03 Endemic nephropathy [37] †
http://www.ncbi.nlm.nih.gov/protein/. Table 2 List of leptospiral proteins excreted in hamster urine during Leptospira infection Spot no. Accession no.† Locus tag* Protein annotation MW (kDa) pI Predicted location# 32 gi:45599159 LIC10012 conserved hypothetical protein 61792 9.27 Unknown gi:45599713 LIC10580 ABC transporter, atp-binding protein 71297 9.3 Cytoplasmic membrane gi:45601755 LIC12676 conserved hypothetical protein 76551 5.75 Cytoplasm gi:45602095 LIC13023 conserved hypothetical protein 51182 8.23 Cytoplasm gi:45602258 LIC13191 conserved hypothetical protein ISRIB research buy 65453 6.51 Cytoplasm gi:45602297 LIC13229 conserved hypothetical protein 68742 9.21 Unknown gi:45602365 LIC13300 3-hydroxyacyl-CoA dehydrogenase 47865 8.65 Cytoplasm gi:45602427 LIC13362 chloride channel 67352 8.07 Cytoplasmic membrane † http://www.ncbi.nlm.nih.gov/protein/.
* http://aeg.lbi.ic.unicamp.br/world/lic/. #The proteins were predicted with PSORTb (http://www.psort.org/psortb/). Identification of HADH in hamster urine As mentioned Selleck Oligomycin A in the previous section, candidate leptospiral proteins in urine were selected based on the results of LC/MS/MS analysis. In order to identify leptospiral proteins that are excreted in hamster urine during infection, recombinant proteins for each selected protein were made. The proteins were screened by immunoblotting with anti-L. ABT263 interrogans pAb. Among them, only HADH reacted to the antibody. The amino acid sequence of HADH are shown in the Additional file 1: Table S1 and had a coverage of 27%. The rHADH was purified with TALON® Metal Affinity Resin (Clontech) and its expression was confirmed with coomassie brilliant blue (CBB) staining (Figure 4A) and immunoblotting by anti-His Idelalisib chemical structure (C-term)
antibody (Figure 4B). The anti-L. interrogans pAb also recognized the rHADH (Figure 4C). Figure 4 SDS-PAGE and immunoblotting of recombinant leptospiral HADH. (A) The rHADH with His-tag was produced by E. coli and purified by cobalt resin. In total, 1 μg of the protein was run by SDS-PAGE and CBB staining. (B) Anti-His-tag antibody and (C) anti-L. interrogans pAb detected the protein. Sharp signs indicate recombinant protein bands of 52 kDa. These experiments were repeated three times, and the representative data are shown in this figure. Detection of HADH in infected hamster urine with antiserum We produced anti-rHADH antiserum in rabbits, and examined its reactivity to rHADH by immunoblotting. The rabbit antiserum recognized the recombinant protein (data not shown). We then performed immunoblotting of urine samples as in Figure 2B and the antiserum reacted with the post-infection samples (Figure 5A). The reacted protein increased after the seventh day of infection (Figure 5B). The protein was found to be excreted in the urine before leptospires were shed (Figure 1A). Figure 5 Immunoblotting of infected hamster urine by anti-HADH antisera.