We identified the major protein band recognized by IgG as TcTUB. low levels (1, 2). Several studies indicate the importance of antibodies in protection against infection (3, 4, 5, 6), but the precise role of humoral immunity in host defense remains incompletely understood. Purified proteins induce protection in mice challenged with live parasites. These antigens include cruzipain (7), antigens which are targeted by the host antibody diversity in the course of infection. Antibody diversity can be analyzed by an immunoblot technique which detects global antibody reactivity against whole protein extracts (13). This method detects autoantibodies produced in autoimmune diseases (14, 15) and identifies repertoire changes linked to resistance against infection (16). Microtubules are cytoskeletal structures composed of / tubulin heterodimers that are found in eukaryotic cells and are abundant in trypanosomatid parasites from the order Kinetoplastide (17). These structures have important functions in cell division, maintenance of cellular morphology, motility, intracellular transport, and signal transduction (18). In trypanosomes microtubules have two -tubulin isoforms and one -tubulin isoform (19) and are found underneath the plasma membrane (the subpellicular microtubules), in the flagellum, and as a component of mitotic spindle apparatus (20). The microtubules function as a perfect target for many compounds with trypanocidal activity, blocking tubulin activity (21). Therefore, a microtubule component is a BTB06584 suitable target to be considered as an effective vaccine candidate to protect from trypanosome infections (22, 23). It was previously shown that mice vaccinated with native tubulin purified from were protected against infection (22). Previous report showed that mice vaccinated with the microtubule-associated protein (MAP) p52 of inhibit the growth of trypanosomes in culture (25). Given the paucity of information regarding the subject, this work addresses the identification of prominent antigens targeted by IgG antibodies during infection. Here, we describe that acute and chronic infection of BALB/c mice induced limited changes in the antibody diversity. Using a proteomic approach, we identified -tubulin (TcTUB) as one of the major antigens targeted by antibodies. The -tubulin gene was BTB06584 isolated, cloned, and expressed. Recombinant TcTUB was recognized by sera from infected mice, and immunization of na?ve mice with a single dose of recombinant Rabbit Polyclonal to OR2T11 TcTUB induced protection against infection. These results indicate the importance of selecting candidate vaccines antigens from analysis of unbiased Ab reactivities from infected mice. Materials and Methods Mice, Parasite, and Infection Male wild-type (WT) BALB/c and Fas-L mutant BALB/c.(mice (26) were produced at the National Institutes of Health, Bethesda, MD, USA by serially backcrossing the gene onto a BALB/c background for 15 generations. All mouse studies followed the guidelines set by the National Institutes of Health, United States. The study was approved by the Research Ethics Committee of Federal University of Rio de Janeiro (protocol 062/14). Protocols for animal were approved by the Institutional Ethical Committees in accordance with international guidelines. All animal experimentation was performed in accordance with the terms of the Brazilian guidelines for the animal welfare regulations. Mice were infected with intraperitoneal injection (i.p.) with 105 chemically induced metacyclic forms of clone Dm28c (17, 18). Chemically induced and insect-derived metacyclic forms induce a similar infection as demonstrated previously (18). Acute infection was evaluated after 23C33?days of infection, while chronic infection was evaluated after 150?days. Recombinant -Tubulin Genomic DNA was extracted from Dm28c epimastigotes (1??108; Rapidprep isolation kit, BTB06584 Pharmacia) and used as template for amplification of gene (19) by touchdown PCR (27). Touchdown PCR was carried out in 50?L of 20?mM TrisCHCl (pH 8.8), 2?mM MgSO4, 10?mM KCl, 10?mM (NH4)2SO4, 0.1% (v/v) Triton X-100, 0.1?mg/mL nuclease-free BSA, 100?ng DNA, 0.5?mM dNTPs, 0.4?M of primers TcTubF (5-ATCATATGCGTGAGATTGTGTGCG) and TcTubR (5-ATGAATTCTTAGTACTGCTCCTCCTC), and a mixture of 2.0?U Pfu (Fermentas) and.