Abstract： Objective:To investigate the epitope and immunogenicity of the Mycobacterium tuberculosis antigen Rv0446c, and to propose candidate antigens and epitopes for immunodiagnosis and vaccine development of tuberculosis. Methods:The bioinformatics softwares TE predict and IEDB MHC Class Ⅱ Binding Prediction were used to predict the T cell epitopes of Mycobacterium tuberculosis antigen Rv0446c. ELISPOT assay was used to examine the immunogenicity of these epitopes in subjects registered to Foshan Fourth People’s Hospital and Fuzhou Chest Hospital between January 2019 and December 2020, including 50 with tuberculosis, 39 with pulmonary diseases, and 55 healthy volunteers. A total of 60 six-week-old BALB/c mice were randomly divided into 10 groups ( n=6 each) to receive subcutaneous immunization with PBS, keyhole limpet hemocyanin (KLH) , high-dose Mycobacterium tuberculosis antigen Ag85B (50 μg/mouse) , low-dose Ag85B (20 μg/mouse) , high dose P120, low dose P120, high dose P121, low dose P121, high dose P123, and low dose P123 peptides (100 μg/mouse as high dose and 50 μg/mouse as low dose of P120, P121 and P123) three times weekly in two weeks apart. At four weeks after the last immunization, ELISA was used to measure the levels of spleen-derived cytokines IFN-γ, IL-2, IL-4 and IL-10 in each group of mice. Results:Of the seven epitope peptides predicted, ELISPOT assay identified four positive T cell epitope peptides: P120, P121, P122, and P123. The sensitivity and specificity of detection using these peptides in tuberculotic patients were 30.0%, 18.0%, 6.0%, 22.0% and 96.8%, 98.9%, 100%, 96.8%. Compared with the PBS control group and KLH group, P120 led to higher levels of IFN-γ, IL-4, IL-10, P121 led to higher levels of IFN-γ and IL-4 (all P<0.05) , and P123 led to higher level of IFN-γ (all P<0.05) generated in mice. The level of IL-2 stimulated by P120, P121, and P123 in mice were higher than those by PBS group and lower than those by low-dose or high-dose Ag85B (all P<0.05) , but did not differ from those by KLH (all P>0.05) . Conclusion:Rv0446c protein and its T cell epitope exhibit high immunogenicity and immunoreactivity that may stimulate an intense cellular immune response in the host, and therefore can be used for clinical diagnosis of tuberculosis and development of novel tuberculosis subunit vaccines.