WO2007113009A2 - Polynucléotides et polypeptides destinés à la détection de rickettsia helvetica - Google Patents

Polynucléotides et polypeptides destinés à la détection de rickettsia helvetica Download PDF

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WO2007113009A2
WO2007113009A2 PCT/EP2007/003062 EP2007003062W WO2007113009A2 WO 2007113009 A2 WO2007113009 A2 WO 2007113009A2 EP 2007003062 W EP2007003062 W EP 2007003062W WO 2007113009 A2 WO2007113009 A2 WO 2007113009A2
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amino acid
seq
polynucleotide
sequence
antigenic fragment
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WO2007113009A3 (fr
WO2007113009A8 (fr
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Marinko Dobec
Martin Sievers
Tobias Wermelinger
Cyril Tanner
Andreas Steffen Stein
Franz KÄPPELI
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Medica Medizinische Laboratorien Drf Kappeli AG
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Medica Medizinische Laboratorien Drf Kappeli AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)

Definitions

  • the present invention relates to newly identified polynucleotides and polypeptides encoded by such polynucleotides for detecting and/or monitoring the presence of Rickettsia helvetica. More particularly, the present invention relates to polynucleotides encoding an OmpA polypeptide of Rickettsia helvetica and antigenic fragments thereof and the respective polypeptides and antigenic fragments as well as to methods for detecting and/or monitoring the presence of Rickettsia helvetica in a subject and respective kits.
  • Rickettsiae are well known gram-negative pathogenic bacteria.
  • the genus Rickettsia is considered to consist of three groups of strictly intracellular bacteria, namely the typhus (TG), the spotted fever (SFG) and the scrub typhus (STG) groups.
  • the SFG Rickettsiae are mainly transmitted by ticks and at least 17 different species of SFG have been described so far (Bouyer et al., 2001, Int. J. Syst. Evol. Microbiol. 51, 339-347).
  • the species definition of SFG has been based on their serotype as determined by e. g. microimmunofluorescence test (MIF) and cross-adsorption.
  • MIF microimmunofluorescence test
  • Rickettsia helvetica which belongs to the SFG group, has been isolated from the tick Ixodes ricinus collected in Switzerland, France, Slovenia and Sweden (Fournier et al., 2000, Emerging Infectious
  • WO 99/42479 describes the use of an antigenic amino acid sequence from the 17 kDa outer membrane protein of R. helvetica for serological testing of an infected patient. Unfortunatley, the 17 kDa outer membrane protein is very conserved among Rickettsiae and the sequence used by the applicant of WO 99/42479, has 100 % identity to the corresponding sequence of the 17 kDa outer membrane protein of R. sibirica (Genbank Accession number: AAO38436), R. conorii (Genbank Accession number: AAL03825), R. japonica (Genbank Accession number: Q52764) and R. rickettsii (Genbank Accession number: ABA01551), which makes it difficult to discriminate R.
  • other proteins and/or the respective genes have been used to identify Rickettsiae using serological testing or PCR amplification such as 16S rRNA, citrate synthase, the envelope proteins OmpA and OmpB (for review see La Scola and Raoult, 1997, J. Clin. Microbiol., 35, 2715-2727) or the PS120 protein encoding gene D as described by Sekeyova et al., 2001, Int. J. Syst. Evol. Microbiol. 51, 1353-1360.
  • helvetica is a frequent human pathogen as can be seen from the occurence of R. helvetica infections described above, there is a need of the identification of new tools and methods which allow for highly specific detection of R. helvetica and reliable discrimination of this pathogenic bacteria from other Rickettsiae of the SFG group.
  • the object of the present invention is therefore to provide new tools and methods which allow for highly specific detection of R. helvetica using serological as well as polynucleotide amplification procedures and which reliably discriminate this pathogen bacteria from other Rickettsiae.
  • This object and other objects as will be apparent from the following description have been achieved by providing new polynucleotides, polypeptides and antigenic fragments thereof and new primers and probes which can be used for detecting and/or monitoring the presence of Rickettsia helvetica in a subject.
  • R. helvetica harbours a gene encoding an OmpA polypeptide which has not been identified yet and which contains various antigenic fragments specific for R.
  • the present invention provides a polynucleotide encoding an OmpA polypeptide of Rickettsia helvetica selected from the group consisting of: a) polynucleotides encoding the OmpA polypeptide of Rickettsia helvetica comprising the amino acid sequence as shown in SEQ ID NO: 1 ; b) polynucleotides comprising the nucleotide sequence as shown in SEQ ID NO: 2; c) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polynucleotide as defined in (a) or (b); d) polynucleotides which are at least 80 % identical to a polynucleotide as defined in (a) or (b).
  • polynucleotide encoding an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica selected from the group consisting of: a) polynucleotides encoding an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica comprising at least 5 consecutive amino acids located from amino acid 1 to amino acid 800 of the amino acid sequence as shown in SEQ ID NO: 1; b) polynucleotides comprising a nucleotide sequence selected from SEQ ID NOS: 3-17; c) polynucleotides which are at least 60 % identical to a polynucleotide comprising a nucleotide sequence selected from SEQ ID NOS: 3-17. d) polynucleotides which are at least 90 % identical to a polynucleotide comprising a nucleotide sequence selected from SEQ ID NOS: 3-17.
  • an OmpA polypeptide of Rickettsia helvetica having the amino acid sequence encoded by the polynucleotide encoding an OmpA polypeptide of Rickettsia helvetica as defined above as well as an OmpA polypeptide of Rickettsia helvetica selected from the group consisting of: a) polypeptides comprising the amino acid sequence as shown in SEQ ID NO: 1; b) polypeptides comprising an amino acid sequence which is at least 80 % identical in amino acid sequence to the amino acid sequence as defined in (a).
  • an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica having the amino acid sequence encoded by the polynucleotide encoding an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica as defined above and an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica selected from the group consisting of a) an antigenic fragment comprising at least 5 consecutive amino acids located from amino acid 1 to amino acid 800 of the amino acid sequence as shown in SEQ ID NO: 1; b) an antigenic fragment comprising an amino acid sequence selected from SEQ ID NOS: 18- 32; c) an antigenic fragment comprising an amino acid sequence which is at least 60 % identical to an amino acid sequence selected from SEQ ID NOS: 18-32; d) an antigenic fragment comprising an amino acid sequence which is at least 80 % identical to an amino acid sequence selected from SEQ ID NOS: 18-32; e) an antigenic
  • a further object is to provide an antibody against the polynucleotides, the OmpA polypeptides or the antigenic fragments of the present invention as defined above, for detecting and/or monitoring the presence of Rickettsia helvetica in a subject as well as a method for detecting and/or monitoring the presence of Rickettsia helvetica in a subject, comprising assaying a biological fluid of the subject with a compound selected from the polynucleotides, the polypeptides, the antigenic fragments and the antibody as well as with the primers and/or probes of the present invention and a respective diagnostic kit.
