Influence Of L7/L12 Protein From Brucella On The MAPK Signal Pathways

Brucella is a small gram-negative bacillus that is parasitic in the cells of the host,and it can infect humans and animals.Brucellosis,caused by Brucella,is a zoonotic disease that seriously endangers human health and the development of animal husbandry.Currently,the studies have reported that many Brucella proteins can cause a series of immune responses.Among them,the L7/L12 protein is recognized as the dominant antigen of T cell immunity,and the gene encoding the L7/L12 protein is relatively conservative in a variety of species.At present,the ribosomal protein L7/L12 is used for the vaccine to prevent diseases,however,its pathogenic mechanism is not clear.In this studying,primers encoding L7/L12ribosomal protein genes were designed,and the genome of the S2 vaccine strain was used as the template.The L7/L12 gene was linked with the prokaryotic expression vector p ET30a and the eukaryotic expression vector p EGFP-N1,After L7/L12 ribosomal protein acts on DC cells and 293t cells,the node molec?les of the MAPK signaling pathway are detected to explore the influence on the MAPK,which has laid the foundation for the following study of the immunological mechanism of Brucella.(1)The gene,obtained by PCR amplification,was connected to the prokaryotic expression vector p ET30a plasmid,and transformed into E.coli BL21(DE).After induction with different temperature,inducer concentration and induction time,the best induction conditions were screened and large quantities of soluble recombinant protein was obtained.The recombinant protein,purified by Ni column,was analyzed purity and reactogenicity by SDS-PAGE and Western blotting.The res?lts showed that the protein was highly expressed in a soluble form with the conditions of 1 m M IPTG and 16?overnight.The size of the protein,detected by SDS-PAGE,was 18KD that was consistent with the predicted value.The purity of the recombinant protein reached more than 93%,and it has a certain degree of reactogenicity.(2)The separate bone marrow cells,from mouse hind limb bones,were successf?lly induced and differentiated into DCs with IL-4 and GM-CSF.The DC cells were stim?lated with LPS and L7/L12 protein for 24 h,then,the stim?lated DCs were collected.The surface co-stim?latory molec?les were detected with flow cytometry,and the changes of the inflammatory factor and MAPK signaling pathway node molec?lar were detected with RT-QPCR.The expression of CD40,CD80 and other antigen molec?les on the surface of stim?lated BM-DC detected by flow cytometry was significantly higher than that of the blank control group(P<0.05).RT-QPCR res?lts showed that the inflammatory cytokines TNF,IL-1?,and IL-12 were extremely significantly higher than the blank control group(P<0.01).After the action of the Brucella L7/L12 ribosomal protein,the expression of MAPK and JNK were up-reg?lated,but the p38 and ERK molec?les were only slightly up-reg?lated.These prove that L7/L12 protein can stim?late the differentiation and maturation of dendritic cells,and promote DCs to present antigens to immature T lymphocytes.Meanwhile,L7/L12 protein can stim?late T lymphocytes to activate and secrete TNF-?and IL-1?,and promote T cells to differentiate into Th1 cells.The L7/L12ribosomal protein plays an important role in the process of the immune effect.(3)The gene,encoding L7/L12 ribosomal protein,connected to the eukaryotic expression vector p EGFP-N1 plasmid.Then,the p EGFP-N₁-L7/L12 recombinant plasmid were transferred into 293 t cells with liposome 2000.The conversion res?lts were observed with a fluorescence microscope.The RNA of the 293t cells were extracted and reversed into c DNA.The ordinary PCR was used to test whether the effective expression of recombinant plasmid.Western blotting was used to analyze the reactogenicity of the protein,and RT-QPCR was used to detect the node molec?lar changes in the MAPK signal pathway.The res?lts showed that the transfection efficiency was above 80%.L7/L12 target gene bands can be seen after electrophoresis Western blot analysis clearly shows the reaction bands of antigen and antibody on the PVDF membrane.RT-QPCR res?lts show that The RNA expression of ERK and JNK node molec?les in the MAPK signaling pathway has increased significantly,which indicates that the recombinant protein can be effectively expressed in293t cells and can transmit signals to the nucleus,which can?ltimately promote cell proliferation,inhibit cell apoptosis,and reg?late cell cycle.Conclusion:(1)Induced recombinant protein can stim?late the differentiation and maturation of dendritic cells;(2)L7/L12 ribosomes Protein can stim?late the inflammatory response of dendritic cells;(3)L7/L12 ribosomal protein affects the MAPK signal pathway of dendritic cells and 293t cells c?ltured in vitro.