Secreted antigen 85 (Ag85) protein is present around the cell wall surface and needed for the survival of in macrophages and is considered a virulence factor. of immunoglobulin G was found in group V with 62.495.4327 ng/ml. The highest mean number of CD8+ T-cells, NK T-cells, granzyme-B and perforin was found in group IV with 4.32%, 1.03%, 35.111.7789 pg/ml and 6.190.2235 pg/ml, respectively. The results of One-Way ANOVA test showed that there were significant differences in immunoglobulin responses, with p 0.05. The Felbamate expressions of granzyme-B and perforin were higher in mice treated with combination of BCG and recombinant proteins. Conclusions: Ag85 protein can be combined with the BCG vaccine to improve protection against contamination. and BCG culture filtrate is the Ag85 complex. Secreted antigen 85 (Ag85) protein is present around the cell wall surface and needed for the survival of in macrophages and is considered a virulence factor. Three members of Ag85 protein with molecular weights of 30-32 kDa: Ag85A, Ag85B and Ag85C that are encoded by three paralogous genes: and in intracellular parts of host cells and helps the bacteria defend themselves against the immune system while facilitating the formation of tubercles (Launois et al., 2011; Nr4a1 Piubelli et al., 2013 Zarif et al., 2013). Expressions of protein Ag85 by and genes can stimulate proliferation and differentiation of B cells and T- cells. T cells can be differentiated into Th1, Th2, Treg and Tc cells. B cells are activated by Ag85 antigens which will stimulate the production of IgM or IgG antibodies against on a cellular basis (Jiang et al., 2015; Rizzi et al., 2012; Metcalfe et al., 2016). Ag85 protein of can induce macrophages and dendritic cells to produce Macrophage Inflammatory Protein (MIP) such as MIP-1 (CCL4) and secretes IL-15. MIP-1 and IL-15 can increase Tc cell and NK cell activation and proliferation. Activated Tc cells and NK cells can kill cells infected by by producing chemical mediators, such as granzyme-B and perforin (Wang et al., 2010; Felbamate Kuo et al., 2013; Silva et al., 2013). Ag85A (32 kDa) and Ag85B (30 kDa) proteins are potential candidates for tuberculosis vaccines, because they are the main proteins secreted by and show strong immunogenic properties (Yuk and Jo., 2014). This study aimed to determine the ability of Ag85A and Ag85B proteins in activating the response of antibodies, granzyme-B and perforin in Balb/c mice. Materials and Methods Animal models In this research, twenty-five (n=25) male Balb/c mice (8-10 weeks old, 20-30 g) Felbamate were obtained and kept at the animal experimental laboratory of LPPT Universitas Gadjah Mada. Ethical clearance for use of animals in research was obtained from the Ethical Clearance Commission of the Faculty of Veterinary Medicine, Universitas Gadjah Mada (Approval Number: 0021/EC- FKH/Eks/2018). The mice were kept in standard conditions as demanded by humane protocol and each cage contained five Balb/c mice. Preparation of Ag85 recombinant protein Ag85A and Ag85B genes were inserted into pET SUMO plasmid (InvitrogenTM ChampionTM pET SUMO Cat Protein Expression System No. K300-01) and were transformed into (DE3). Ag85 recombinant genes were expressed in liquid LB medium made up of kanamycin (50 g/ml), and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). Ag85A and Ag85B recombinant proteins were then purified by Ni-NTA purification system kit (InvitrogenTM NovexTM Cat No.K950-01). The purified proteins were cleaved, using protease to separate the SUMO taq protein in SUMO and Ag85 recombinant proteins, and stored at -200C. Immunization of Balb/c mice Immunogenicity of Ag85 recombinant proteins of was measured in Balb/c mice that had been immunized by Ag85A and Ag85B proteins. The expressions of the Balb/c mice antibodies and cellular immune responses Felbamate were analyzed. The animals.