  • a vector comprising at least one copy of the polynucleotides of the present invention as well as a prokaryotic host cell transformed with such polynucleotides and/or such vector as well as primers for amplifying DNA encoding an OmpA polypeptide of Rickettsia helvetica.
  • polypeptide As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.
  • polypeptide As used herein, the terms “polypeptide”, “peptide”, “protein”, “polypeptidic” and “peptidic” are used interchangeably to designate a series of amino acid sequence connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent sequence.
  • Antigenic fragments of amino acids sequences or “antigenic fragments of a polypeptide” or “antigenic fragments thereof refer to an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the respective sequence of the polypeptide from which they derive and which has antigenic properties.
  • An antigenic fragment will generally include at least about 5, preferably between 5 and 100, most preferably between 10 and 50, in particular between 10 and 30 or more consecutive amino acid residues of the full-length molecule, that comprise an epitope, provided that the fragment in question retains immunoreactivity in the assays described herein, i.e. the fragment binds an antibody produced in a subject in response to the presence of R. helvetica.
  • epitope refers to a sequence of at least about 3 to 5, preferably about 5 to 10 or 15 amino acids, which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence.
  • length of the fragment which may comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes.
  • An epitope for use in the present invention is not limited to a polypeptide having the exact sequence of the portion of the parent protein from which it is derived.
  • Useful antigenic fragments are designed using computer-assisted analysis of amino acid sequences which identify probable surface exposed, antigenic regions.
  • Species specific antigenicity refers to the antigenicity of an antigenic fragment of a polypeptide which is specific to a particular bacteria species such that the polypetide or the antigenic fragment does not cross-react with other species of the same genus of the bacteria, i. e. shows no genus specific antigenicity.
  • Species specific antigenicity can be determined e. g. after cross-absorption by a number of methods, including e. g. Western blot, ELISA (enzyme- linked immunosorbent assays, equivalent used herein to Enzyme Immunoassay (EIA)) or MIF (Micro-Immuno fluorescence).
  • antigenic fragments used in the methods of the present invention are selected as to show species specific antigenicity, i. e. the antigenic fragment is species specific to R. helvetica.
  • Conservative amino acid substitutions are herein defined as exchanges for example within one of the following five groups:
  • stringent conditions means hybridization will occur between nucleotide sequences only if there is at least 95% and preferably at least 97% identity between the sequences.
  • a “vector” a “vector expressible in a host” or “expression vector” is a polynucleotide construct, generated recombinantly or synthetically, with a series of specified polynucleotide elements that permit transcription of a particular nucleotide sequence in a host cell.
  • this vector includes a transcriptional unit comprising a particular nucleotide sequence to be transcribed operably linked to a promoter.
  • a vector expressible in a host can be e. g. an autonomously or self-replicating plasmid, a cosmid or a phage.
  • host refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • identity refers to the number of exactly matching residues (expressed as a percentage) in a sequence alignment between the template sequence and the sequence relative. Identity of polynucleotide sequences or amino acid sequences can be measured using sequence analysis software known to the person skilled in the art such as e. g. the BLAST program of the National Center for Biotechnology Information, Bethesda, USA (http://www.ncbi.nlm.nih.gov).
  • cell transfected or “cell transformed” or “transfected/transformed cell” means the cell into which an extracellular nucleotide sequence has been introduced and thus harbors the extracellular nucleotide sequence.
  • the nucleotide sequence might be introduced into the cell so that the nucleotide is replicable either as a chromosomal integrant or as an extra chromosomal element.
  • Antibody refers to a class of plasma proteins produced by the B-cells of the immune system after stimulation by an antigen.
  • Mammal (i.e. human) antibodies are immunoglobulins of the Ig G, M, A, E or D isotype.
  • the term "antibody” as used for the purposes of this invention includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, anti-idiotypic antibodies, as well as chimeric antibodies.
  • the term antibody is used to mean whole antibodies and binding fragments thereof. Binding fragments include single chain fragments, Fv fragments and Fab fragments.
  • an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, addition or repeat of one or more amino acids that does not alter the biological function of the polypeptide.
  • biological function is meant the function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells.
  • biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium.
  • Biological function is distinct from antigenic property.
  • a polypeptide can have more than one biological function.
  • Allelic variants are very common in nature. For example, a bacterial species is usually represented by a variety of strains that differ from each other by minor allelic variations.
  • a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence (and polynucleotide sequence) that is not identical in each of the strains as can be seen e. g. from Ryffel et al. (Ryffel et al., 2003, BMC Infectious Diseases, 3:14) who demonstrated that the average identity of the deduced amino acid sequences of the OspA proteins from different strains of Borrelia valaisiana is about 69,9 %.
  • polypeptides of the present invention include naturally-occurring allelic variants, including mutants or any other non-naturally occurring variants or fragments thereof that retain the inherent characteristics of the respective polypeptide such as species specific antigenicity of at least one antigenic fragment of the polypeptide.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA, preferably it is in the form of DNA.
  • the DNA may be doublestranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the polynucleotide of the present invention may encode for a polypeptide, or for a polypeptide having a signal sequence.
  • the polypeptides, their antigenic fragments or variants thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • nucleotide sequence of polynucleotides as defined herein encoding a polypeptide of Rickettsia helvetica or an antigenic fragment of that polypeptide may be as well a different sequence which sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide or the same antigenic fragment of that polypeptide.
  • the polynucleotides and polypeptides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the present invention provides a polynucleotide encoding an OmpA polypeptide of Rickettsia helvetica selected from the group consisting of: a) polynucleotides encoding the OmpA polypeptide of Rickettsia helvetica comprising the amino acid sequence as shown in SEQ DD NO: 1; b) polynucleotides comprising the nucleotide sequence as shown in SEQ ID NO: 2; c) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polynucleotide as defined in (a) or (b); d) polynucleotides which are at least 80 % identical to a polynucleotide as defined in (a) or (b).
  • Polynucleotides as defined in c) the complementary strand of which hybridizes under stringent conditions to a polynucleotide as defined in (a) or (b) are polynucleotides of which hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • Polynucleotides as defined in d) are preferably at least 85 %, more preferably at least 90 %, most preferably at least 95 %, in particular at least 97 %, most particular at least 99 % identical to a polynucleotide as defined in (a) or (b).
