Objective To evaluate the antigenicity of two proteins of Mycobacteium tuberculosis (M. tuberculosis), Dnak(Rv0350) and MPT83(Rv2873), in order to provide a scientific basis for immuno-logical diagnosis of tuberculosis and research on vaccines. Methods The two antigen proteins, Dnak (Rv0350) and MPT83(Rv2873), were cloned, expressed and purified using the methods of genetic recom-bination and protein purification technology. Blood samples were collected from subjects including tuberculo-sis patients ( TB) , non-tuberculosis patients with other pulmonary diseases ( non-TB) and healthy volunteers (HV). To analyze the immunological properties of the recombinant Dnak (Rv0350) and MPT83 (Rv2873) proteins, they were used as antigens to detect humoral and cellular immunity in the subjects with enzyme linked immunosorbent assay ( ELISA ) and effector T cell enzyme-linked immunospot assay ( ELISPOT ) . Results The recombinant and purified Dnak (Rv0350) and MPT83 (Rv2873) proteins of M. tuberculosis were successfully obtained and used as antigens in the detection of humoral and cellular immunity in the sub-jects. Specific antibodies ( IgG) in the serum samples of 135 TB, 56 non-TB and 94 HV were tested with ELISA. The results showed that the sensitivity, specificity and accuracy of Dnak ( Rv0350 ) protein were 77. 80％ (105/135), 56. 70％ (85/150) and 66. 67％ (190/285). Similarly, the sensitivity, specificity and accuracy of MPT83 (Rv2873) protein were 76. 30％ (103/135), 43. 30％ (65/150) and 58. 95％(168/285). Cellular immunity was tested with the levels of IFN-γproduced by effector T lymphocytes after stimulating peripheral blood monouclear cells ( PBMC) collected form subjects of 59 TB, 65 non-TB and 64 HV with Dnak (Rv0350) and MPT83 (Rv2873) protein antigens. The results showed that the sensitivity, specificity and accuracy of Dnak (Rv0350) and MPT83 (Rv2873) proteins were 66. 10％ (39/59), 62. 79％ (81/129) and 63. 83％ (120/188), and 47. 46％ (28/59), 79. 84％ (103/129) and 69. 68％(131/188), respectively. Conclusions M. tuberculosis Dnak (Rv0350) and MPT83 (Rv2873) proteins have good antigenicity and could stimulate T cells to produce stronger immune responses. The two proteins used in combination might have promising potential in the research of immunodiagnosis of tuberculosis and the development of new anti-tuberculosis vaccines.

Objective:The cellular immunity of 5 Mycobacterium tuberculosis recombinant proteins and their compositions was evaluated.Method:A total of 88 fresh venous blood from peripheral heparin anticoagulant population, 42 of which were from tuberculosis patients treated by The Tuberculosis Prevention and Treatment Center of Changping District, Beijing, and 46 of healthy volunteers were provided by the Infection Diseases of Chinese Center for Disease Control and Prevention. Healthy volunteers without a history of tuberculosis exposure and any clinical signs and symptoms. Using the Mycobacterium tuberculosis standard strain H37Rv DNA as a template, complete genes of the selected 5 recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c by PCR amplified; 5 recombinant proteins were cloned, expressed and purified as stimulants by genetic recombination and protein purification techniques, and the effector T cell enzyme-linked immunospot assay (ELISPOT) was used to detect cellular immunity in the population.Results:The recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c were successfully cloned, expressed and purified; And the sensitivities were 50.00%, 71.43%, 69.04%, 73.81% and 76.19%, and the specificities were 86.96%, 76.09%, 71.74%, 39.13% and 36.96%. In addition, the positive predictive value, negative predictive value, area under the curve and Youden index were 52.46% to 77.78%, 62.96% to 74.47%, 0.511 to 0.754 and 0.129 to 0.475, respectively. Except for Rv1411c and Rv3418c, the number of spot-forming cell (SFC) detected by Rv3874, Rv3875 and Rv2031c in tuberculosis patients was higher than healthy volunteers, and the differences were statistically significant ( P<0.001). Among the 26 compositions composed of 5 recombinant proteins, the sensitivity was 80.95% to 95.24%, and the specificity was 68.89% to 24.44%. As the number of recombinant proteins in the composition increases, the sensitivity gradually increased, but the specificity decreased. Conclusion:The recombinant proteins of Mycobacterium tuberculosis Rv3874, Rv3875 and Rv2031c have strong ability to stimulate T cells to produce immune response, and have certain antigenicity. The efficacy of Rv1411c and Rv3418c alone as diagnostic antigens is not ideal, and the composition composed of multi-component antigens has certain application value. This article provides experimental evidence for the immune diagnosis of tuberculosis and the preparation of new anti-tuberculosis vaccines.

