| In the study of the immunology of tuberculosis(TB),the study on specific antigen and its epitopes plays a very significant role of developing the diagnosis methods and new vaccine of TB. Epitope is a special molecular group or spatial structure that determines the specificity and antigenicity of protein antigens. B cell epitopes play an important role in the immune response, such as activating the humoral immune response and regulating the cellular immune response. So that study on B-cell epitope polypeptide and immunogenic antigen is conducive to the development of the rapid diagnostic reagent and vaccine of tuberculosis.This study was majoring to screen new antigens of Mycobacterium tuberculosis(MTB), predict and synthesis new B-cell epitopes of the new antigens. At the same time MTB proteins with epitopes were selected to clone, express and purify. The B-cell epitopes polypeptides artificially synthesized and rencombinant proteins of MTB were evaluted by serological method for providing the experimental evidences of developing the specific diagnostic reagents and new TB vaccine.New antigens of MTB were screened according to genomics,proteomics, immunomics and bioinformatics, excluding the coding genes of PE/PPE protiens, transposase and the protein antigens of known B epitopes from the whole genome of MTB. B-cell epitopes of the new antigens were predicted by bioinformatics software SEPPA2.0, from which B-cell epitopes polypeptides were optimized to be synthesized.MTB antigens with epitopes were selected according to the relative data in NCBI and IEDB. The gene encoding the proteins were amplified by PCR from the genome of Mycobacterium tuberculosis H37 Rv strain,and cloned into prokaryotic expressing vector and were confirmed by restriction endonuclease digestion and bidirectional DNA sequencing.The recombination proteins were identified by SDS-PAGE and purified by Nickel ion affinity chromatography column. Recombinant proteins which had low purity were purified again by the purification kit.The purified proteins which were the form of inclusion bodies were renatured and concentrated. BCA method was used to determine the concentration of recombination proteins.Chessboard titration method was used to determine the best dilution concentration of recombination proteins, B-cell epitopes polypeptides, the primary and secondary antibodies. B cell epitopes and recombination proteins were evaluted by serological antibodies test with enzyme-linked immunosordent assay(ELISA).In this study, three new proteins(Sod C 、 Mog 、 Rv1566c) were seclected. Thirteen B-cell linear epitopes peptides were predicted,screened and synthesized, in which Sod C had three, Mog had four and Rv1566 c had six B-cell epitopes polypeptides. The purity of each polypeptide was greater than 90% and met the experimental requirements.Mog predicted conformational epitope successfully.Nine known specificity proteins with epitopes were screened. The specificity proteins and conformational epitope Mog had achieved cloning, expression and purification. The sequencing and enzyme cleavage results of this ten gene were totally consistent with the data in NCBI.According to the chessboard titration, the best coated concentration of the 13 B-cell epitopes polypeptide was 3.13 μg/ml. The best coated concentration of Pfk B, Rv2628, Rv2653 c, Mpt83, Mpt70, Lppx, Esx R,Fad D28, Dev S and Mog was 0.46μg/ml, 1.03μg/ml, 1.31μg/ml,1.53μg/ml, 1.20μg/ml, 1.15μg/ml, 5.53μg/ml, 0.85μg/ml, 1.08μg/ml and1.65μg/ml respectively. The best serum dilution of Pfk B and Esx R was1:50 and 1:400 respectively. The best serum dilution of all the 13 B-cell epitopes polypeptides and other protein were 1:100.104 positive sera and 104 negtive sera samples were collected for the serological experiments. The result of serological test of Pfk B,Rv2628, Rv2653 c, Mpt83, Mpt70, Lppx, Esx R, Fad D28, Dev S and Mogshowed that the sensitivity was 73.08%, 54.81%, 58.65%, 94.23%,79.81%, 77.88%, 2.31%, 86.54%, 91.35%, 89.42% and the specificity was 90.57%, 94.34%, 87.74%, 59.43%, 86.79%, 77.36%, 62.26%,82.08%, 83.96% and 69.81% respectively, the area under the ROC curve was 0.882, 0.789, 0.773, 0.825, 0.884, 0.851, 0.822, 0.891, 0.870 and0.901 respectively. The sensitivity of the 3 B-cell epitopes polypeptidess in Sod C was 88.46%, 65.38%, 72.12% and the specificity was 75.96%,91.35% and 89.42. The area under the ROC curve was higher than 0.85.Mog having the biggest Youden index was 0.734. The sensitivity and the specificity of P209 in Mog were higher than those of the other epitopes polypeptides. The sensitivity and the specificity of the 6 B-cell epitopes polypetiedes in Rv1566 c was from 77.88% to 92.31% and 69.23% to85.58% respectively。In summary, in this study we predicted, screened and synthesized 13B-cell linear epitopes polypeptides in 3 new protein antigens. We had cloned, expressed and purified ten proteins to evaluate the antigenicity with serological antibodies test. The serological evaluation results will provide the experimental evidences for developing the specific diagnostic reagents and new TB vaccine. |
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