  • the invention provides a polynucleotide encoding an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica selected from the group consisting of: a) polynucleotides encoding an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica comprising at least 5 consecutive amino acids located from amino acid 1 to amino acid 800 of the amino acid sequence as shown in SEQ ID NO: 1; b) polynucleotides comprising a nucleotide sequence selected from SEQ ID NOS: 3-17; c) polynucleotides which are at least 60 % identical to a polynucleotide comprising a nucleotide sequence selected from SEQ ID NOS: 3-17. d) polynucleotides which are at least 90 % identical to a polynucleotide comprising a nucleotide sequence selected from SEQ ID NOS: 3-17.
  • polynucleotides as defined in a) encode an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica which comprises at least 10 consecutive amino acids located from amino acid 1 to amino acid 800 of the amino acid sequence as shown in SEQ ID NO: 1. More preferably, polynucleotides as defined in a) encode an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica which comprises between 5 and 100, most preferably between 10 and 50, in particular between 10 and 30 consecutive amino acids located from amino acid 1 to amino acid 800 of the amino acid sequence as shown in SEQ ED NO: 1.
  • polynucleotides as defined in a) encode an antigenic fragment of an Omp A polypeptide of Rickettsia helvetica comprising at least 5, usually at least 10, preferably between 5 and 100, most preferably between 10 and 50, in particular between 10 and 30 consecutive amino acids, located from amino acid 1 to amino acid 150 and/or from amino acid 151 to amino acid 350 and/or from amino acid 351 to amino acid 550 and/or from amino acid 551 to amino acid 800 of the amino acid sequence as shown in SEQ ID No 1.
  • polynucleotides as defined in a) encode an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica comprising at least 5, usually at least 10, preferably between 5 and 100, most preferably between 10 and 50, in particular between 10 and 30 consecutive amino acids consecutive amino acids located from about amino acid 49 to about amino acid 100 and/or from about amino acid 173 to about amino acid 309 and/or from about amino acid 393 to about amino acid 521 and/or from about amino acid 597 to about amino acid 776 of the amino acid sequence as shown in SEQ ID No 1.
  • polynucleotides as defined in a) which encode an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica comprising at least 5, preferably at least 10 consecutive amino acids located from about amino acid 87 to about amino acid 100 and/or from about amino acid 194 to about amino acid 218 and/or from about amino acid 295 to about amino acid 309 and/or from about amino acid 406 to about amino acid 420 and/or from about amino acid 494 to about amino acid 518 and/or from about amino acid 507 to about amino acid 521 and/or from about amino acid 657 to about amino acid 675 and/or from about amino acid 697 to about amino acid 711 and/or from about amino acid 758 to about amino acid 776 of the amino acid sequence as shown in SEQ ID No 1.
  • the preferred polynucleotides of the polynucleotides as defined in b) comprise a nucleotide sequence selected from SEQ ID NOS: 3-13, in particular the nucleotide sequences which are identical to each other but are located on different positions of the polynucleotide such as SEQ ID NOS: 3 and 4 and SEQ ID NOS: 9 and 10, respectively. More preferred polynucleotides of the polynucleotides as defined in b) comprise a nucleotide sequence selected from SEQ ID NOS: 3-11. More particular preferred polynucleotides of the polynucleotides as defined in b) comprise a nucleotide sequence selected from SEQ ID NOS: 3-8. Most particular preferred polynucleotides of the polynucleotides as defined in b) comprise a nucleotide sequence selected from SEQ ID NOS: 3, 4, 6, 8 and 11.
  • Polynucleotides as defined in c) are preferably at least 70 %, more preferably at least 80 % most preferably at least 90 %, in particular at least 95 % identical to a polynucleotide as defined in (b), whereas the nucleotide sequence is preferably selected from SEQ ID NOS: 3, 4, 6, 8, 10, 11, 12, 14, and 17 .
  • Polynucleotides as defined in d) are preferably at least 95 % identical to a polynucleotide as defined in (b), whereas the nucleotide sequence is preferably selected from SEQ ID NOS: 5, 7, 9, 13, 15, and 16.
  • Polynucleotides of R. helvetica are usually found in ticks, in particular in Ixodes ricinus ticks.
  • the polynucleotides of this invention were isolated from Ixodes ricinus ticks.
  • the ticks are collected e. g. using the close dragging method and DNA is isolated from the collected ticks by methods known to the person skilled in the art.
  • the identification of the presence of R. helvetica from the collected ticks can be performed by known methods such as e. g. PCR using the gene coding for the intracytoplasmatic PS 120 protein as described in Sekeyova et al., 2001, Int. J. Syst. Evol. Microbiol. 51, 1353-1360
  • the present invention provides an OmpA polypeptide of Rickettsia helvetica having the amino acid sequence encoded by the polynucleotide encoding an OmpA polypeptide of Rickettsia helvetica as defined above.
  • a polypeptide is at least 80 %, preferably at least 85 %, and more preferably at least 90, most preferably at least 95 %, in particular at least 97 %, most particular at least 99 % identical to the amino acid shown in SEQ ID NO: 1.
  • the present invention provides as well an OmpA polypeptide of Rickettsia helvetica selected from the group consisting of a) polypeptides comprising the amino acid sequence as shown in SEQ ID NO: 1 ; b) polypeptides comprising an amino acid sequence which is at least 80 % identical in amino acid sequence to the amino acid sequence as defined in (a).
  • Polypeptides as defined in b) comprise preferably an amino acid sequence which is at least 85 %, more preferably at least 90%, most preferably at least 95%, in particular at least 97 %, most particular at least 99 % identical in amino acid sequence to the amino acid sequence as defined in (a) and that preferably differs from the sequence of reference by conservative amino acid substitutions which do not destroy the species specific antigenicity of the polypeptide.
  • An OmpA polypeptide of Rickettsia helvetica provided by the present invention include naturally-occurring allelic variants, including mutants or any other non-naturally occurring variants or fragments thereof that retain the inherent characteristics of the OmpA polypeptide of Rickettsia helvetica such as species specific antigenicity of at least one antigenic fragment of the polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 1.
  • a further object of the present invention is to provide antigenic fragment of an OmpA polypeptide of Rickettsia helvetica selected from the group consisting of a) an antigenic fragment comprising at least 5 consecutive amino acids located from amino acid 1 to amino acid 800 of the amino acid sequence as shown in SEQ ID NO: 1; b) an antigenic fragment comprising an amino acid sequence selected from SEQ ID NOS: 18- 32; c) an antigenic fragment comprising an amino acid sequence which is at least 60 % identical to an amino acid sequence selected from SEQ ID NOS: 18-32; d) an antigenic fragment comprising an amino acid sequence which is at least 80 % identical to an amino acid sequence selected from SEQ ID NOS: 18-32; e) an antigenic fragment consisting of at least 5 consecutive amino acids of an amino acid sequence selected from SEQ ID NOS: 18-32.
