Studies On Function And Immunology Characteristics Of Mycobacterium Tuberculosis Membrane Protein RV0849

Tuberculosis (TB) is a worldwide infectious disease caused by Mycobacterium tuberculosis (MTB). According to the World Health Organization (WHO), there are more than 2 billion people infected with MTB, accounting for one third of the world's population. Among them, one in ten will be suffered from active tuberculosis in their lives. In recent years, the emergence of multi-drug-resistant TB, as well as Co-infection of MTB and HIV, cause TB more difficult to control. BCG is the only TB vaccine used in the human body approved in many countries. However, after inoculated by newborn, its immune protection declines with the increase of age. In addition, BCG is only effective in preventing primary tuberculosis but of no use in patients who have been infected. Therefore, it is high time to develop the researches of new drugs and vaccines to combat TB.Membrane protein play crucial roles in bacteria, such as, cell-cell interaction, ion and nutrient transport, cell signaling, and pathogenicity-related cellular reactions. Among them, the functions of bacterial drug efflux pumps are very important, which is also a cause for multi-drug resistant. Besides, as the membrane proteins directly exposed to the cell surface, they interact with the corresponding membrane receptor of the host immune activity cells and stimulate the formation of cellular and humoral immunity, so their good immunogenicities are expected. Therefore, genes encoding membrane proteins, especially those efflux pump genes are considered to be the main targets of new drug and vaccine.H37Rv0849, a gene coding conserved membrane protein in Mycobacterium tuberculosis, function as a drug efflux pump, belonging to major facilitator superfamily. TMHMM analysis demonstrates it contains 12 transmembrane domains and has a good immunogenicity. Reports on this protein's function and the immune properties were not seen yet.Based on this, in order to characterize the roles and functions of Rv0849, it was amplified from Mycobacterium tuberculosis H37Rv strain genomic DNA and recombinant plasmid pMV0849 was constructed with shuttle plasmid pMV261 after endonuclease digestion, finally recombinant bacterium SM::pMV0849 was acquired with Mycobacterium smegmatis(SM) as a vector. By analyzing the ability of transportation of variously kinds of agents and flurescence dyes with the recombinant strains, it can be deduced that Rv0849 may participate in the transport but has little ability of drug efflux. In addition, by evaluating the effect of immunity response using C57BL/6 mice inoculated by SM::pMV0849, it is found that Rv0849 considerably induced mice's humoral immunity and stimulated the production of tumornecrosis factor-α(TNF-α) and IFN-γ, which were secreted by lymphocytes and participating in cellular immunity. The results give us a hint that Rv0849 might be a new immune antigen. To sum up, our researches provide evidences for further study on anti-TB vaccines, MTB efflux pumps and its mechanism.