strain BP (3, 4) was used throughout this study. was expressed in under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected like a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic activation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human being donors. While ScMp65 was considerably nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+ T-cell clones were generated using a mannoprotein portion as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant Mp65 but not to ScMp65. Therefore, the recombinant Mp65 of retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies. Despite the identified importance of cell-mediated immunity (CMI) in the protecting response against the human being opportunistic fungus (33, 34), few antigenic focuses on of this response have so far been characterized. They include heat shock proteins, enolase, and a number of as-yet-uncharacterized mannoproteins, some with adhesive function (1C3, 6, 7, 9, 11, 15C18, 26, 36). The recognition of these antigens and an understanding of the mechanisms whereby they elicit and regulate CMI is an obvious prerequisite for generation of molecules with potential immunoprophylactic or immunotherapeutic activity, and even for use as immunodiagnostic reagents. We have analyzed a 65-kDa mannoprotein (here designated CaMp65) of strain generated a strong and protecting CMI which was promptly exposed by CaMp65 activation of splenocytes in vitro, as well as by a delayed-type hypersensitivity response to this antigen in vivo (10, 27). Moreover, a moderate yet significant level of safety was conferred upon mice by immunization having a mannoprotein portion comprising CaMp65 as a major antigenic, CMI-inductive component (27C28). This safety was clearly enhanced from the concomitant administration of interleukin-12 (IL-12) as an adjuvant (10). Because of these interesting and potentially useful properties, we have recently tackled the biochemical characterization of BDA-366 CaMp65. A strong homology in the protein level was found between this protein and the glucanase or transglycosidase family of cell wall proteins of (8, 20, 21). Interestingly, the least similarity between CaMp65 and the candida proteins was found in probably the most antigenic peptides of the N-terminus regions of CaMp65 (21). The availability of the amino acid sequences of several tryptic and chymotryptic fragments of CaMp65 and the founded sequence homology with cell wall proteins have allowed us to clone the relevant genes of both and and to communicate them in induces in vitro an intense CMI response by peripheral blood mononuclear cells Rabbit Polyclonal to HDAC7A (phospho-Ser155) (PBMC) of healthy subjects and derived CD4+ T-cell clones, having a magnitude comparable to that previously demonstrated by the native antigen (10, 20, 37). These data show that CaMp65 provides a appropriate reagent for studies of defense. MATERIALS AND METHODS Microrganisms, growth conditions, and mannoprotein draw out. strain BP (3, 4) was used throughout this study. It was cultivated in YPD (2% glucose, 1% candida draw out, 2% Bacto Peptone; Difco, Detroit, Mich.), Winge (0.3% candida draw out, 0.2% glucose; Difco), or revised Lee (4, 31) press, as specified in single experiments. XL1-Blue cells [(F HL1-Blue MRF ([F [F cells were usually cultivated in L broth (1% tryptone, 0.5% yeast extract, 0.5 NaCl; pH 7.0), Luria-Bertani (LB) plates (1% tryptone, 0.5% yeast extract, 0.5 NaCl, 1.5% agar; pH 7.0) or top agarose (1% tryptone, 0.8% NaCl, 0.6% agarose; Boehringer, Mannheim, Germany) supplemented when necessary with BDA-366 ampicillin (100 g/ml), kanamycin (50 g/ml), or tetracycline (12.5 g/ml) (Boehringer). The mannoprotein portion MPF2 was acquired by ethanol precipitation followed by gel filtration of a crude extract of autoclaved BDA-366 cells, as reported elsewhere (37). Oligonucleotides and PCR. Ca33, Ca34, Ca64, and Ca65 oligonucleotides were purchased from Pharmacia. Their sequence and specificity are demonstrated in Table ?Table1.1. PCR reactions with or purified DNA were performed as previously explained (26) by using primer pairs Ca33-Ca34 and Ca64-Ca65, respectively. Briefly, the reactions were carried out on a Gene AMP PCR System 9600 Apparatus (Perkin-Elmer/Cetus Corp., Norwalk, Conn.) inside a volume of 100 l comprising 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 M concentrations of each deoxynucleotide, 50 pmol of each.