Surface proteins of leptospira
a surface protein and bacteria technology, applied in the field of membrane associated proteins of leptospirosis bacteria, can solve the problems of high mortality rate, failure to thrive, and major problem of leptospirosis
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example 1
[0027] A strain of Leptospira, referred to as “Fiocruz L1-30,” was used. The strain was isolated from a patient with severe leptospirosis, contracted during an epidemic in 1996. See Ko et al., Lancet 354:820-5 (1999), incorporated by reference. Leptospires were detected during dark field microscopy examination of a culture of tween-albumin media that had been inoculated with patient blood, following “Guidelines for the Control of Leptospirosis.”WHO Offset Publ. (1982) incorporated by reference. The strain was identified as Leptospira interrogans, serovar Copenhageni, via biochemical and sera typing analysis. See Ko, et al., supra, Barocchi, et al., J. Clin. Microbiol 39:191-195 (2001). A culture of the organism was then prepared in media with 10% glycerol, and stored at −70° C. Virulence capacity was determined by inoculating 28 day old weaning hamsters. Kidneys were removed from anesthetized animals, approximately 7 days after infection, macerated and were used to inoculate Tween-a...
example 2
[0032] The purified DNA described in example 1 was sequenced, using commercially available reagents, and well known methods.
[0033] Specifically, commercially available products were used to carry out Taq dye deoxy terminator cycle sequencing reactions, using M13 reverse and forward matching primers, which flanked the inserts of the clones. Reaction products were analyzed on a commercially available genetic analyzer.
[0034] There were a total of 289,963 shotgun genomic sequences. Open reading frames were obtained from these, and assembled using phred / phrap software, as described by Ewing, et al., Genome Res 8(3):186-194 (1998) incorporated by reference. The assembly yielded 2,042 contigs. The “Glimmer” program of Delcher, et al., Nucl. Acids Res 27:4636-4641 (1999), incorporated by reference, was applied to the contigs, and yielded 5826 putative open reading frames. Each of these ORFs contained at least 90 base pairs, and overlapped other ORFs by 30 base pairs, or 10% of the ORF siz...
example 3
[0047] The expression vectors resulting from the preceding examples were used to express the relevant proteins. E.coli strains BL21(DE3) or BL21SI were used under inducing conditions, including 1 mM IPTG, or 300 mM NaCl, respectively, using standard methods. Proteins were then analyzed on 10-20% SDS-PAGE gels, under denaturing conditions. Each of FIGS. 1-22 presents an SDS-PAGE pattern for the proteins.
[0048] In addition to the SDS-PAGE work, the proteins were purified, by using Ni2+ chelating sepharose. Either proteins were mixed with charged beads, or were applied onto columns in a balanced salt buffer (0.1M Tris / 0.3 M NaCl, pH 8.0). Any impurities were washed away using the same buffer, with a low concentration of imidazole (20-60 mM). Proteins of interest were then eluted, using a buffer containing imidazole at a concentration of from 0.75 to 10.M.
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