Screening And Funcitonal Study On CD4~+T Cell Epitopes From Proteins Encoded By The Specific RD Regions Of Mycobacteirum Tuberculosis

Tuberculosi（sTB）infected by Mycobacterium tuberculosis is a kind of infectiondesease. It has high infection rates and mortality. TB is serious harm to human health.Our country accounted for16%of new and relapse cases of TB that were notified in2010. Although these two years the number of TB cases declined, but the humanimmunodefici ency virus and M.tuberculosis infect in common and a broad range ofmultiple drug resistance tuberculosis(MDR-TB) emerged put forward a difficultproblem for the treatment of TB. As the effici ency of the TB testing and bacillusCalmette-Guérin（BCG）immune effect declined, a new vaccine for TB is urgentneeded.A lot of research prove that CD4+T cells play important roles in the immuneresponse against TB. The region of difference(RD) become the researchers’ focus ofconcern, because a lot of proteins in the region show strong immunogenicity.Therefore, analysis of promiscuous epitopes on the region of difference of encodedproteins in M.tuberculosis is our goal in this research.In order to find more new T cell epitopes for TB from RD on M.tuberculosis.The sequence of amino acids from the proteins coded by the gene of RD were got onthe website NCBI. With the help of the NetMHCII and ProPred which are onlineprediction software for T cell epitopes. Qualification on HLA-Ⅱ type susceptiblegene HLA-DRB1*0803、HLA-DRB1*1101、HLA-DRB1*1401、 HLA-DRB1*1501HLA-DRB1*1502、 HLA-DRB1*1601, we choosed18polypeptides with the lengthof9mer as our research subjects. Peripheral blood mononuclear cells from10healthyvolunteers and30TB cases (20sputum smear-negative tuberculosis patients,10sputum smear-positive tuberculosis patients) were used to assay the screened18peptides., using the technology of ELISPOT assay, ELISA assay and flow cytometrytechnology(FCM). Finally, through the technology of inducing mature dendritic cellsin vitro, we identified the role of DC in the use of T cell epitopes.Peptides ESAT-6(69-77)、FP10(76-84)、 RV3876(aa556-564)、 RV0222 (149-157) showed high immunogenicity and high ability in CD4+T cell prolifertionresponse. We can see that7/25,6/25,8/25,4/25PBMC from tuberculosis patientsshowed significant IFN-γ production in the presence of P3, P6, P15, P18.4/25,3/25PBMCs from tuberculosis showed lower IL-10production in the presence of P3, P15.4/15,2/15,3/15,4/15PBMCs from sputum smear-negative showed enhancedsecretion of TNF-α. The content of TNF-α in serum had significant difference,which could guide on tuberculosis. T cell epitope worked with DC showed their highbility in PBMCs proliferation and IFN-γ secretion.CD4~+T cells, especially Th1cells play key roles during the progress of M.tuberculosis infection, IFN-γand TNF alpha are important Th1type inflammato ry cellfactors, which level of secretion are qualified as standard for tuberculosis vaccineimmune efficacy. T cell epitops selected by the study could significantly enhance thesecretion of IFN-γ and TNF alpha, especially in mature dendritic cells. Therefore,the four peptides are promising T cell epitopes for TB vaccine and new detectionreagent.