Zumarraga, K. This study indicates the 27-kDa antigen has an adverse effect on the safety afforded by identified vaccines. We are currently studying how the 27-kDa antigen modulates the mouse immune response. The Th1-type immune response is believed to be necessary for safety against mycobacterial pathogens, such as and (9, 36, 43). Several mycobacterial lipoproteins have been shown to be an important result in in the activation of humoral and cellular immune reactions to mycobacteria (43). One such protein, the 19-kDa antigen, is able to access the major histocompatibility complex class I pathway in macrophages, which is important in order to induce a protecting Th1-type immune response against (33). The 19-kDa lipoprotein was also shown to induce antimicrobial activity and sponsor defense mechanisms induced through toll-like receptors (10, BMS303141 39). Furthermore, lipoproteins were successfully used as potential protecting antigens against different pathogens (11, 18). These observations show that lipoproteins might be appropriate candidates for mycobacterial vaccines. Recently, the 27-kDa lipoprotein was recognized and shown to be identified by the sera of cattle naturally infected with (7). This lipoprotein is definitely exclusively present in the species complex and forms an operon having a 55-kDa multidrug-resistant pump. Subcellular fractionation of specifically located the native 27-kDa lipoprotein in the membrane portion (6, 7). In the present study we cloned the gene encoding the 27-kDa lipoprotein from and indicated its recombinant form in The immunogenicity and the potential protecting efficacy of this antigen, delivered as subunit or as DNA vaccines, were analyzed in the and BCG challenge models in BALB/c mice. MATERIALS AND METHODS Mice. Specific-pathogen-free female BALB/c mice aged 5 to 6 weeks older were purchased from Harlan (Jerusalem, Israel). Animals were managed under specific-pathogen-free conditions during the experiments. Plasmids. H37Rv DNA was prepared as explained previously (5). pQE70 vector (Qiagen, Hilden, Germany) was used to clone the gene and communicate the recombinant 27-kDa protein in In this system, a histidine tail is definitely added to the C terminus of the indicated protein. The primers that were used to amplify the gene encoding the 27-kDa protein from H37Rv DNA contained gene was termed pRHB27. For DNA vaccination, the gene from your pRHB27 plasmid was cloned into the pcDNA3.1 vector (Invitrogen, San Diego, Calif.) under the control of the CMV promoter to generate the pcIHB27 vector. The pcIHB27 vector was then transfected to the XLI strain and purified using the Endo-free Giga prep kit (Qiagen). In both cases, the gene was cloned with its transmission PRPF38A peptide sequence. Sequence of the 27-kDa clone was confirmed to be right in an automated sequencer (ABI; Perkin-Elmer, Applied Biosystems). Manifestation of the pcIHB27 vector in HeLa cells was tested following transfection with FuGENE 6 (Boehringer Mannheim Corp.) according to the manufacturer’s instructions. The vector pRHB27 was used as a negative control. Isolation of mRNA was performed with the SV total RNA isolation system (Promega), and reverse transcription (RT)-PCR was carried out with the Access RT-PC system (Promega). Purification of the 27-kDa recombinant protein indicated in SG13009 strain harboring the pRHB27 plasmid was cultivated in Luria-Bertani medium comprising kanamycin (10 g/ml) (Sigma Chemical Co.) and ampicillin (100 g/ml) (Biochemie GmbH, Kundl, Austria). At an optical denseness at 600 nm (OD600) of 0.7, the synthesis of the 27-kDa protein was induced by adding a 1 mM concentration of isopropyl–d-thiogalactopyranoside (IPTG) (Ornat, Rehovot, Israel). The incubation continued for 3 h at 37C. Then cells were harvested and lysed, and the recombinant 27-kDa protein was purified using a nickel-nitrilotriacetic acid BMS303141 column (Qiagen, Hilden, Germany). The purified protein was dialyzed against saline and then transferred to a column comprising immobilized polymyxin B (Sigma Chemical Co.) to remove endotoxin. The amount of lipopolysaccharide (LPS) in the protein fraction was measured quantitatively with the amebocyte lysate assay (Biowhittaker, Walkersville, Md.) and found out to be 0.11 endotoxin unit (EU) per g of antigen. The purified protein was analyzed BMS303141 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. Metabolic labeling with [3H]palmitate. strain SG13009 harboring the pRHB27 vector was cultivated in 10 ml of Luria-Bertani medium comprising ampicillin (100 g/ml) and 5 Ci of 27-kDa antigen was separated by SDS-PAGE, and the bands corresponding to the recombinant protein were excised. The BMS303141 mass spectrometry was carried out with Qtof2.