  • An antigenic fragment as defined in a) comprises preferably at least 10 consecutive amino acids located from amino acid 1 to amino acid 800 of the amino acid sequence as shown in SEQ ID NO: 1. More preferably, an antigenic fragment as defined in a) comprises between 5 and 100, most preferably between 10 and 50, in particular between 10 and 30 consecutive amino acids located from amino acid 1 to amino acid 800 of the amino acid sequence as shown in SEQ BD NO: 1.
  • an antigenic fragment as defined in a) comprises at least 5, usually at least 10, preferably between 5 and 100, most preferably between 10 and 50, in particular between 10 and 30 consecutive amino acids located from amino acid 1 to amino acid 150 and/or from amino acid 151 to amino acid 350 and/or from amino acid 351 to amino acid 550 and/or from amino acid 551 to amino acid 800 of the amino acid sequence as shown in SEQ ID No 1.
  • an antigenic fragment as defined in a) comprises at least 5, usually at least 10, preferably between 5 and 100, most preferably between 10 and 50, in particular between 10 and 30 consecutive amino acids located from about amino acid 49 to about amino acid 100 and/or from about amino acid 173 to about amino acid 309 and/or from about amino acid 393 to about amino acid 521 and/or from about amino acid 597 to about amino acid 776 of the amino acid sequence as shown in SEQ ID NO: 1.
  • an antigenic fragment as defined in a) which comprises at least 5, preferably at least 10 consecutive amino acids located from about amino acid 87 to about amino acid 100 and/or from about amino acid 194 to about amino acid 218 and/or from about amino acid 295 to about amino acid 309 and/or from about amino acid 406 to about amino acid 420 and/or from about amino acid 494 to about amino acid 518 and/or from about amino acid 507 to about amino acid 521 and/or from about amino acid 657 to about amino acid 675 and/or from about amino acid 697 to about amino acid 711 and/or from about amino acid 758 to about amino acid 776 of the amino acid sequence as shown in SEQ ID No 1.
  • Preferred antigenic fragments as defined in b) comprise an amino acid sequence selected from SEQ ID NOS: 18-28 in particular the amino acid sequences which are identical to each other but are located on different positions of the polypeptide such as SEQ ID NOS: 18 and 19 and SEQ ID NOS: 24 and 25, respectively. More particular preferred antigenic fragments as defined in b) comprise an amino acid sequence selected from SEQ ID NOS: 18-27 and 30-32.
  • Antigenic fragments as defined in c) are preferably at least 70 %, more preferably at least 80 %, most preferably at least 90 %, in particular at least 95 % identical to the amino acid sequences as defined in (b), whereas the amino acid sequence is preferably selected from SEQ ED NOS: 18-27 and 30-32.
  • Antigenic fragments as defined in d) are preferably at least 90 %, in particular at least 95 % identical to the amino acid sequences as defined in (b), whereas the amino acid sequence is preferably selected from SEQ ID NOS: 28, 29. Most particular preferred antigenic fragments as defined in b), c), d) and e), comprise antigenic fragments, whereas the amino acid sequence is selected from SEQ ID NOS: 18, 19, 21, 23 and 26.
  • the antigenic fragment as defined in (e), consists preferably of at least 10 consecutive amino acids of an amino acid sequence selected from SEQ ID NOS: 18-32
  • the antigenic fragments of an OmpA polypeptide of Rickettsia helvetica of the present inention are selected, such as that they retain their inherent characteristics such as species specific antigenicity.
  • the antigenic fragment may recombinantly produced or may be produced by chemical synthesis.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide.
  • the polypeptide of the present invention is preferably a recombinant polypeptide produced by using a polynucleotide of the present invention.
  • the polypeptide may comprise additional amino acids which are fused to the polypeptide, such as a signal sequence or a sequence which is employed for purification of the polypeptide.
  • the present invention further provides an antibody against the polynucleotides, the OmpA polypeptides or the antigenic fragments thereof as defined above for detecting and/or monitoring the presence of Rickettsia helvetica in a subject.
  • the preferred antibody of the present invention is an antibody against the OmpA polypeptides or the antigenic fragments as defined above.
  • Antibodies of the present invention can be polyclonal antibodies, monoclonal antibodies, anti- idiotypic antibodies, as well as chimeric antibodies, preferably monoclonal antibodies.
  • Antibodies generated against the polypeptides and the antigenic fragments of the present invention can be obtained by direct injection of the polypeptides or the antigenic fragments into an animal or by administering them to an animal, preferably a nonhuman. In this manner, even a sequence encoding an antigenic fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to detect or to isolate the polypeptide or its antigenic fragments from tissue or a biological fluid containing that polypeptide.
  • a more preferred antibody of the present invention is a monoclonal antibody against an antigenic fragment of an OmpA polypeptide of the present invention, in particular a monoclonal antibody against an antigenic fragment comprising an amino acid sequence selected from SEQ ID NOS: 18-32.
  • any technique which provides antibodies produced by continuous cell line cultures can be used.
  • Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBVhybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • a further aspect of the present invention is to provide a method for detecting and/or monitoring the presence of Rickettsia helvetica in a subject, comprising assaying a biological fluid of the subject with a compound selected from a) a polynucleotide encoding an OmpA polypeptide of Rickettsia helvetica as defined above; b) a polynucleotide encoding an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica as defined above; c) an OmpA polypeptide of Rickettsia helvetica as defined above; d) an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica as defined above; e) an antibody against the polynucleotides, the OmpA polypeptides or the antigenic fragments of an OmpA polypeptide of Rickettsia helvetica as defined above.
  • Procedures for detecting and/or monitoring the presence of Rickettsia helvetica in a subject according to the method of the invention by assaying a biological fluid of a subject may be conventional, and will admit of design and selection by the skilled person who understands the present disclosure, mindful of well accepted principles of diagnostic and immunodiagnostic methodology and interpretation. Such principles are set forth, for example, in Harlow, E., and Lane, D., "Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988), Rose, Noel R., et al., eds., Manual of Clinical Laboratory
  • Typical examples of assays which can utilize the polynucleotides of the invention are polynucleotide amplification procedures which permit amplification of a target polynucleotide such as polymerase chain reaction (PCR), nested PCR or real time PCR.
