## 1 Introduction
tuberculosis_ and _M. bovis_ BCG vaccine [40]. Ag85B also belongs to a group of three proteins known as complex 85, which is a component of the cell wall in all species of mycobacteria [41]. This antigen has been extensively studied and tested during the production of DNA, protein and recombinant subunit vaccines [42; 43; 44; 45]. Glycosylation is a post-translational modification (PTM) and refers to the covalent linkage between carbohydrates and a polypeptide, lipid, polynucleotide or organic compound [46; 47; 48; 49]. In Mtb, this PTM is involved in various cellular mechanisms, including pathogenesis, virulence and antigenic properties such as the stimulation of the immune response against various pathogens [31; 39; 50]. The use of mycobacterial glycoproteins is an option for the development of a TB vaccine. Studies have shown that glycosylation plays a key role in the T-cell antigenicity, suggesting that the use of glycosylated proteins in vaccines could enhance the induction of a protective immune response [51]. This effect has been associated, for example, with the native glycosylation pattern of APA (Rv1860), also known as 45/47 kDa. When the pattern is altered, a lower stimulation of T-lymphocyte responses is observed [52; 53]. However, there is little research about the role of glycosylation on recombinant antigens and its importance in the immunological response against TB. The first glycosylated proteins in _M. tuberculosis_ were described in the 1970s and 1980s, and recent quantitative glycoprotein analyses have revealed 101 differentially glycosylated sites in 67 proteins in four _M. tuberculosis_ strains [54]. Most proteins that have been described for Mtb are O-mannosylated proteins. However, the presence of other types of glycosylation, glycan types, glycan occupancy and glycan length are poorly understood. Most of the identified glycosylated proteins/lipids in mycobacteria are located in the cell wall or are secreted. These glycoproteins are involved in several biological mechanisms, such as cell adhesion, invasion, cellular signaling, intracellular bacterial survival, antigenicity, pathogenicity and virulence, among others [50; 54]. [PERSON] et al. (2013) have shown that mannosylation of the adhesin APA in _M. tuberculosis_ is essential for the human T-cell activation in a murine model of subcutaneous infection. In addition, the differences in cytokine production, including IFN-\(\gamma\), the tumor necrosis factor (TNF)-\(\alpha\) and IL-2, have been demonstrated following the immunization with the APA protein, independent of the mannosylated state [27]. The importance of glycosylation for various _M. tuberculosis_ proteins and the effects of glycosylation on the interactions with the immune response need to be further explored to understand their implications for vaccine development [51]. Recently, our research group studied the _M. microti_ strain ATCC 11,152 in a murine model of pulmonary TB, and we observed that the bacterial burden of _M. tuberculosis_ was significantly reduced, resulting in a lower percentage of pneumonia in lung tissue after it was challenged with _M. tuberculosis_ H37 Rv (data not shown). Based on these results and the positive outcomes of recombinant vaccines, we decided to use _M. microti_ as a vehicle for the overexpression of the _M. tuberculosis_ antigens PstS-1 and Ag85B. These antigens have been widely studied and are known to stimulate T- and B-lymphocyte proliferation and elicit a T-helper (Th)1-type response in animal models [37; 38; 55; 56; 57]. These antigens are encoded in the genome of _M. microti_, but little is known about the in vitro and in vivo expression of the two antigens in this strain. In the present study, we developed two recombinant strains of _M. microti_, overexpressing these PstS-1 and Ag85B antigens. The protein expression of these antigens was performed in a constitutive vector and analyzed with SDS-PAGE and 2D-PAGE.