Study On The Diagnosis Of Tuberculosis By Using The Antigen Epitopes Of Mycobacterium Tuberculosis In RD Region

Tuberculosis(TB)caused by Mycobacterium tuberculosis infection remains a major threat to human health.Tuberculosis is the ninth leading cause of death in the world,ranking first among infectious diseases and surpassing AIDS.Bacillus Calmette-Guerin Vaccine(BCG)is the only world-accepted immunization method.However,studies in recent years have shown that the relative protection rate of BCG is only 0%-80% and cannot effectively protect the body.At the same time,the existing detection methods for tuberculosis cannot meet the clinical needs for specificity and sensitivity.Therefore,effective vaccines and rapid test kits with better specificity remain two important tasks in the fight against tuberculosis.There is a Region of Difference(RDs)between BCG and tuberculosis genomes.BCG lost some genes of Mycobacterium tuberculosis and Mycobacterium bovis RD1-RD16 during long-term passaging.There are 129 open reading frames,some of which may be The virulence is related and may be related to the low protection rate of the BCG vaccine.In recent years,studies on Mycobacterium tuberculosis have been mainly concentrated in the RD1 region,and the study of 15 other differential regions is not comprehensive.Therefore,the potential T and B cell epitopes of RD1-RD16 region are to be predicted and analyzed in this study.The predominant epitope peptides were used to establish an ELISA method for diagnosis of tuberculosis using dominant epitope peptides,and preliminary studies were conducted on the influence of dominant epitope peptides on TLR4/MyD88 signaling pathways.1.A systematic analysis of 129 proteins in the RD1-RD16 region was performed using bioinformatics methods.A comprehensive analysis of the RD region protein of Mycobacterium tuberculosis was performed by analyzing the secondary structure,hydrophobicity,and glycosylation sites of the protein.And screened the candidate B-cell epitopes and T-cell epitopes against the homology and spatial structure of the proteins,and finally obtained 117 predominant B-cell epitopes;dominant T-cell epitopes.18 articles.2.According to the result of epitope screening,one T-cell epitope peptide and one B-cell epitope peptide were synthesized.B cell epitope peptide sequence: P1: LKTQIDQVESTAGSLQ GG KWDATATELNNALQNL GG TVCGFRSRLYA;T cell epitope peptide sequence: P2: SLDLLDPIY GG KTDAATLAQ GG MTEQQWNFA GG ASDNNAGDY.3.Using the B-cell epitope synthesis peptide P1 as the coating antigen,indirect ELISA was used to detect the specific antibodies in the serum of tuberculosis patients,and a method for the diagnosis of tuberculosis using antibodies as the target was established.It was determined that the optimal coating concentration of P1-ELISA was 5 ?g/mL,the optimal dilution of serum was 1:400,the optimal working concentration of enzyme-conjugated secondary antibody was 1:5000,and the optimal working time of substrate was 15 minutes.Sensitivity experiments determined that the highest dilution of serum was 1:2000 with strong specificity.4.T-cell epitope synthesis peptide P2 and adjuvant immunization C57 mice were immunized,and 60 C57 mice were equally divided into three groups with 20 in each group.Group A: CFA+P2 1:1 mixture,Group B: IFA +P2 1:1 mix,group C: PBS group.One week after the immunization,the spleens were taken and the mRNA expression levels of TLR4/MyD88 signaling pathway related factors were detected by fluorescence quantitative technique.The results showed that the expression levels of MyD88,TRAF6,JUN,IL-6,IL-12 and TNF-a in group A were significantly different from those in PBS control group(P<0.05);the expression levels of FOS and IL-8 were significantly different from those in PBS control group.Difference(P<0.01);MAP2K4 expression was significantly different from PBS control group(P<0.001).In group B,the expression of TLR4,TRAF6,MAP2K4,FOS,IL-6 was significantly different from PBS control group(P <0.05);MyD88,JUN,IL-12 expression levels and PBS control group were significantly different(P <0.01).The experimental results show that the synthetic peptide P1 can be used as a candidate antigen for the diagnosis of tuberculosis;the synthetic peptide P2 can regulate the mRNA expression level of TLR4/MyD88 signaling pathway related factors.