Study On The Diagnostic Value Of Six Protein Antigens And The Antigenicity Of UspC Protein From Mycobacterium Tuberculosis

Tuberculosis is still one of the major infectious diseases that seriously threaten human health in the world.In order to control the severe epidemic of tuberculosis and finally achieve the goal of ending tuberculosis,it is urgent to screen and evaluate more immunodominant antigens or antigen combinations for the immunological diagnosis of tuberculosis and new tuberculosis vaccines in order to improve the diagnostic efficacy and promote the development of effective tuberculosis vaccines.Based on the above two standpoints,two related experiments were carried out in this research.On the one hand,six M.tuberculosis-specific protein antigens with 100%coverage of T cell epitopes were selected and their independent and combined immunological diagnostic value were assessed.On the other hand,the antigenicity of Rv2318(UspC),a potential immunodominant antigen,was preliminarily studied.We evaluated the immunological diagnostic value of six MTB proteins,Rv0288,Rv1980c,Rv2029c,Rv2031c,Rv3874 and Rv3875,and explored potential diagnostic compositions.First,the six M.tuberculosis-specific proteins were expressed using the E.coli expression system,and the six recombinant proteins were purified by affinity chromatography.Then,venous blood samples were collected from 43 patients with tuberculosis(including sputum positive/negative pulmonary tuberculosis and extra pulmonary tuberculosis)and 38 healthy volunteers to isolate the peripheral blood mononuclear cells for ELI SPOT assay and obtain the serum for ELISA test.Finally,test results were analyzed by statistical methods such as Mann-Whitney non-parametric T test,Dunn's multiple comparison test,ROC analysis and Spearman correlation analysis.The results showed that except Rv2029c the other five antigens(Rv0288,Rv1980c,Rv2031c,Rv3874,and Rv3875)all had good immunodiagnostic value in cellular immunity,and their AUC values which reflects the diagnostic accuracy were 0.852,0.814,0.852,0.874 and 0.872,respectively.Only Rv2031c had little immunodiagnostic value in humoral immunity,with AUC being 0.637.The diagnostic performance analysis of antigen combinations demonstrated that Rv3874-Rv3875 and Rv2031c-Rv3874 were excellent dual-antigen diagnostic combinations,Rv2031c-Rv3875-Rv3874 and Rv0288-Rv3875-Rv3874 were great three-antigen diagnostic compositions.The four-antigen combination Rv0288-Rv3875-Rv3874-Rv2031c had a better diagnostic value than all other combinations.To conclude,the five MTB proteins,Rv0288,Rv1980c,Rv2031c,Rv3874 and Rv3875,all exhibited good diagnostic performance in cellular immunity,and the four-antigen composition Rv0288-Rv3875-Rv3874-Rv2031c was the antigen combination with the most potential in cellular immunodiagnosisIn order to screen out more immunodominant antigens,this study also preliminarily explored the antigenicity of Rv2318(UspC),a less studied protein up to now.First,according to the T cell epitope information of Rv2318 protein from IEDB,two regions rich in epitopes were selected,and their sequences were spliced by overlapping PCR to finally construct the recombinant plasmid of Rv23 1 8P.The expression and purification of the complete Rv2318 protein and the epitope splicing protein Rv2318P were performed.Then,the venous blood samples from volunteers(including tuberculosis patients and healthy volunteers)were collected for ELISPOT and ELISA to evaluate the immunoreactivity of Rv23 18 and Rv231 8P.Finally,the immunogenicity of Rv2318 and Rv2318P was evaluated in BALB/c mice model.The results of in vitro and in vivo experiments demonstrated that Rv2318(UspC)protein had strong immunoreactivity and immunogenicity,and could induce the body to produce strong cellular and humoral immune responses.So,it could be a candidate antigen for TB vaccines with great potential.At the same time,a comparative analysis of Rv2318 and Rv2318P showed that Rv2318P could effectively retain the immunogenicity and immunoreactivity of the intact Rv2318 protein,even better than it.Therefore,Rv2318(UspC)protein has strong antigenicity.and may be a potential vaccine candidate antigen and Rv2318P may be a great surrogate for Rv2318.The protective efficacy of both Rv2318 and Rv2318P should be further explored to confirm their potential as a vaccine component and the effectiveness of epitope splicing method.In summary,the five protein antigens-Rv0288,Rv1980c,Rv2031c,Rv3874 and Rv3875 all had excellent diagnostic value in cellular immunology.Among their combinations,three antigen combinations Rv2031c-Rv3875-Rv3874 and Rv0288-Rv3875-Rv3874 and four antigen combination Rv0288-Rv3875-Rv3874-Rv2031c were potential excellent diagnostic compositions for IGRAs.The Rv2318(UspC)protein and its epitope splicing protein Rv2318P had good antigenicity and were potential candidate antigens for tuberculosis vaccine.Epitope splicing may be one of the ways of antigen optimization.