Study on the Antigenicity and Immunogenicity of ErfK Protein of Substrate in the Twin-arginine Translocation System of Brucella melitensis
In order to analyze the immune response of hosts that were induced by virulence related substrate protein L,D-transpeptidase ErfK of twin-arginine translocation system(Tat system)of Brucella melitensis,bioinformatics analysis was conducted for ErfK gene sequence(WP_002963714.1)of Brucella melitensis M28 strain,based on which,primers were designed to construct an expression vector pET-30a(+)-ErfK,and target protein was obtained through IPTG induction,affinity chromatography and nickel column purification.The antigenicity of recombinant ErfK protein(rErfK)was identified using SDS-PAGE and Western blot.rErfK protein was immunized into mice,serum and spleen were collected on 15,30 and 45 days after immunization,and the cellular and humoral immune responses of the immunized mice were detected.The bioinformatics prediction results showed that ErfK protein was hydrophilic without transmembrane structure,and several T and B cell antigen epitopes existed.25kDa protein bands could be obtained by SDS-PAGE and Western blot,and the recombinant protein could react with Brucella positive serum,proving a good antigenicity.Immunity test of mice showed that,compared to the control group,the content of CD8+T cells in spleens of immunized mice was significantly increased(P＜0.05),so was the transcription level of Th1-type cytokines of IL-2,TNF-α and IFN-γ as well as Th2-type cytokine of IL-4(P＜0.05).Furthermore,the toal and specific IgG level in the serum of mice could be greatly induced by rErfK immunization(P＜0.01).In conclusion,Brucella rErfK protein expressed by E.coli prokaryotic expression system could induce mice to produce good humoral and cellular immune response.The development of reagents and subunit vaccines based on ErfK protein for brucellosis was provided with a reference by the study.
Brucellasubstrate protein L,D-transpeptidase ErfK of Tat systemantigenicityimmunogenicity
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