  • the polynucleotides of the invention can be used as a basis to select probes and/or primers for amplification which are specific for a target DNA of R. helevtica encoding an OmpA polypeptide or an antigenic fragment of an OmpA polypeptide.
  • Particluar useful to amplify DNA of R. helevtica are the primers comprising the sequences SEQ ID NO: 33 (forward primer) and SEQ ID NO: 34 (reverse primer), which are encompassed by the present invention.
  • Typical examples of assays which can utilize the polypeptides, the antigenic fragments thereof and the respective antibodies of the invention are serological assays like enzyme- linked immunosorbent assays (ELISA), immunofluorescence assays (IFA), Western immunoblot assays, cross-absorption assays.
  • serological assays like enzyme- linked immunosorbent assays (ELISA), immunofluorescence assays (IFA), Western immunoblot assays, cross-absorption assays.
  • the types of assays which can be used by practising the method of the present invention and which can be incorporated in kit form are many, and include, for example, indirect assays or direct assays, with indirect assays presently preferred as well as competitive and non-competitive assays, with non-competitive assays presently preferred.
  • These kinds of assays comprise as well as immunometric or sandwich immunoassays.
  • immunometric assay or "sandwich immunoassay,” it is meant to
  • the polypeptides, the antigenic fragments thereof and the respective antibodies of the invention will be useful in other variations and forms of assays which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.
  • the method is a serological assay selected from enzyme- linked immunosorbent assays (ELISA), immunofluorescence assays (IFA), Western immunoblot assays and cross-absorption assays and, wherein the biological fluid of the subject is assayed with a compound selected from the polypeptides, the antigenic fragments thereof and the respective antibodies of the invention as defined above.
  • the serological assay is an ELISA and the compound used is an antigenic fragment of the present invention.
  • Antibodies produced in relation to the presence of Rickettsia helvetica in a subject which can be detected and/or monitored by the method of the invention are of the isotypes IgG, IgA and IgM.
  • As biological fluid usually diluted serum is used, wheras serum samples are diluted between 1:5 and 1:1000, preferably between 1:10 and 1:500, more preferably between 1:10 and 1 : 100.
  • Assays of this type used for monitoring and/or detecting Rickettsiae are summarised e. g. in La Scola and Raoult, 1997, J. Clin.
  • the polypeptides, the antigenic fragments thereof and the respective antibodies of the invention can be either covalently or physically bound to that solid support, by techniques such as covalent bonding via an amide, ester or disulfide linkage, or by adsorption.
  • suitable solid support immunoadsorbents and methods for immobilizing polypeptides, antigenic fragments thereof and antibodies thereon can be bound directly to the solid support or can be bound via a binding pair such as the biotin/streptavidin system.
  • antigenic fragments they are preferably biotin labelled, whereas a spacer, such as amino hexanoic acid, might be introduced between the fragment and biotin.
  • a spacer such as amino hexanoic acid
  • solid supports which can be used as immunoadsorbents for the method and the kit of the present invention.
  • Well known materials which may be employed include glass, polystyrene, polypropylenes dextran, nylon, agarose, dextran, acrylamide, Nitrocellulose, PVDF and other materials, in the form of tubes, beads, membranes and microtiter plates formed from or coated with such materials, and the like.
  • Another object of the present invention is to provide a diagnostic kit for detecting and/or monitoring the presence of Rickettsia helvetica in a subject, comprising a compound selected from a) a polynucleotide encoding an OmpA polypeptide of Rickettsia helvetica as defined above; b) a polynucleotide encoding antigenic fragment of an OmpA polypeptide of Rickettsia helvetica as defined above; c) an OmpA polypeptide of Rickettsia helvetica as defined above; d) an antigenic fragment of an OmpA polypeptide of Rickettsia helvetica as defined above; e) an antibody against the polynucleotides, the OmpA polypeptides or the antigenic fragments of an OmpA polypeptide of Rickettsia helvetica as defined above; optionally with reagents and/or instructions for
  • kit may comprise further labels and labelled compounds known to the skilled person.
  • a kit may comprise as well a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as plates, vials, tubes and the like, each of said container means comprising the separate elements of the assay to be used.
  • the kit of the present invention may further comprise reagents for carrying out an assay as described above.
  • the kit includes reagents for carrying out an ELISA, more preferably a noncompetitive ELISA, most preferably an indirect non-competitive ELISA.
  • Such reagents are well known to the person skilled in the art and comprise e. g. Immobilization-buffer, Blocking-buffer, Incubation-buffer, Wash-buffer, Substrate-buffer and/or Secondary- Antibody-Conjugate.
  • the subject as referred herein is usually a human or an animal, preferably a human.
  • the method as well as the kit can be applied to a biological fluid of a subject such as serum or endothelial cells of bloss vessels.
  • the biological fluid is obtained as a sample from the subj ect and assayed in vitro.
  • the polynucleotides of the present invention are usually located on a vector. As well encompassed by the present invention is therefore a vector comprising at least one copy of the polynucleotides of the present invention.
  • the vector according to the invention is preferably an autonomously or self-replicating plasmid, a cosmid or a phage.
  • a wide variety of host/vector combinations may be employed in expressing the polynucleotides of this invention.
  • Useful vectors for example, may consist of segments of chromosomal, non-chromosomal and/or synthetic nucleotide sequences. Suitable vectors include vectors with specific host range such as vectors specific for e. g. E.
  • a useful vector for expression in E. coli is e. g. pBAD/HisB (Invitrogen) which is preferred. Further useful plasmids are well known to the person skilled in the art and are described e.g. in "Cloning Vectors" (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985).
  • a vector provided herewith which contains SEQ ID NO: 2 is pUC(OmpA).
  • the polypetides or the antigenic fragments of the present invention can be produced in various prokaryotic or eukaryotic host cells which can be transformed with the respective polynulceotides.
  • a further object of the present invention is a prokaryotic or eukaryotic host cell transformed with the polynucleotides and/or the vector of the present invention. Transformation of appropriate host cells with a vector comprising a polynucleotide according to the invention is accomplished by well known methods that typically depend on the type of vector used. With regard to these methods, see for example, Maniatis et al. 1982, Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory and commercially available methods.
  • prokaryotic host cells are useful in expressing the nucleotide sequences of this invention. These hosts may include strains of gram-negative cells such as E. coli and Pseudomonas, or gram positive cells such as Bacillus.
  • Useful eukaryotic host cells are e. g. insect cells which can be used together with a baculovirus expression system.
  • the host cell is a prokaryotic host cell, more preferably a gram-negative cell, most preferably an E. coli cell.
