Effect Of B.melitensis Omp31 Protein To Microglia And Its Molecular Mechanism

Objective The purpose of this study was to clone Brucella melitensis omp31 gene, construct its prokaryotic expression vector p ET-30a-omp31, express and purify recombinant fusion protein and analyze the capability that Omp31 induced microglia to produce proinflammatory cytokines TNF-α, IL-6, HMGB1 and express TLR4 m RNA. Investigate the effect of B.melitensis Omp31 Protein to Microglia and its molecular mechanism by observing the correlation between them. At the same time, this study explored the effect of Omp31 on observing apoptosis.Methods PCR amplification was performed using primers based on omp31 gene sequence from Genbank, B.melitensis genomic DNA as template. Then, omp31 gene was cloned into the prokaryotic expression vector p ET-30 a. Constructed recombinant plasmid p ET-30a-omp31 and identified by restriction enzyme digestion and sequencinganalysis. The recombinant plasmid was transfected into Ecoli competent cell BL21(DE3) to express recombinant protein and the recombinant protein was purified. Microglia was stimulated by different concentrations Omp31 fusion proteins, while setting the control group. Levels of inflammatory cytokines TNF-α, IL-6 and HMGB1 were measured by ELISA and levels of TLR4 m RNA relative expression were measured by RT-q PCR. At the same time, using flow cytometry instrument to analyze its impact on apoptosis of microglial cells. The comparison between the groups mean using one way ANOVA analysis, P<0.05 was denote difference was significant.Results The prokaryotic expression vector of Brucella melitensis omp31 gene was constructed successfully which were identified by double digestion and Sequence analysis. The recombinant plasmid was transfected into Ecoli competent cell BL21(DE3) to express recombinant protein and the recombinant protein was purified. Then, we obtained Omp31 fusion protein and molecular weight of about 31 k Da. Microglia was stimulated by different concentrations Omp31 fusion proteins and we found that Omp31 fusion proteins cound induce microglia to produce proinflammatory cytokines TNF-α, IL-6, HMGB1 and express TLR4 m RNA, the level of TNF-α, IL-6 is higher than the control group, the difference was statistically significant(P<0.05). the level of HMGB1 and TLR4 m RNA expression in1.0ug/ml group,2.5ug/ml group and 5.0ug/ml group is higher than the control group, the difference was statistically significant(P<0.05). The level of TNF-α and HMGB1 reach a peak at 2.5ug/ml and the level of IL-6 reach a peak at 5.0ug/ml, After that, the level of inflammatory cytokines is deceased gradually with the protein concentration increased. The trend of TLR4 m RNA expression is first increased and then decreased with the different of Omp31 fusion proteins. In addition, Omp31 fusion proteins can inhibit apoptosis of microglia by flow cytometry.Conclusions Compared with the control group, secreting of HMGB1 and the expression level of TLR4 m RNA were significantly increased while the secreting of TNF-α, IL-6 was significantly upregulated. This shows that HMGB1/ TLR4 singnaling pathway is activated and this singnaling pathway may mediate the pathological process of Omp31 caused the central nervous system damage. In addition, Omp31 fusion proteins may inhibit apoptosis of microglia.