The Effect Of HupB On T Lymphocyte Factor Release And Its Potential Application In The Diagnosis Of Tuberculosis

Objective: In this study,the HupB protein of Mycobacterium tuberculosis H37 Rv was taken as the research object to investigate the regulatory role of HupB protein on T lymphocyte differentiation,which also provides a reliable theoretical reference for the further application of HupB protein.Methods: Bioinformatics analysis of HupB: Ex PASY Prot Param,Prot Scaleon Ex PASY,TMHMM Server v.2.0,SOPMA,Vaxijen v2.0 and other online tools were used to analyze the physicochemical properties,hydrophobicity,transmembrane region,secondary structure and antigenicity of HupB proteins.The phylogenetic tree and protein interaction of protein HupB were analyzed by MEGA X 10.1.7,STRING database and other analysis tools.Expression,purification and identification of HupB protein: Pgex-6p-1-rv2986 c recombinant plasmid was constructed based on the gene sequence of Mycobacterium tuberculosis H37 Rv HupB protein.The recombinant plasmid was transformed into E.coli DH5 and induced to express in BL21(DE3).SDS-PAGE was used to analyze the expression of HupB protein in E.coli.The recombinant protein HupB was purified by GST Agarose and its relative molecular weight was determined by SDS-PAGE.Removing endotoxin from the protein by Et Eraser TM HP.The secondary structure of recombinant protein was analyzed by circular dichroism spectroscopy.Regulatory effect of Mycobacterium tuberculosis H37 Rv HupB protein on T lymphocyte differentiation: 11 patients with tuberculosis,10 patients with non-tuberculosis pulmonary disease and 10 healthy people were selected from Wuhan medical treatment center for control.PBMC were isolated from patients’ venous blood,and HupB proteins were co-cultured with PBMC cells for 48 h.ELISA was used to detect levels of IFN-γ、IL-2、IL-4、IL-6、IL-10、IL-12p70、IL-17A、TGF-β1 and TNF-α.Diagnostic value of HupB-specific Interleukin-6 in tuberculosis: The cytokines released after HupB protein stimulated PBMC in TB patients were analyzed and hupb-specific cytokines were screened.40 patients with pulmonary tuberculosis,27 patients with non-tuberculous pulmonary disease,and 45 healthy people were selected to the specificity of IL-6 that is released by PBMC stimulated by HupB,and optimize experimental conditions.The diagnostic value of TB was evaluated by the ROC curve,the optimal cut-off value was calculated according to the Youden’s index,and the Negative/Positive diagnosis was conducted on the experimental samples.The present laboratory diagnostic methods of tuberculosis were compared to explore its potential application value.Results: The HupB protein of Mycobacterium tuberculosis is composed of 214 Amino acids,which is a stable,hydrophilic protein with no transmembrane region,and has high antigenicity.Among them,the secondary structure is mainly composed of α-helix(51.40%).In terms of evolutionary relationship,the proteins encoded by Mycobacterium tuberculosis H37 Rv and those encoded by Mycobacterium canettii have the closest relationship and belong to the same evolutionary branch.Protein interaction analysis showed that HupB protein mainly interacts with five proteins,Mih F,Phe T,Top A,Pol A and Dna A,distributed in the cell wall,cytoplasm and plasma membrane,can bind to DNA,and participate in biological processes such as intracellular macromolecule metabolism and nucleic acid metabolism.The recombinant plasmid p GEX-6p-1-rv2986 c was successfully constructed and the recombinant protein HupB was obtained with the protein concentration of 853 μg/m L.The molecular weight of the recombinant protein of 51 k Da,which was consistent with the theoretical value.The protein endotoxin content is less than 0.3 EU/m L,which can be used for subsequent experiments.The secondary structure of recombinant HupB at 25℃ included α-helix,32.1% β-sheet,and 54.5% random coil.Cytokine assay results showed that there was no significant difference between IFN-γ、IL-2、IL-4、IL-10、IL-12p70、IL-17A、TGF-β1 and TNF-α in PBMC of patients with tuberculosis,patients with non-tb pulmonary disease and healthy people stimulated by HupB(P>0.05).However,in the tuberculosis group,the amount of IL-6 released by PBMC after stimulation with HupB was 788.1±608.0 pg/m L,while the amount of IL-6 released by PBMC without stimulation with HupB was 72.29±20.38 pg/m L,showing a significant difference(P<0.0001).Meanwhile,the amount of IL-6 released by PBMC in the non-tb pulmonary disease group was 259.3±99.81 pg/m L after stimulation by HupB.After stimulation with HupB,the amount of IL-6 released by PBMC in the healthy group was 53.51±9.019 pg/m L,which was significantly different from that in the tuberculosis group(P<0.0001).The ROC curve of HupB-specific Interleukin-6 was analyzed in patients with pulmonary tuberculosis as the experimental group,and in patients with non-tuberculosis pulmonary diseases as the control group.The area under the ROC curve(AUC)value was greater than 0.9,and the P value was less than 0.0001,which was statistically significant.The optimal cut-off value was obtained based on the Jorden index,and the diagnostic sensitivity,specificity and accuracy of the experimental samples were 88.89%,86.57% and 87.5%,respectively.Conclusions: 1.IL-6 is an inflammatory factor,and the secretion of IL-6 increases after HupB protein stimulates PBMC in TB patients,indicating that IL-6 may be involved in the process of MTB infection,which provides a reference for further study of the pathogenesis of MTB.2.HupB protein has high antigenicity and interacts with a variety of proteins involved in DNA binding and nucleic acid metabolism.Combined with HupB protein to induce PBMC in tuberculosis patients,HupB protein has potential applications in diagnosis,immunotherapy and prevention,which is helpful to further explore the mechanism of MTB and immune system interaction.3.The release of IL-6 by HupB protein-induced PBMC in pulmonary tuberculosis patients is higher than that in non-tuberculous pulmonary disease patients and healthy people,which has potential application value in the evaluation of auxiliary diagnosis and treatment effect of tuberculosis.4.HupB protein-specific Interleukin-6 reaction can be used as a potential diagnostic method for tuberculosis and used in clinical research.