  • a further object of the present invention are the above mentioned primers for amplifying DNA encoding an OmpA polypeptide of Rickettsia helevtica selected from the group consisting of: a) a primer comprising the sequence SEQ ID NO: 33, an oligonucleotide complementary to SEQ ID NO: 33, an oligonucleotide consisting of 10- 50, preferably 15-30, nucleotides in length which hybridizes to SEQ ID NO: 33 or its complement under stringent conditions, an oligonucleotide which is at least 60 %, preferably at least 70 %, more preferably at least 80 %, most preferably at least 90 % in particular at least 95 %, identical to the polynucleotide comprising the nucleotide sequences SEQ ED NO: 33; b) a primer comprising the sequence SEQ ID NO: 34, an oligonucleotide complementary to SEQ ID NO: 34, an oligonucleotide consisting of
  • primers and/or probes for amplifying a target DNA sequence of the OmpB gene of Rickettsia helevtica selected from the group consisting of: a) a primer comprising the sequence SEQ ED NO: 35, an oligonucleotide complementary to SEQ ED NO: 35, an oligonucleotide consisting of 10- 50 nucleotides, preferably 15-30, in length which hybridizes to SEQ ED NO: 35 or its complement under stringent conditions, an oligonucleotide which is at least 60 %, preferably at least 70 %, more preferably at least 80 %, most preferably at least 90 % in particular at least 95 %, identical to the polynucleotide comprising the nucleotide sequences SEQ ID NO: 35; b) a primer comprising the sequence SEQ ID NO: 36, an oligonucleotide complementary to SEQ DD NO: 36, an oligonucle
  • primers and/or probes for amplifying a target DNA sequence of the OmpB gene of Rickettsia helevtica database sequences such as GenBank or EMBL, fragments described in the literature or isolated fragments of the respective microorganism can be used.
  • the primers and/or probes for amplifying a target DNA sequence of the OmpB gene of Rickettsia helevtica as defined above allow species-specific detection and/or monitoring of R. helvetica.
  • the present invention thus further comprises a method for detecting and/or monitoring the presence of Rickettsia helvetica in a subject, comprising assaying a biological fluid of the subject with primers and/or probes for amplifying a target DNA sequence of the OmpB gene as defined above.
  • assays which can utilize the primers and probes are polynucleotide amplification procedures which permit amplification of a target polynucleotide such as polymerase chain reaction (PCR), nested PCR or real time PCR, which are preferred.
  • PCR methods which permit amplification of a target DNA sequence are well known to the person skilled in the art.
  • a PCR method is used as amplification method e. g. a DNA polymerase such as Taq DNA polymerase (Perkin Elmer) or AmpliTaq (PE Applied Biosystems), Hot GoldStar Taq polymerase (Eurogentec), AmpliTaq Gold polymerase (PE Applied Biosystems), SureStart Taq(Stratagene) or Platinum Taq (Gibco) can be used.
  • the treatment to denature into a single-stranded DNA is usually a thermal treatment.
  • PCR conditions used for amplification are known to the person skilled in the art.
  • the presence and amount of the amplified target DNA sequences can be detected by e. g. a method in which the size of the amplified target DNA sequences is confirmed by electrophoresis, by hybridizing the amplified target DNA sequences with a labelled probe having a sequence complementary to the sequence of the target amplified or by real-time PCR, wherein the amplified target DNA sequences are detected during amplification e. g. by measuring fluorescence during amplification using a variety of fluorescent dyes emitting at different wavelengths. Different fluorescent dyes and non-fluorescent quenchers depending on real-time PCR the system used can be coupled with the probe prior to amplification e. g.
  • a fluorescent dye such as FAM (6-carboxyfluorescein), HEX(6-carboxy-2', 4,4',5', 7,7'hexachlorofluorescein), TET (6-carboxy-4,7,2 ⁇ 7'-tetrachloro-fluorescein), JOE (6- carboxy4', 5'-dichloro-2', 7'-dimethoxy fluorescein) or Texas-Red'can be coupled at the 5'-end of the probe and a fluorescent quencher such as TAMRA (6-carboxy-N, N, N', Ntetramethylrhodamine) or a non- fluorescent quencher such as DABCYL(4- ( (4- (dimethylamino) phenyl) azo) benzoic acid) can be coupled at the 3'-end of the probe.
  • a fluorescent quencher such as TAMRA (6-carboxy-N, N, N', Ntetramethylrhodamine
  • the present invention further comprises a diagnostic kit for detecting and/or monitoring the presence of Rickettsia helvetica in a subject, comprising the primers and/or probes for amplifying a target DNA sequence of the OmpB gene of Rickettsia helevtica as defined above.
  • Ixodes ricinus ticks were collected using the cloth dragging method. We collected females, males and nymphs in the region of Zuerich and Luzern in Switzerland. Ticks were identified to species level and development stage and stored at 4°C in tubes with high humidity.
  • DNA extraction Prior to DNA isolation, ticks were disinfected in 70% ethanol, dried and mechanically crushed. Lysis of the tick samples was carried out in the presence of 6.7 % saccarose solution, 0.2 % proteinase K, 20 mg/ml Lysozyme and 10 ng/ml RNase A for 16 h by 37°C. 0.5 M EDTA and 20 % SDS was added and further incubated for 1 h at 37°C. Extraction was performed twice with 80 % Phenol (1:1, v:v) and Methylenchloride/Isoamylalcohol (24: 1 , v:v). DNA was precipitated with isopropanol. The DNA-pellet was washed with 70 % ethanol and centrifuged at 16000 x g for 15 min. After removal of the ethanol the pellet was dried at 50°C and dissolved in 25 - 50 ⁇ l distilled water.
  • DNA isolated according to example 1 from male and female ticks was used for PCR.
  • a specific 593-bp region of the Rickettsia helvetica 16S ribosomal RNA gene was amplified by nested PCR.
  • First-round PCR amplified a fragment of 736-bp of the 16S ribosomal RNA gene of Rickettsia using primers 16SRf 5' ACGCTATCGGTATGCTTAACAC 3 'and 16SRr 5' GGACTACCAGGGTATCTAATCC 3 '.
  • First-round PCR was performed in 25- ⁇ l reaction mixture containing 2 ⁇ l of isolated DNA, in Thermocycler (MWG, M ⁇ nchen, Germany) with the following PCR profile: initial 10 min denaturation step at 95°C, followed by 40 cycles consisting of denaturation at 95°C for 30s, primer annealing at 50°C for 30s and polymerase extention at 72°C for 90s.
  • Primers 16SRhv 5' GTGGGAATCTACCCATCAGTAT 3' and 16SRhr 5' CGTCAGTTGT AGCCC AGATGAT 3 ' amplified a specific 593 bp fragment of the PCR product generated in the first-round PCR.
