Construction Of GL00487 Gene Deletion Mutant Of Rimerella Anatipestifer And Analysis Of Its Biological Characteristics

Riemerella anatipestifer(RA)infection is a contagious disease of domestic ducks,geese,turkeys,and various other birds,and it is a major disease confronting the duck industry worldwide.It accounts for significant economic losses due to high mortality,weight loss,condemnations,downgrading,and salvage.There are at least 21 serotypes,but the cross-protection between serotypes is very low or absent.At present,there are commercial oil emulsion inactivated vaccines in China for the prevention of this disease,but inoculation of oil emulsion inactivated vaccines has a large stress response to ducklings,and more importantly,the meat ducks,such as Cherry valley ducks and Beijing ducks,which are the largest in domestic breeding.When ducks are on the market at 40-45 days of age,the inactivated oil emulsion vaccine may still remain in the ducks,which is a potential risk to people's health.The above-mentioned risks do not exist in vaccination of attenuated live vaccines,and the production cost is much lower than that of oil emulsion inactivated vaccine.The key points of constructing gene-deleted live vaccines is the selection of deleted target genes.In our preliminary studies,dozens of genes involved in the virulence of R.anatipestifer strain WJ4 were identified by STM using 17 different tagged Tn4351 transposon mutagenesis libraries.GL00487 gene(In this study,it is referred to as 0487 gene)is one of them.The GL00487 gene encodes an aminotransferase.This study will construct a 0487gene deletion mutant strain and evaluate its feasibility as a target gene for constructing a gene deletion live vaccine.1.Construction of 0487 gene deletion mutant of strain WJ4 and the complementary strainThe upstream and downstream homologous arms of the 0487 gene are amplified and linked into the 313 suicide plasmid,generating 313-0487-LR,and then Escherichia coli S17-1(313-0487-LR)was used as the donor,and the wild type WJ4 was used as the recipient,the recombinant suicide plasmid 313-0487-LR was introduced into the wild type WJ4 by conjugation,generating the deletion mutant WJ4?0487.For constructing the complemented strain,the amplified 0487 ORF was linked into the E.coli-R.anatipestifer shuttle expression plasmid p RES2 to construct complementary plasmid p RES2-0487,and then E.coli S17-1(p RES2-0487)was used as the donor,and the deletion strain WJ4?0487was used as the recipient,the plasmid p RES2-0487 was introduced into WJ4?0487 by conjugation,generating the complemented strain c WJ4?0487.2.Analysis of biological characteristics of the deletion mutant WJ4?0487.The results in this study showed that the growth curves and protein hydrolysis ability of WJ4?0487 were not significantly different from those of wild type WJ4,and the adhesion and invasion abilities of the deletion mutant WJ4?0487 to Vero cells were significantly lower than those of the wild type WJ4.In addition,the results of duck pathogenicity test showed that the LD50 of WJ4?0487 to ducklings was more than 10¹⁰CFU,and moreover,the bacterial loads in blood,liver and brain tissues of the deletion mutant WJ4?0487-infected ducklings were significantly lower than those of the wild type WJ4-infected ducklings.These results indicated that 0487 was involved in the virulence of R.anatipestifer.Two weeks after immunization with WJ4?0487,the ducklings were challenged with highly virulent strain YL4.The results showed that the challenge protection rates of the 10⁷,10⁸,10⁹ and 10¹⁰ CFU dose immunization groups were 50%,40%,88.9% and 100%.