenuated vaccine strain that can provide effectively protection for RA. First, we cloned a potential phoP gene in RA. To clarify the gene, phoP gene sequence was ligated into pYA3337 plasmid and transformed into Salmonella UK-lΔphoP. The result showed that RA phoP restored the phenotype of UK-1ΔphoP strain. Also, bioinformatics prediction showed RA phoP didn’t have signal peptides and transmembrane domain. Next, PhoP protein was expressed in prokaryotic cell and the polyclonal antibody was prepared. phoP gene was ligated into pET32a(+) and transformed into Rosetta, The protein was expressed by IPTG-induction and purified according to Histidine purification of Kit. Then rabbits were immunized with the PhoP protein and the polyclonal antibody was collected post four times immunization. The agar diffusion test showed that the antibody titer was up to 1:16. This work laid foundation to the research on phoP regulatory network and protein interaction in RA.In addition, we constructed RA-CH1 ΔphoP mutant strains and systemically measured the phenotypic characteristics, immunogenecity, and protection potency of mutant strain. First, the homologous arms of phoP were amplified by PCR, which were fused by crossover PCR and then we used Spec cassette to generate recombinant plasmid pRE112-Left-Spec-Right, the RA-CH1ΔphoP mutant strain were constructed by recombinant suicide plasmid-mediated homologous recombination under the selection pressure of spectinomycin and kanamycin. Next, bacterial phenotypic analysis showed that RA-CH1 ΔphoP mutant strain grew slower in vitro, susceptibility to ampicillin was decreased and gentamicin sensitivity was rised, colonization in brain and liver were reduced as well. In contrast, the ability of adherence and invasion to Duck embryo fibroblast cells was similar between two strains. Importantly, the virulence of the mutant strain was decreased significantly, the lethal dose (LD50 value) of mutant strain was more than 100-fold higher than that of parent strain in 9-day-old Cherry Valley ducklings. Finally,10-day-old ducklings were immunized with RA-CH1Δphop mutant strains by intramuscular injection, then ducks were challenged with 150×LD50 wild strain after 14 days. ELISA testing showed that immunized ducks produced significantly higher antibody level than unimmunized ducks 2 weeks and 3weeks post immunization. Moreover, all immunized ducks were alive and showed normal growth rates.Thus, our study suggested that RA-CHl AphoP was attenuated, could induce strong antibody response and provide protection against the challenge of virulent strain in ducks.