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All spectra were searched against Spectra were analyzed with TurboSEQUEST, version 3.1 (28). All spectra were searched against a database of deduced open reading frames (ORFs) of the B. hermsii genome, including the chromosome (GenBank accession number CP00048), plasmid sequences reported previously by Dai et al. (29), and the 174-kb large plasmid reported previously by Campeau Miller et al. (GenBank accession number HM008709) (16). DNA extractions and PCR. Borrelia genomic DNA was isolated from harvested cultures, blood, or tissue with a Qiagen (Valencia, CA) DNeasy Blood and Tissue kit, as described previously (30). The DNA target was amplified with Phusion high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA). For the partial or full-length bha007 sequence, the forward and reverse primers were 5=-ATGCAATTGAAAAAACAATGTT T-3= and 5=-CCTATGCCTGTTCTTTTTGC-3= or 5=-ATGCAATTGAAA AAACAATG-3= and 5=-TTATTTTAAGGCTTTCTTGAT-3=, respectively. The PCR conditions were as follows: 3 min at 95 followed by 30 cycles of 1 min at 95 , 1 min at 54 (partial) or 56 (full length), and 1 min at 75 and then a final extension step for 7 min at 75 . PCR products or cloned inserts in plasmid vector pCR2.1 in E. coli Top10F cells were sequenced over both strands by Genewiz (South Plainfield, NJ). Quantitative PCR using a Rotorgene 6000 thermal cycler (Qiagen, Valencia, CA) and primers and probe for the 16S rRNA gene of B. hermsii was performed as previously described (30). PCR results were normalized for total DNA concentrations of the samples; a positive result was 5 gene copies per g tissue DNA. Recombinant protein. The protein-encoding bha007 sequence was modified by the replacement of the signal peptide with a histidine tag at the N terminus and substitution of E. coli codon usage. Gene synthesis, protein expression in E. coli, and protein purification of the Borrelia recombinant BHA007 protein were performed by GenScript (Piscataway, NJ). Protein purity was 70 , as estimated by using a Coomassie bluestained PAGE gel. Recombinant Vlp7, FlaB, and factor H-binding protein A (FhbA) of B. hermsii strain HS1 were cloned with a replacement of their signal peptide by a 6 histidine tag, expressed in E. coli, and purified as described previously (31). Recombinant protein purity was estimated to be 80 to 90 . Protein concentrations were determined by a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Microagglutination assay. Serum or antibody preparations were 2-fold serially diluted 1:5 in BSK II medium, with a final spirochete cell density of 108 spirochetes/ml. After incubation for 1 h at 37 , 10 l of the suspension was viewed under a coverslip by phase-contrast microscopy at a 400 magnification. A positive agglutination reaction at a given dilution was 50 of the cells in the suspension in aggregates of 2 cells; the titer was the highest dilution of serum with a positive reaction. Monoclonal antibody H5332 for OspA (32) was a positive control for the agglutination of B. burgdorferi, and monoclonal antibody H4825 (see above) was a positive control for the agglutination of Vtp B. hermsii. Immunofluorescence assays. Thin smears on glass slides of blood from B. hermsii-infected mice were fixed in methanol, dried, and then blocked for 1 h at 22 with PBS plus 2 bovine serum albumin (BSA) and 0.05 Triton X-100.