| Brucellosis is a zoonotic disease which caused by Brucella. It is widely popular in theworld and endanger the public health and safety. Brucella can invade the body by a variety ofways such as the digestive tract, skin, mucous membranes or respiratory system, it poses aserious threat to livestock and human health. Macrophages are the major cells in whichBrucella proliferation in the body. On the early stage of Brucella infection, macrophagesproduce pro-inflammatory cytokines (TNF-α, IL-12, IL-6) and chemokines initiate theimmune response to clear Brucella. But in the macrophages which Brucella proliferation, theTNF-α production secreted by macrophages is significantly decrease and the ability ofmacrophages to remove bacteria was also significantly reduced. Finally Brucella can survivein macrophages and replication. Studies have found that Brucella outer membrane protein isclosely related to the regulation of macrophage cytokine secretion. Therefore, this studyconstruct the prokaryotic recombinant plasmid pET-32a-omp25and eukaryotic recombinantplasmid pCI-neo-omp25，then detect the effects of Omp25protein on morphology and growthactivity of macrophage, finally discuss the effects and the molecular machanism of omp25protein on TNF-α secretion in macrophages. The study results are as follows:1. Construction of prokaryotic and eukaryotic plasmid which express Omp25proteinSpecificity primers P1, R1and R2was synthesized and the optimization of the omp25gene was amplified, then the omp25gene was inserted into the vector pCI-neo and pET-32aby double digestion of BamH I and Sal I, and constructed the recombinant expression plasmidpCI-neo-omp25and pET-32a-omp25. The recombinant vector PCI-neo-omp25wastransfected into macrophages3D4/21, then Omp25protein was identificated by SDS-PAGEand Western blot. Recombinant pET-32a-omp25vector was transformed into BL21whichwas induced by IPTG to expressed Omp25protein. The Omp25protein was purified byHisTrap HP affinity chromatography and identificated by SDS-PAGE and Western blot.2. The effects of Omp25protein on morphology and growth activity of macrophage pCI-neo-omp25was transfected into macrophages3D4/21and the purified Omp25protein was added to the cultureof3D4/21. Observation by microscope showed that Omp25protein had no significant effect on cell morphology. MTT results indicated that Omp25protein had slightly but not significantly inhibitory effect on the growth activity of3D4/21cells.3. The effects of omp25protein on the ability of secretion TNF-α in macrophagesELISA results showed that Omp25protein suppressed the expression of the TNF-α.Quantitative RT-PCR results indicated the mRNA of TNF-α was suppressed by Omp25protein. Dual luciferase assay data illustrate that Omp25protein suppressed the transcriptionalactivity of NF-κB and the activity of TNF-α promoter. This results provide evidence that theOmp25protein of B. suis function to suppress the TNF-α by inhibiting the TNF-α promoterand the transcriptional activity of NF-κB, then help B. suis to escape from the immunesystem.This study successfully constructed Omp25protein prokaryotic and eukaryoticrecombinant plasmid. Prokatyotic recombinant plasmid was transformed into E.coli BL21,and eukaryotic recombinant plasmid was transfected into macrophage3D4/21, this plasmidssuccessfully expressed Omp25protein. Intracellular and estracellular test confiemed thatOmp25protein has not siginificantly effects on morphology and growth activity ofmacrophage. The molecular machanism of inhibitory effects of Omp25protein on TNF-αsecretion in macrophage was Omp25protein firstly inhibited the activity of NF-κB, therebyinhibited the activity of TNF-αpromoter,finally inhibited the transcription levels of TNF-αinmacrophage. |
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