  • the thermal cycling programm of the second-round PCR consisted of an initial 10 min denaturation step at 95°C, followed by 40 cycles consisting of denaturation at 95°C for 30s, primer annealing at 55°C for 30s and extention at 72°C for 60s.
  • the Qiagen HotStarTaq Master Mix according to Qiagen protocols was used for PCR.
  • DNA electrophoresis was carried out in 1 % agarose gels containing ethidium bromide, and DNA fragments were visualized under ultraviolet light.
  • the sequence of the PCR product was confirmed by DNA sequencing.
  • the sequence obtained from the PCR product is 100% identical to the 16S rRNA sequence of R. helvetica strain C9P9 (Genbank Accession No.: L36212).
  • a second specific PCR method was developed for detection of R. helvetica based on the PS 120 gene (Accession No: AF163009, Sekeyova et al., 2001, Int. J. Syst. Evol. Microbiol. 51 (PT 4), 1353-1360).
  • Isolated DNA used in Example 2 from male and female ticks containing Rickettsia helvetica was used for PCR.
  • Specific primers PS120_for TGATCCTGTTTTAGAGGCTCATTCAGAAG and PS120_rev CTTTAGCTATATCTAAAGGATCACGAAAAGTACTT were used to amplify a PCR product of 128 bp.
  • the Primers were specific for R. helvetica.
  • a primer annealig temperature of 60°C was used for PCR.
  • the resultant PCR product of 128 bp was sequenced using Big dye terminator chemistry (Applied Biosystems) and DNA sequencer ABI PRISM 310.
  • the sequence obtained from the PCR product is 100% identical to the PS 120 gene sequence of R. helvetica (Genbank Accession No.: AF163009). This sequence is unique for R. helvetica.
  • Isolated DNA used in Example 2 from male and female ticks containing Rickettsia helvetica was used for PCR.
  • PCR amplification reactions were performed using the oligonucleotid primer pair ompB_f (5' aatctagatgttgttggtattc 3') and ompB R.h. NT_r (5' attatgcagcaaccgcagc 3').
  • the primers were designed with the OmpB sequence of R. helvetica (Genbank Accession No. AF123725) without the signal sequence and without the autotransporter domain beta- domain, which is described by Uchiyama, T.; 1999, Microbiol. Immunol., 43, 1061-1065.
  • the Triple Master PCR System (Eppendorf, 954140261) was used. Two microlitres of the DNA preparation was amplified in a 50 ⁇ L reaction mixture, containing 20OnM of each primer, 200 ⁇ M dNTP mix (Roche), 5 ⁇ L High Fidelity Buffer with Mg2+ and 0.75 ⁇ L Triple Master Polymerase Mix. The PCR was subjected to one initial denaturation step at 94°C for 2 min and then amplified in 35 cycles of 94°C for 20 sec, 54°C for 20 sec and 68°C for 3 min, followed by a single 7-min extension step at 68 0 C.
  • the resulting PCR product of 3919bp was purified using the QIAquick PCR purification kit (Qiagen) and directly ligated in the vector pCRT7/NT-TOPO (Invitrogen) by using the pCRT7 TOPO TA Expression Kit (Invitrogen).
  • the ligation-mix was transformed in competent E. coli TOP 1OF' cells and the transformants were selected and analysed for the integration of the ompB PCR product in the vector pCRT7/NT TOPO by sequencing.
  • the sequencing reactions were carried out twice in both directions using 4 ⁇ L of the resultant recombinant plasmid construct, named pCRT7/NT TOPO-ompB, l ⁇ L of the PCR primer T7_f(5' taatacgactcactataggg 3'), T7_r (5' ctagttattgctcagcggtgg 3') respectively and 2 ⁇ L BigDye terminator cycle sequencing mix (Applied Biosystems).
  • the cycle sequencing was started by one initial denaturation step at 96 0 C for 2 min and then amplified in 28 cycles of 96°C for 10 sec, 5O 0 C for 5 sec, 45°C for 5 sec, 40 0 C for 5 sec and 60°C for 3 min.
  • helvetica, 3 'end, 245 nucleotides aatctagatgttgttggtattcctgggatagtaacaggtgattctttaacttatgttgtacaagcaggcggtccttgcactgctacggctaccg gaggaaatgataacggtggtaatgctacagtacttttcggttcgattaacatgttacaaaacggtgtttttgcagttaacggtgcagatatag ctattggttctgtttccggtactgcaggtcaattattaacagttaatattgcaggaaaaaa
  • ompB R. helvetica, 5 'end, 328 nucleotides aacgtagttactaatgatatcgcaaatggtccgtttggaggtgcacctggtgtaggtcagaacgttacaacatttgtaaatgcaactaatacc gcagcatataataatcttcttttagctaaaaatagtgctgattctgctaactttgtcggaactattactaccgatacaagtgcagccgtaactaatgcacacacaattagatatagctaaagatatccaagctcaacttggtaacagattaagtgcgcttagatatttaggtactcctgaaactgctgaaactgctgaaat gactgggctgaaat gactgggctgaaat gactgggctg
  • Both primers were designed based on alignments using the program ClustalW of homologous OmpA sequences of Rickettsia species available in the NCBI Genbank.
  • PCR was performed in a Primus-HT thermocycler (MWG BIOTECH) with 1 ⁇ g, or less, of template DNA in a total reaction volume of 50 ⁇ l with 2.5 U TripleMaster Polymerase Mix ( Triple Master PCR System Eppendorf, 954140261), 200 ⁇ M dNTP, 200 nM of forward and reverse primer, respectively. Cycling parameters was 94°C for 2 min followed by 35 cycles of 94°C for 20 sec, 48 0 C for 20 sec and 68°C for 6 min followed by a final 7 min extension step at 68°C.
  • the PCR product after purification using QIAquick PCR purification kit (Qiagen) was digested with Sad and EcoRI restriction enzymes and ligated after a second purification step with Sad and EcoRI digested purified expression vector pBAD/HisB (Invitrogen).
  • the ligation mix was used to transform E. coli TOP 1OF'. Transformants were selected and analysed for the integration of the OmpA-PCR-product in the vector pUC18.
  • the resultant recombinant plasmid was named pUC(OmpA).
  • pUC(OmpA) was partially sequenced using M13rev 5' (CAG GAA ACA GCT ATG AC) and M13vor (TGT AAA ACG ACG GCC AG) primers using Big dye terminator chemistry (Applied Biosystems) and DNA sequencer ABI PRISM 310.
