Bioinformatics Analysis The Epitopes Of Antigens Form Mycobacterium Tuberculosis And Evaluation Of The Immunogenicity

Objective：In the Tang Dynasty, there had formed a said of "TB bug" of Tuberculosis (TB)in Chinese medicine. Robert Koch found the Mycobacterium Tuberculosis(MTB) in1882,demonstrating that the "TB bug" which is the MTB is the pathogeny of TB. To realize theTB further in Chinese medicine, we use the modern techniques and methods to research forMTB. The study takes bioinformatics and immunology to evaluate the antigen of RD12andRD5from MTB, laying the foundation of integrated traditional Chinese and westernmedicine in TB, sequentiallyMethods: The amino acid sequences of the antigens from RD12and RD5wereobtained by online NCBI database.The homology between Mycobacterium tuberculosiscomplex and human proteins was analyzed by BLAST.The distribution of CD8+T cellepitopes was predicted by SYFPEITHI、NETCTL、BIMAS、NetMHC databases; thedistribution of CD4+T cell epitopes was predicted by NetMHC databases. Rv3117fromMycobacterium Tuberculosis, which unique exists in pathogenic MycobacteriumTuberculosis H37Rv standard strains while absenting in the live attenuated BCG strains andthe nonpathogenic mycobacteria mc2155strains, were expressed in E.coli. and purified,removed the endotoxin in the target protein. The endotoxin and target protein were measuredby the methods of chromogenic substrate limulus reagent and BCA, respectively. The proteinwas used to immune C57BL/6mice, anti mycobacterium tuberculosis Rv3117IgG antibodyin the mice serum were abtained and detected by Enzyme-linked immunosorbent assay; testthe SFC of specific IFN-γ which produced in the spleen lymphocytes of C57BL/6miceinduced by specific antigen Rv3117using Enzyme-linked Immunospot Assay. Serum fromclinical tuberculosis patients were used to detect the level of anti mycobacteriumtuberculosis Rv3117IgG antibody. While serum from healthy participants as controls. The PBMCs separated from the whole blood obtained from the clinical tuberculosis patients andhealthy participants, then test the SFC of specific IFN-γ which produced in thelymphocytesby by specific antigen Rv3117using Enzyme-linked Immunospot Assay.Results: We select13and23candidate T cell epitopes of RD12and RD5through acomprehensive analysis，respectively. The Rv3117has strong enough immunogenic to detectthe level of anti mycobacterium tuberculosis antigens IgG antibody from serum of65clinical tuberculosis patients and59healthy controls, compared with CFP-10and ESAT-6which have been recognized by the people. The results show that specific antigen Rv3117from Mycobacterium Tuberculosis can cause significant differences(p<0.05) in serum ofIgG levels between65cases of tuberculosis patients and59cases of healthy controls. Inaddition, the sensitivity of IgG antibody which induced by Rv3117(26.20%) is equal withCFP-10（24.60%） and ESAT-6（24.60%） in the tuberculosis patients; in the healthycontrols, the specificityof IgG antibody which induced by Rv3117（98.30%） is higher thanCFP-10（94.90%） and ESAT-6（96.60%）.The results of Enzyme-linked Immunospot Assay shows that0.10μg/mL,1.00μg/mLand10.00μg/mL of Rv3117could all cause specific IFN-γ produced in the spleenlymphocytes of C57BL/6mice that were immuned by Rv3117, while differentconcentrations of Rv3117produce little specific IFN-γ in the spleen lymphocytes ofC57BL/6mice immuned by CFP-10, the two groups have significantly different (p<0.05).Conversely different concentrations of CFP-10could all cause specific IFN-γ produced inthe spleen lymphocytes of C57BL/6mice which immuned by CFP-10, while differentconcentrations of CFP-10produce little specific IFN-γ in the spleen lymphocytes ofC57BL/6mice which immuned by Rv3117.In order to perfectly reflect the cellular immune response of antigen. We use the finalconcentration of20.00μg/mL of Rv3117to stimulate the PBMCs of tuberculosis patientsand the healthy controls, there is no statistical significance（p>0.01） in distingμishing thelevel of specific IFN-γ produced by PBMCs from tuberculosis patients and the healthycontrols. Moreover we analysis the level of specific IFN-γ produced by PBMCs intuberculosis patients（n=17） which stimulated with different concentration, the results showthat there is no specificIFN-γ（p>0.05） producing in PBMCs from tuberculosis patientswhich was stimulated by1.00~20.00μg/mL of recombinant Rv3117.Conclusion: With modern techniques and methods to understand the TB "TB bug" hypothesis in traditional Chinese medicine is a necessary means. This study use thebioinformatics and immunology to evaluate the antigen of RD12and RD5from MTB, andevaluate the antigen Rv3117. Although those are not immunological parameters as antiTuberculosis treatment, as antigen which exists only in pathogenic MycobacteriumTuberculosis H37Rv standard strains encoded in the region of differentiation(RD) ofMycobacterium Tuberculosis, further study for the function of the antigen could contributeto the development of new vaccines and tuberculosis drugs and explore the pathogenicmechanism of tuberculosis, and provide research basis to prevent、diagnosis and treat TB inIntegrated Traditional Chinese and Western Medicine.