Objective:The cellular immunity of 5 Mycobacterium tuberculosis recombinant proteins and their compositions was evaluated.Method:A total of 88 fresh venous blood from peripheral heparin anticoagulant population, 42 of which were from tuberculosis patients treated by The Tuberculosis Prevention and Treatment Center of Changping District, Beijing, and 46 of healthy volunteers were provided by the Infection Diseases of Chinese Center for Disease Control and Prevention. Healthy volunteers without a history of tuberculosis exposure and any clinical signs and symptoms. Using the Mycobacterium tuberculosis standard strain H37Rv DNA as a template, complete genes of the selected 5 recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c by PCR amplified; 5 recombinant proteins were cloned, expressed and purified as stimulants by genetic recombination and protein purification techniques, and the effector T cell enzyme-linked immunospot assay (ELISPOT) was used to detect cellular immunity in the population.Results:The recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c were successfully cloned, expressed and purified; And the sensitivities were 50.00%, 71.43%, 69.04%, 73.81% and 76.19%, and the specificities were 86.96%, 76.09%, 71.74%, 39.13% and 36.96%. In addition, the positive predictive value, negative predictive value, area under the curve and Youden index were 52.46% to 77.78%, 62.96% to 74.47%, 0.511 to 0.754 and 0.129 to 0.475, respectively. Except for Rv1411c and Rv3418c, the number of spot-forming cell (SFC) detected by Rv3874, Rv3875 and Rv2031c in tuberculosis patients was higher than healthy volunteers, and the differences were statistically significant ( P<0.001). Among the 26 compositions composed of 5 recombinant proteins, the sensitivity was 80.95% to 95.24%, and the specificity was 68.89% to 24.44%. As the number of recombinant proteins in the composition increases, the sensitivity gradually increased, but the specificity decreased. Conclusion:The recombinant proteins of Mycobacterium tuberculosis Rv3874, Rv3875 and Rv2031c have strong ability to stimulate T cells to produce immune response, and have certain antigenicity. The efficacy of Rv1411c and Rv3418c alone as diagnostic antigens is not ideal, and the composition composed of multi-component antigens has certain application value. This article provides experimental evidence for the immune diagnosis of tuberculosis and the preparation of new anti-tuberculosis vaccines.

Objective To investigate the cross-reactive immune responses to Mycobacterium vac-cae (M. vaccae), Mycobacterium tuberculosis (M. tuberculosis, H37Rv) and Mycobacterium bovis Bacillus Calmette-Guerin ( BCG) for providing reference for the development of new vaccines with M. vaccae. Meth-ods M. vaccae (ATCC95051), M. tuberculosis (H37Rv) and BCG (China strain) were cultured on L-J solid media and harvested. Total bacterial protein antigens prepared by ultrasonic disruption were used to im-munize BALB/c mice. IgG antibodies in serum samples were detected with enzyme-linked immunosorbent assay ( ELISA) to evaluate humoral immune responses. Cellular immunity was assessed by detecting various cytokines with cytokine release assay ( CRA) . Results The mice that were respectively immunized with the three mycobacterial antigens could produce high titers of antibodies ( IgG) and high levels of IFN-γand IL-2, but low levels of IL-4 and IL-10. Results of the cross reactivity tests showed that ATCC95051, H37Rv and BCG were able to cross-react with the immunized mice, and all of them induced high levels of IFN-γ, IL-2 and IgG antibodies. Conclusions The three Mycobacteria mainly elicited Th1 immune responses. There were cross-reactive immune responses to M. vaccae, M. tuberculosis and BCG, which might provide ref-erence for using M. vaccae in the development of new anti-tuberculous vaccines.

