Abstract: Objective  To evaluate the immune protective effect of resuscitation promoting factor (Rpf) DNA vaccine on M. tuberculosis (Mtb) infected mice. Method  Rpf D and Rpf E plasmid DNA vaccines were construct. 180 BALB/c mice with 4 weeks of age were divided into 6 groups, 30 mice each group were immunized with empty plasmid, Rpf D plasmid DNA, Rpf E plasmid DNA, BCG and saline, respectively. All of mice were infected by aerosol with Mtb H37Rv. Serum anti-Rpf D or E antibodies and IFN-γ were detected. The lymphocyte proliferation assay and cytotoxicity test were performed, IFN-γ, IL-12 and IL-2 levels were tested after immunization with RpfD,E DNA vaccine. Histopathologic examination of the lung and CFU count were performed.  Results  (1) The antibody levels against RpfD and E were as follow：Rpf D group 0.32±0.1，Rpf E group 0.39±0.1,BCG group 0.02±0.01，empty plasmid group 0.01±0.0，saline group 0.09±0.04，blank group 0.03±0.01.The antibody levels in RpfD group and E group were significantly higher than those in BCG group，empty plasmid group,  saline group, and blank group (F value: Rpf D：（45.6，43.2，45.1，45.7）；Rpf E：（51.6，53.6，51.0，52.2）; P<0.01).  Serum IFN-γ levels: Rpf D group (43.9±24.8）pg/ml，Rpf E group (45.9±21.0）pg/ml,BCG group (21.0±11.0）pg/ml，empty plasmid group (7.9±4.9）pg/ml，Saline group (4.7±2.1）pg/ml，blank group (5.8±4.7）pg/ml，Serum IFN-γ levels of RpfD group and E group were higher than empty plasmid group, saline group, and blank group (F value: Rpf D：15.6，17.8，17.3；Serum IFN-γ levels of Rpf E：17.5，21.1，20.7; P<0.01). (2) The lymphocyte proliferation assay using cell counting kit-8 (CCK-8) showed: Rpf D group 176.0±4.2，Rpf E group 183.0±4.3,BCG group 101.0±1.1，empty plasmid group 100.0±6.8，saline group 104.0±8.4，blank group 116.0±2.2, which in vaccine groups were significantly higher than the other controls (F value: Rpf D：9.5，10.1，10.0，9.0；Rpf E：11.2，12.9，11.7，10.3，P<0.05). Cytotoxicity test showed: Rpf D group 32.0±3.2，Rpf E group 30.0±4.2,BCG group 16.0±5.9，empty plasmid group 3.3±1.5，saline group 6.7±0.5，blank group 7.3±3.5. The proliferation index in RpfD group and RpfE group was higher than the other control groups (F value Rpf D：8.8，14.5，13.7，11.9；Rpf E：8.1，12.5，11.6，11.1; P<0.05). (3) Cytokines in the supernatant of lymphocytes stimulated by Rpf D and Rpf E:IL-2 level: Rpf D group 9.5±2.4，Rpf E group 9.2±1.2,BCG group 2.4±2.1，empty plasmid group 1.2±0.3，saline group 1.8±1.0，blank group 1.5±0.7. The IL-2 levels in RpfD group and RpfE group were higher than the other control groups (F value Rpf D：12.5，14.6，13.5，13.9；Rpf E：12.0，13.6，13.1，13.2; P<0.05). IFN-γ level: Rpf D group 22.2±5.7，Rpf E group 28.7±14.4,BCG group 16.1±10.1，empty plasmid group 9.8±1.6，saline group 13.2±2.1，blank group 15.7±2.9. The IFN-γ level in RpfD group and RpfE group were higher than the other control groups（F value Rpf D：7.8，12.6，8.7，8.3；Rpf E：11.3，16.4，14.7，14.2; P<0.05）. Conclusion  RpfD and RpfE DNA vaccines can induce immune responses including humoral and cellular immunogenicity in Mtb infected BALB/c mice. Rpf might be used as the vaccine candidate.

Key words: Mycobacterium tuberculosis, Bacterial proteins, Cytokines, Vaccines,DNA