Construction Of GL000622 Gene Deletion Mutant Of Rimerella Anatipestifer And Analysis Of Its Biological Characteristics

Riemerella anatipestifer（R.anatipestifer）mainly infects ducks,turkeys,geese and other birds,causing highly pathogenic and contact R.anatipestifer disease.It is also a major disease that harms the world’s duck industry,causing great economic losses to the duck industry.However,little is known about the molecular pathogenic mechanism of this bacterium.The results in our previous studies showed that the R.anatipestifer mutant in which transposon Tn4351 was inserted into gene GL000622 of strain WJ4,significantly reduced the pathogenicity of ducklings.This transposon encodes a serine protease secreted by IX secretion system.Here,a mutant strain with GL000622 gene deletion was constructed by homologous recombination with suicide plasmid.The biological characteristics of wild strains and deleted strain were analyzed.The results show that the virulence of R.anatipestifer were weakened if GL00622 gene is deleted.1.Construction of GL000622 gene deletion strain and the complemented strainThe left and right homologous arms of GL000622 gene were amplified by PCR and ligated into the 313 suicide plasmid.The recombinant suicide plasmid p313-622-LR was constructed and transferred into S17-1 competent cells.Then,the donor strain S17-1（p313-622-LR）was transferred into the recipient strain WJ4 by conjugation transduction,and the deletion strain WJ4ΔGL000622 was obtained by screening and identification.Gl0000622 ORF was amplified by PCR,and ligated into the E.coli-R.anatipestifer shuttle expression vector p RES2,and the complementary plasmid p RES2-GL000622 was constructed.The constructed p RES2-GL000622 was transferred into S17-1 E.coli competent cells.Then the p RES2-GL000622 was transferred into the deletion strain WJ4ΔGL000622 by conjugation transduction.The complemented strain c WJ4ΔGL000622 constructed was identified.2.Biological characteristics analysis of deletion strain WJ4ΔGL000622After the wild type WJ4 strain and the deletion strain WJ4ΔGL000622 were expanded,the protease hydrolysis activity was detected by the defatted plate method.Vero cell adhesion test was performed on 10⁷CFU bacterial solution to analyze bacterial invasion.The 7-day-old ducklings were inoculated with different gradient diluted bacterial solutions,and the LD₅₀and bacterial load in tissues were analyzed.The results showed that compared with wild type WJ4 strain,the growth curve of the deleted strain WJ4ΔGL000622 had no significant difference,but the hydrolytic protease activity of the deleted strain was significantly reduced,and the ability to adhere and invade Vero cells was also significantly reduced.The experimental results of ducklings infection showed that the median lethal dose of WJ4ΔGL00622 was more than 10¹⁰CFU,and the bacterial load in blood,brain and liver of ducks infected with WJ4ΔGL00622 was significantly lower than that of ducks infected with WJ4 in wild strains.In summary,we compared and analyzed the biological characteristics of the wild type WJ4 and the deletion mutant WJ4ΔGL000622 by constructing the gene deletion and reversion mutant strain of R.anatipestifer GL00622.The activity of the GL000622deletion strain with hydrolytic protease,adhesing and invasing Vero cells were significantly lower than those of the wild strain,and its mortality rate and bacterial load of ducklings were also lower.This study confirmed that GL000622 gene was related to the virulence of R.anatipestifer,which laid a foundation for further analysis of molecular mechanism of R.anatipestifer pathogenic and vaccine development.