Preliminary Studies On The Molecular Mechanism That The Rv2185c And Rv3763Protein Of MTB Regulated NF-κB

TB is a zoonotic and chronic wasting disease caused by mycobacterium tuberculosis (MTB). It is the world’s single pathogen infection which leads to the highest disease mortality. The advent of multi-drug resistant strains of tuberculosis and the widespread of HIV has made more complex and difficult to prevent and control tuberculosis in recent years.NF-κB (nuclear factor-KB) is a transcription factor that presents in a variety of cell types, which regulates the transcription of many genes in the immune response, inflammation, and plays an important role in apoptotic.MTB pathogenic mechanism is complex. Secreted proteins、lipoproteins and transmembrane proteins mediate the interaction of bacteria with the host, damaging the host tissues and causing a cascade of reactions, which play important roles in MTB from the pathogenic process and impacting of the host immune defense.This study intends screening MTB secreted proteins, membrane proteins and lipoproteins, which regulate NF-κB, based on NF-κB signaling pathway, in order to analyze the regulation and biological significance of NF-κB signaling pathways, then clarify tuberculosis pathogenesis and lay the foundation of immune mechanisms. The main research works were as following:1. Selection of the target gene and construction of recombinant eukaryotic expression plasmidsMTB H37Rv strain secreted proteins, membrane proteins and lipoproteins were found through bioinformatics and tuberculosis proteomics.160proteins (125secreted proteins,10membrane proteins and15lipoproteins) were selected as candidate genes.Methods of PCR and restriction were used to insert the candidate genes into the eukaryotic expression vector pcDNA3.1/V5-His B, and successfully constructed the eukaryotic expression plasmid.2. Screening MTB proteins that can regulate NF-κB activity160eukaryotic expression plasmids were transfected into cells, respectively. Then the MTB proteins that could regulate NF-κB activity were screened out by luciferase reporter system from them. As the result, MTB Rv2185c protein (secreted protein) can significantly induce NF-κB activation and also have a dose-dependent, cell-specific. MTB Rv3763protein (lipoprotein) can significantly inhibit the activity of NF-κB induced by TNF-a, and also have a dose-dependent but no cell-specific. Further constructed MTB Rv2185c, Rv3763different regions deletion mutants, and studied their ability to regulate NF-κB activity. Only the full length MTB Rv2185c induced NF-κB activity, and the first30amino acids of Rv3763is essential for inhibiting NF-κB activity induced by TNF-α.3. MTB Rv2185c promotes the phosphorylation of P65The eukaryotic expression plasmid MTB Rv2185c transfected into cells, then the changes in the phosphorylation of p65were detected by Western-blot method after30hours. The result indicated MTB Rv2185c can promote the phosphorylation of P65.4. MTB Rv3763inhibits the phosphorylation of p65induced by TNF-αThe eukaryotic expression plasmid MTB Rv3763transfected into cells, and stimulate cells6hours with TNF-α after24hours. Then the changes in the phosphorylation of p65were detected by Western-blot method. The result indicated the Rv3763inhibited the phosphorylation of p65induced TNF-α.