Screening And Identification Of Immunodominant CTL And Th Epitopes Of Mycobacterium Tuberculosis In Xinjiang Kazakh People

Objective:To screen the immunodominant CTL and Th epitopes of Mycobacterium tuberculosis in Xinjiang Kazak population and study their immunogenicity,so as to provide basis for the research and development of recombinant BCG vaccine and the design of new tuberculosis epitope vaccine suitable for different regions and ethnic groups.Methods:（1）Obtain the amino acid sequences of five kinds of Mycobacterium tuberculosis immune dominant antigen proteins（Rv3425,Rv3873,Rv1980c,Rv0288,Rv2450c）from Uni Prot website,and analyze their homology with human proteome online using the Blast tool of NCBI website.（2）The online tools Prot Param and DNAStar software were used to comprehensively evaluate the five dominant antigens of Mycobacterium tuberculosis.（3）Four online software,Net MHC,Net MHCpan,SYFPEITHI and RANKPEP,were used to predict the HLA-B*5101 restricted CTL cell epitopes of five proteins;The HLA-DRB1*1301 restricted Th cell epitopes of five proteins were predicted using Net MHCⅡ,Net MHCⅡpan and IEDB software.（4）Five CTL epitopes and five Th epitopes were screened by analyzing the evaluation results of five proteins and the prediction results of CTL epitopes and Th epitopes;Combined with the tertiary structure of five proteins predicted by Alpha Fold,the selected CTL and Th epitopes were analyzed using Py MOL software;CTL and Th epitope peptides were synthesized by chemical method for in vitro experiments.（5）CCK8 method was used to detect the optimal stimulation concentration of CTL epitope peptides and screen out candidate CTL epitopes;The level of CD8+T cytokines secretion after peripheral blood mononuclear cells（PBMC）stimulated by candidate CTL epitope peptides was detected by ELISA test,and its ability to induce CD8+T cell proliferation was detected by flow cytometry.（6）CCK8 method was used to detect the optimal stimulation concentration of Th epitope peptides and screen out candidate Th epitopes;ELISA test was used to detect the secretion level of CD4+T cytokines after PBMC stimulated by candidate Th epitope peptides,and flow cytometry was used to detect its ability to induce CD4+T cell proliferation.Results:（1）Five HLA-B*5101 restricted CTL epitopes were screened out by analyzing the results of bioinformatics analysis of five immunodominant antigen proteins of Mycobacterium tuberculosis,which were P₅（Rv3804c）,P₂₀₁（Rv3875）,P₁₅₃（Rv1980c）,P₈（Rv3804c）,and P₉（Rv2450c）;Five HLA-DRB1*1301restricted Th cell epitopes were screened as P₆₈（Rv3804c）,P₁₃₈（Rv3875）,P₁₁₀（Rv1980c）,P₆₃（Rv3804c）,and P ₁₅₇（Rv2450c）.（2）The purity of CTL and Th epitope peptides synthesized by chemical method were more than 95%,which can be used for subsequent in vitro experiments.（3）The optimal concentration of CTL epitope peptide in vitro is 40μg/ml,three candidate epitopes(P₂₀₁,P₁₅₃,P₈)were selected from five CTL epitopes,and all three candidate epitopes can stimulate CD8+T to secrete IFN-γ,TNF-α,Gr B,and can induce CD8+T cell proliferation.（4）Th epitope polypeptide in vitro,the optimal concentration is 40μg/ml,three candidate epitopes(P₆₈,P₁₃₈,P₆₃)were selected from five Th epitopes,and all three candidate epitopes can stimulate CD4+T to secrete IFN-γ,TNF-α,IL-2,and to inhibit IL-10.They cannot stimulate the secretion of IL-17,and can induce the proliferation of CD8+T cells.Conclusion:（1）The HLA-B*5101 restricted immune dominant CTL epitopes of Mycobacterium tuberculosis in Xinjiang Kazak population were screened and identified:P₂₀₁、P₁₅₃and P₈.（2）The HLA-DRB1*1301 restricted immune dominant Th epitopes of Mycobacterium tuberculosis in Xinjiang Kazak population were screened and identified:P₆₈,P₁₃₈ and P₆₃.This will lay the foundation for the development of tuberculosis specific diagnostic reagents and specific and efficient tuberculosis vaccine.