Effects Of Mycobacterium Tuberculosis HupB On Antigen Presented Cells And T Cells Function And Immunization

Objective:To investigate the effects of HupB an antigen from Mycobacterium tuberculosi-s on antigen presented cells and polarization of na(?)ve CD4~+T cells immune responses.Materials and Methods:we expressed and purified HupB and then the mass spectrome-ter technology was used to test the molecular weight.The expressed and purified HupB to stimulate RAW264.7 macrophage.Real-time PCR was used to test the expression of NF-κB m RNA,ELISA was used to test the secretion level of IL-6 and TNF-αin culture supernatant.And then we used the same method to treat the mice bone marrow-derived dendritic cell.Flow cytometry was used to detect the expression level of CD80,CD86 and MHC-II surface molecules,ELISA was used to test the secretion level of IL-6,IL-12p40,TNF-αand IFN-γin culture supernatant.After mixed lymphocyte reaction the secretion level of IFN-γwas tested by ELISA and the q RT-PCR was used to test the expression of the main transcription factors T-bet、GATA3、ROR-γt and Fox P3 m RNAs in Th1,Th2,th17and Treg.Results:Data showed that the molecular weight is 24.79KDa.It is consistent with the NCBI data and the molecular weight on the plasmid.Compared with the untreated macrop-hage the expression level of NF-κB increased significantly.ELISA showed that the secretion of IL-6 enhanced obviously but the level of TNF-αwas not significant.Compared with the untreated dendritic cells expression level of CD80,CD86 and MHC-II increased significantly.Moreover,ELISA showed that the secretion of IL-6,IL-12 p40,TNF-αand IFN-γenhanced obviously but the level of IL-10 was reduced.After mixed lymphocyte reaction,q RT-PCR results showed that the expression level of specific transcription factor T-bet increased.ELISA showed that the secretion of IFN-γenhanced obviously.Conclusions:(1)HupB through the NF-κB signaling pathways activated RAW264.7macrophages,and released IL-6 to induce host protective immunity.(2)HupB can activite mice dendritic cells to present antigen,contributes to Th1 type polarization of na(?)ve CD4~+T cell and increase the secretion level of IFN-γ.This experiment provide a reference to explore the MTB proteins-host interaction mechanism,and it can be used to diagnosis and treatment of reagents for TB.