Isolation And Identification Of Enterococcus Faecalis From Bear And Immunization Of Efaa Gene Expressed Protein In Mice

E.faecalis is a Gram-positive pathogen with a serotype D,which can cause a wide range of human diseases,including sepsis,urinary tract infections,endocarditis and meningitis,etc.Coccidia-induced diseases account for 80-90%of human intestinal infections.Enterococcus faecalis is also a resident organism in animals,which can spread horizontally between humans and animals.More and more reports of the incidence and death of faecal enterococci are mainly piglets arthritis,quail chicken sepsis,rabbits Diarrhea,lamb encephalitis and bovine mastitis.E.faecalis of bear was isolated and evaluated,construction of recombinant expression plasmid of EfaA gene of Enterococcus faecalis was carried out and the immunogenicity of the protein was determined.E.faecalis of bear was identified by morphology,biochemical characteristics,susceptibility test,serology and molecular biology experiments,moreover the pathogenicity of the strain was identified by PCR.The EfaA gene was selected as the target gene,cloning the target gene and analyzing the target gene via bioinformatics.The EPCA gene was subcloned into pGEX-4T-1 prokaryotic expression vector to construct pGEX-4T-FfaA prokaryotic expression plasmid into BL21(DE3)competent cells.The expression was induced by IPTG.The optimal induction time was screened and the biological characteristics for the expression protein was analyzed.The antibody titer was measured in mice serum after inoculation of BALB/c mice.IgG1,IgG2a,NF-? and IL-4 levels were measured by ELISA kit.The results showed that the pathogen was Enterococcus faecalis by the separation,identification and NCBI comparison.Four pathogenic genes were obtained by PCR and the EfaA gene of Enterococcus faecalis was obtained.After induction with IPTG,the expressed protein was about 59 Ku,after purification,the concentration was 3 mg/mL and the protein purity was above 85%.The purified protein was analyzed by Western-blot and showed good reactivity.After BALB/c mice inoculated by E.faecalis,the antibody titer was 1:1 024,and the levels of IgG1,IgG2a,IFN-y and IL-4 were significantly higher than control group in the third immunization(P<0.01).The results showed that the EfaA protein of Enterococcus faecalis could produce a good immunoprotective effect in mice,and could achieve the purpose of preventing Enterococcus faecalis infection.The polyclonal antibody against EfaA protein was successfully prepared and would be the foundation for the development in order to prepare Escherichia coli EfaA Gene serological diagnostic reagents,monoclonal antibodies and vaccines.