Study On New Human T Cell Epitopes And Antigenicity Of The Specific Proteins Of Mycobacterium Tuberculosis

Tuberculosis(TB)caused by Mycobacterium tuberculosis(MTB) is a chronic respiratory infectious disease, which greatly threatens human health in the world. In recent years, because of the occurrence and prevalence of multi-drug resistance TB(MDR-TB) and extensively drug-resistant TB(XDR-TB) and human immunodeficiency virus(HIV) coinfection, old TB has once again become one of the serious global public health challenges. At present, the gold standard for the diagnosis of TB is still the culture of Lowenstein Jensen, there is no rapid and effective availlable method. Meanwhile, studies have confirmed that the protection of BCG is not ideal, especially has no protection for adults. The study of specific proteins and its epitopes plays an important role on the research of novel diagnostic strategies and effective vaccines of TB.In this study, two different methods were used for screening of specific antigens and its human T cell epitopes of MTB to select the suitable antigens and epitopes for providing the basis of diagnostic agents and vaccines. Excluding the PE/PPE proteins, transposons and known epitopes, the human T cell epitopes of all the rest associated proteins of MTB were predicted by means of the bioinformatics systematically and roundly, from which high score epitopes were selected and synthesized, then those were tested with the initial and large scale clinical verification trials by enzyme-linked immunospot assay(ELISPOT). At the same time, the gene of the predicted antigens owning concentrative epitopes and foregone specific proteins selected in this study was amplified by PCR from the genome of Mycobacterium tuberculosis H37 Rv strain, and constructed recombinant plasmids by molecular cloning methods. Then the recombinant plasmids were transformed into the prokaryotic expression system to obtain recombinant proteins with Ni-NTA metal affinity chromatography. If the recombinant protein expressed by inactive inclusion body, it was restored to the natural conformation with dialysis renaturation. The enzyme-linked immunosorbnent assay(ELISA) with the optimal concentration of the antigen and dilution of humen serum samples was established to evaluate the antigenicity of the recombinant proteins.2726 T cell epitopes of 273 protein antigens were predicted by the two softwares(TEPredict and IEDB) simultaneously, from which according to that the score was greater than 5, 50 epitopes were chosen to synthesize and purify by biological company. These epitopes were located in the five antigens, Rv1798, Rv0197, Rv0658 c, Rv2942 and Rv3239 c. Ultimately, 32 peptides of 5 antigens were confirmed for the next research. Using separated peripheral blood mononuclear cells(PBMC) from 10 tuberculosis patients with bacteriological positive, the T cell immune reactivity of every epitope were examined by ELISPOT. 6 positive peptides were discovered through preliminary test. The T cell immune reactivity of the positive peptides was verified with 50 TB patients and 60 pulmonary non-tuberculosis patients and 60 healthy people, respectively. The sensitivity of single peptide Rv1798-1, Rv1798-2, Rv0197, Rv0658 c, Rv2942 and Rv3239 c was 12.0%, 18.0%, 20.0%, 8.0%, 10.0% and 4.0%, respectively, and the specificity was all exceed 99%. The result sum of two polypeptide of Rv1798 antigen showed that the sensitivity was 24.0% and the specificity was 100%. Therefore, the combination of polypeptide for diagnostic value of TB, could be much more sensitive than that of a single polypeptide.At the same time, ten specific antigens were chosen in molecular cloning and prokaryotic expression. This study successfully constructed ten recombinant plasmids with the encoding gene of corresponding antigens. We got 8 purified recombinant proteins Gro ES, Lpq H, Pst S3, PPE18, PPE19, Gln A1, Plc A and Ecc A5(the other two recombinant plasmids could not express) for further evaluating the diagnostic value by enzyme-linked immunosorbent assay(ELISA). The results showed that the antigen Gro ES, PPE18, PPE19, Gln A1 and Ecc A5 had higher diagnostic value, of which the area under curve(AUC) was greater than 80%, and the sensitivity was 72.1% 76.8%, 59%, 66.8% and 66.8%, as well as the specificity was 81.4%, 78.4%, 92.2%, 78.4% and 83.3%, respectively. The AUC of antigen Lpq H, Pst S3 and Plc A was also higher than 70%, while the sensitivity was 66.3%, 52.1% and 75.3%, and the specificity was 68.6%, 88.2% and 65.7%, respectively.In summary, this study was the first time used TEPredict and IEDB two software simultaneously to predict the T cell epitopes of MTB associated the antigens systematacially, verified the 6 positive polypeptides for human T cell epitope of MTB. Also 8 recombinant proteins of Gro ES, Lpq H, Pst S3, PPE18, PPE19, Gln A1, Plc A and Ecc A5 were expressed and purified, which had good antigenicity and be expected to become the new antigen candidates for immunological diagnosis of MTB infection and vaccines.