Functional Identification Of Fur In Riemerella Anatipestifer CH-1 And The Evaluation Of The Fur Mutant Strain As A Vaccine Candidate

Riemerella anatipestifer（RA）is a Gram-negative bacteria that mainly infects a variety of poultry such as ducks,and huge economic losses to the duck industry.This study analyzed the function of ferric uptake regular—Fur in RA and evaluate the potential of this mutain as a attenuated vaccine.The main results of this study are as follows:1.Expression,purification and polyclonal antibody preparation of FurThe fur gene was amplified and cloned on the expression plasmid p ET32a that these recombinant plasmids were transformed in E.coli Rosetta for prokaryotic expression;The Fur proteins containing His-tag were purified by affinity chromatography;There purified Fur proteins were inoculated into Kun Ming mices and collected serum to obtain the polyclonal antibody.2 Consturction of fur deletion mutant and complementation strain of RA CH-1Firstly we amplified the left and right flanking sequence of fur gene and the spc R cassette,and then ligated the three PCR fragments using the overlap PCR.The fused fragments were transformed into RA CH-1 by the principle of homologous recombination.Then we selected the mutant RA CH-1Δfur by spectinomycin resistance.The entire fur gene that amplifed from RA CH-1 genome was ligated into the plasmid pLMF03.We also transformed the plasmid pLMF03::fur into RA CH-1Δfur to construct the complementation strain RA CH-1Δfur pLMF03::fur.Finally,q PCR was used to verify the mutain had no extreme effect and Western-blot was used to detect the normal expression of fur in the complementation strain.3.Functional study of fur geneThe Fur protein of RA CH-1 can inhibit the transcription of the iron uptake genes B739＿0103 and B739＿0173,and can directly bind to the promoter of B739＿0173.In addition,H₂O₂can inactivate the regulatory ability of Fur.After the fur was deleted,the growth of the mutain was impaired and the intracellular iron concentration was also higher than that of the wild strain.The H₂O₂sensitivity and intracellular reactive oxygen species also increased,indicating that the fur is critical to Iron ion dynamic of RA,bacterial growth and oxidant tolerance.Finally,the mutain was reduced by more than 10⁴times,and the colonization ability was also significantly reduced.4.Evaluation of RA CH-1Δfur as attenuated vaccine candidateFirstly,the immunological safety of the mutain was evaluated.The results showed that the mutain did not affect the health of the ducks and was cleared after 96 hours of immunization,indicating that the mutain was very safe.Secondly,the immunoprotective power of the mutain was detected.The results showed that when the immunized ducks injected with high doses of wild-type strains,their resistance is as high as 100%,the growth status and physical condition of the ducks were unaffected and the wild-type strains were cleared faster,indicating that the mutain had a very high protective force.Finally,cel ular immunity and humoral immunity were examined.It showed that the cytokines were not disorders and the ducks had very high antibody levels.Overall,RA CH-1Δfur has a very high potential as a live attenuated vaccine.