Objective:To evaluate the humoral and cellular immunoreactivity of recombinant Mycobacterium tuberculosis ( M. tuberculosis) PstS1 and HspX protein antigens in order to provide reference for immunodiagnosis of tuberculosis and screening of candidates for vaccine antigens. Methods:Purified recombinant M. tuberculosis PstS1 and HspX proteins were obtained using molecular cloning expression and Ni 2+ affinity chromatography. Blood samples and epidemiological data of healthy individuals and patients with M. tuberculosis infection were collected. Specific IgG antibodies and IFN-γ-producing antigen-specific T cells were respectively detected by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT) with the recombinant proteins used as antigens. The humoral and cellular immunoreactivity of the recombinant PstS1 and HspX proteins were assessed with statistical analysis of data. Results:Both the recombinant PstS1 and HspX proteins could induce the secretion of IFN-γ by more specific effector T cells in patient with M. tuberculosis infection, and the differences between the infection and healthy control groups were statistically significant ( P<0.05). The specificity and sensitivity of the recombinant PstS1 and HspX as the diagnostic antigens of ELISPOT were 92.11% (35/38) and 65.96% (31/47), and 68.42% (26/38) and 91.49% (43/47), respectively. The two proteins also possessed some humoral immunoreactivity, but statistically significant difference was only observed in the HspX-specific antibody level between the two groups ( P<0.05). Conclusions:Both the recombinant PstS1 and HspX proteins had good cellular immunoreactivity and were the immunodominant antigens of cellular immunity. They performed well in cellular immunodiagnosis and were good potential candidate antigens for anti-tuberculosis vaccines.

Objective To investigate the single nucleotide polymorphism (SNP) of toxinantitoxin-chaperone (TAC) system of Mycobacterium (M.) tuberculosis with different genotypes and its biological significance.Methods A total of 183 clinical M.tuberculosis isolates were collected for spoligotyping.The sequences of higA,higB and Rv1957 were obtained by using PCR and DNA sequencing.The sequences were compared for possible mutations.Functional consequences of nonsynonymous SNPs were predicted by using I-Mutant 2.0 servers.Results Among the 183 M.tuberculosis isolates,138(75.41％) belonged to the Beijing family,while 45(24.59％) belonged to the non-Beijing family.A total of 149(81.42％) isolates showed polymorphisms in the TAC system.We discovered 6 nonsynonymous SNPs and 2 synonymous SNPs.All the synonymous mutations occurred in higA gene,while nonsynonymous SNPs were found in the higA,higB and Rv1957 genes either.All the synonymous mutations and 4 nonsynonymous SNPs were restricted to the Beijing family strains and only 2 nonsynonymous SNPs were observed in the non-Beijing family strains.Of the 6 nonsynonymous SNPs studied,4 were predicted to have ability to affect the stability of respective protein.Conclusion The SNPs in the coding sequences of TAC system in clinical isolates can be relatively high and the Beijing family strains are with higher polymorphism,which might benefit to adapt to different host environment.

Objective: Autoregressive integrated moving average (ARIMA) model was used to predict the incidence of tuberculosis in China from 2018 to 2019, providing references for the prevention and control of pulmonary tuberculosis. Methods: The monthly incidence data of tuberculosis in China were collected from January 2005 to December 2017. R 3.4.4 software was used to establish the ARIMA model, based on the monthly incidence data of tuberculosis from January 2005 to June 2017. Both predicted and actual data from July to December 2017 were compared to verify the effectiveness of this model, and the number of tuberculosis cases in 2018-2019 also predicted. Results: From 2005 to 2017, a total of 13 022 675 cases of tuberculosis were reported, the number of pulmonary tuberculosis patients in 2017 was 33.68% lower than that in 2005, and the seasonal character was obvious, with the incidence in winter and spring was higher than that in other seasons. According to the incidence data from 2005 to 2017, we established the model of ARIMA (0,1,2)(0,1,0)₁₂. The relative error between the predicted and actual values of July to December 2017 fitted by the model ranged from 1.67% to 6.80%, and the predicted number of patients in 2018 and 2019 were 789 509 and 760 165 respectively. Conclusion The ARIMA (0, 1, 2)(0, 1, 0)₁₂ model well predicted the incidence of tuberculosis, thus can be used for short-term prediction and dynamic analysis of tuberculosis in China, with good application value.