Study On The Apoptosis Regulation Of Brucella Out Membrane Protein OMP31

Objective: Bacteria of the genus Brucella are Gram-negative facultative intracellular that cancause human and various mammals acute or chronic infection. Macrophages have been shown toconstitute an important site for Brucella intracellular alive and replication. Not only can theBrucella parasitize in macrophage, but aslo it can inhibit the macrophages’ apoptosis. Brucellaouter membrane protein OMP31plays a significant role in its virulence and intracellularparasitism, therefore the research tends to construct the omp31deletion mutant of Brucellamelitensis16M, taking macrophages as the research object to investigate the use of OMP31inmacrophages’ apoptosis in different horizon.Methods: The upstream and downstream of the omp31gene and SacB gene were amplified byPCR from Brucella melitensis16M and Bacillus subtilis.The correctly identified sequence wasconnected to the suicide vector pGEM-7zf+by using restriction enzyme digestion, the subclonepGEM-7zf+-Δomp31-SacB was further obtained and transformed into Brucella melitensis16M byelectroporation. The16MΔomp31was screened out by two times homologous recombination thentested its hereditary stability and analyzed the mutant biology characteristics. To analyze its effectson apoptosis of murine macrophages RAW264.7were infected with16MΔomp31strain andparental strain at different time points by flow cytometry; To measure the murine Macrophagerelease Cytochrome C (CytC) and tumor necrosis factor-α(TNF-α) by double antibody sandwishELISA method; To check out TNF-α, Bax, Bcl-2mRNA expression levels at different time pointsby quantitative Real-time PCR assay.Results:(1)Brucella melitensis16M Δomp31mutant strain was successfully generated andreversion was not observed in15generations. The conventional bacteria identification testsconfirmed that the mutant was Brucella melitensis biotype1;(2)Analysis with flow cytometryshowed that the mutant promoted the apoptosis of infecton macrophages significantly;(3)ELISAresults showed the mutant strain can induce more CytC than parent strain all the time excpet2h;(4)Quantitative Real-time PCR showed the mutant strain and parent strains the mRNAexpressions of TNF-α were up-regulated all the time and Bax were so did excpet4h, while theexpression of Bcl-2was down-regulated excpet2h.Conclusion:(1)16MΔomp31has been successfully constructed, the mutant can be inheritedstably and its biological characteristics shows no changes;(2)The outer membrane protein OMP31is involved in Bruclla inhibiting the macrophages’s apoptosis;(3)omp31can regulate theexpression of TNF-α、CytC、Bax、Bcl-2which belongs to apoptosis pathways, thus realize thecontrol of macrophages’s apoptosis, the study can be laid a foundation of the anti-apoptosismechanism and selection the medical therapy for the patients with chronic Brucellosis.