Attenuating Effect And Applied Research Of Salmonella Enteritis CirA Gene Deletion Strain

S.Enteritidis brings serious harm to aquaculture and human health.At present,except antibiotics,there is no effective way to control salmonellosis.Vaccine,especially live attenuated vaccine,is a research direction.The selection of vaccine target is also the core of Salmonella vaccine research.Based on the important role of the colistin receptor protein CirA in bacterial physiology,the cirA gene was targeted in this study.C50336?cirA was constructed by?-Red homologous recombination technology,and its biological characteristics,LD₅₀ and immune characteristics were evaluated.The specific implementation is as follows:1.Analysis of rCirA protein in S.EnteritidisDesigned targeted primers to amplify the cirA gene fragment,and the recombinant plasmid pEGX-6p-1-cirA.was successfully constructed,The mice were immunized with rCirA protein expressed by E.coli BL21 expression system,and the antibody IgG was detected in the serum of immunized mice.The immune effect was evaluated with C50336.The results showed that the target protein band appeared at 70ku by SDS-PAGE analysis,and it was precipitated expression;the IgG level of immune mice continued to rise;the protection rate of the challenge test was only 20%,indicating that rCirA protein has a certain effect on mice Immune protection.2.Construction and biological characteristics analysis of cirA gene deletion strain of S.EnteritidisIn this study,we used?-Red homologous recombination technology to construct the cirA gene deletion and complement strains of S.Enteritidis.The study found that the growth rate and biochemical characteristics of the cirA gene deletion strain had little effect on the parent strain;Compared with the parent strain,the ability of biofilm formation,motility,adhesion and invasion of infected Caco-2 cells and the survival rate in macrophage RAW264.7 were significantly decreased;the effect of cirA gene deletion on the virulence factors of S.Enteritidis was detected by RT-PCR,and the data support the above findings.It is suggested that CirA protein is a multifunctional protein involved in S.Enteritidis infection.3.Analysis of pathogenicity and immune characteristics of S.Enteritidis cirA gene deletion strainCompared with the parent strain,the LD₅₀ of C50336?cirA in BALB/c mice increased about 8000 times;C50336?cirA was used to challenge mice immunized twice with C50336 strain.The results showed that the protective rate of the immunized mice was 100%;The results of pathological section showed that compared with the mice challenged by C50336 strain,the immune lesions of mice immunized with C50336 strain were reduced,and the lesions of various organs were reduced;the spleen index and serum antibody IgG levels of the immunized mice were measured.The results showed that the spleen index of the immunized mice was significantly different from that of the control group,and the antibody IgG level continued to rise.It has laid the foundation for the further development of live attenuated vaccines against S.Enteritidis.