  • the full-length sequence was determined by Microsynth (Balgach, Switzerland) using the DNA sequencing service.
  • the OmpA protein sequence of Rickettsia helvetica obtained has the following identity with ompA sequences of other Rickettsia: 66% identity with partial ompA sequence of R. slovaca (ompA sequence of R. slovaca 13 -B, comprising 1060 amino acids, Genbank Accession No.: AAC35190);
  • Antigen design tool http://www.genscript.com/cgi- bin/tools/antigenic prediction.pl, GenScript Corporation
  • Predicting antigenic peptides http://bio.dfci.harvard.edu/Tools/antigenic.pl, MIF: Molecular Immunology Foundation
  • Peptides were synthesized by the Thermo Electron Corporation, UIm, Germany. The amino terminus of the peptides was biotin labeled and a spacer was introduced between each peptide and biotin.
  • microtiter plate wells coated with streptavidin HBC (Reacti-BindTM Streptavidin High Binding Capacity [HBC] Coated Plates, Pierce) were washed with 3 x 200 ⁇ L of wash buffer (10OmM sodium phosphate, 15OmM NaCl, pH 7.2, 0.05% Tween-20) followed by the addition of lOO ⁇ L of the biotinylated peptide (l-10ng/ ⁇ L)/well. Plates were incubated for 1 hour at room temperature by shaking with 4500rpm. After the incubation time each well was washed with 3 x 200 ⁇ L of wash buffer.
  • wash buffer 10OmM sodium phosphate, 15OmM NaCl, pH 7.2, 0.05% Tween-20
  • lOO ⁇ L of human blood serum (1:100 diluted in blocking buffer [10OmM sodium phosphate, 15OmM NaCl, pH 7.2, 0.05% Tween-20, 0.1% BSA]) was added to each well and the microwell plate was incubated for 30min at room temperature by shaking with 4500rpm. After this second incubation step each well was washed with 3 x 200 ⁇ L of wash buffer.
  • lOO ⁇ L of goat anti human horse radish peroxidise-labeled secondary antibody specific for IgG or IgM (IgG 1 :3000 diluted in blocking buffer and IgM 1 :20'000 diluted in blocking buffer, respectively) was added to each well and the microwell plate was incubated for 30 minutes at room temperature by shaking with 4500rpm. After the incubation step with the diluted serum sample, each well was washed with 3 x 200 ⁇ L of wash buffer. The microwell plate was developed by adding lOO ⁇ L of TMB-ELISA substrate solution (1-StepTM Ultra TMB- ELISA, Pierce) to each well and each plate was incubated for 15 minutes at room temperature.
  • TMB-ELISA substrate solution (1-StepTM Ultra TMB- ELISA, Pierce
  • each microtiter plate contained human serum controls ranging in reactivity from non-reactive (negative control) to highly reactive (positive control).
  • Human sera used are: 411 : negative control (no infection with Rickettsia); Fl: Patient, 1127: Patient, 1126: Patient, 4290410 (26): Patient with R. conorii infection, 2339140: Erythema migrans patient with prevalence of IgG and IgM antibodies against R. conorii as found by Indirect fluorescence antibody assay (IFA) for the detection of IgG and IgM antibodies against R. conori, 3147357: Erythema migrans patient with prevalence of IgG and IgM antibodies against R.
  • IFA Indirect fluorescence antibody assay
  • R. conorii as found by Indirect fluorescence antibody assay (IFA) for the detection of IgG and IgM antibodies against R. conori, 4234352: negative control (no infection with Rickettsia), 4255924: Patient, presumably infected with R. conorii and/or R. africae.
  • Peptides specific for R. helevtica used are: RaI 94 (TTHKLASITVDGGVGVGAISLAATN), Ra87 (VNNKVPALFTVNKN), Ra657 (AAGKLIAGGLVTGTAVLTT),
  • Peptides Rsp971, Rsp954, RspompB, RspompA were used which react with antibodies in serum samples directed against Rickettsia species.
  • the TaqMan probe and the primer sequences for detection of Rickettsia helvetica based on the ompB gene were designed with the software program Primer Express (Applied Biosystems).
  • a 162 bp fragment of the ompB gene of R. helvetica (AF123725, region 2983 to 3144) is amplified with the forward primer Rh_pmpB_F: GATTTCGACGGTAAAATTACC and reverse primer Rh_ompB_R: GCT ACCGAT ATTACCTACAG.
  • the TaqMan probe Rh_ompB_P: ACTCTACTGCTACAA GTATGGTTGCTACAG was designed.
  • the fluorescent dyes at the 5' end was FAM and at the 3' end TAMRA, respectively.
  • Standard curve was generated with serial dilutions from 108 to 101 copies of the plasmid which contains the vector (pCR2.1, Invitrogen) with the 162 bp fragment of OmpB. Slope, Y-intercept and correlation coefficients of the standard curve are: -3.634, 40.129 and 0.9937, respectively.
  • DNA from whole blood was isolated according to the instructions of Machery-Nagel, CH- Oensingen, Genomic DNA from Blood (April 2005/Rev. 04).
  • the 30- ⁇ l (final volume) real-time PCR mixture contained 15 ⁇ l Master Mix (Applied Biosystems) 900 nM of each primer, 250 nM of the TaqMan probe and 5-10 ⁇ l of the template DNA or diluted plasmid standard.
  • Real-time PCR was performed using ABI 7000 (Appllied Biosystems).
  • the amplification programm consisted of initial heating at 95°C for 15 min, followed by 40 cycles of heating at 95°C for 20s, primer annealing and polymerization at 60°C for 1 min.
  • the copy numbers of detected Rickettsia helvetica based on the developed ompB real-time PCR assay in ticks is in the range of 10 to 10'0OO per tick, corresponding to CT-values of 37.3 and 27, respectively.

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Abstract

La présente invention concerne des polynucléotides nouvellement identifiés et des polypeptides encodés par lesdits polynucléotides, destinés à détecter et/ou à surveiller la présence de Rickettsia helvetica. Plus particulièrement, la présente invention concerne des polynucléotides codant pour un polypeptide OmpA de Rickettsia helvetica et des fragments antigéniques dudit polypeptide, ainsi que les polypeptides et les fragments antigéniques respectifs, ainsi que des procédés permettant de détecter et/ou de surveiller la présence de Rickettsia helvetica chez un sujet. L'invention concerne également des trousses les contenant.
PCT/EP2007/003062 2006-04-04 2007-04-04 Polynucléotides et polypeptides destinés à la détection de rickettsia helvetica Ceased WO2007113009A2 (fr)

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SE515216C2 (sv) * 1998-02-20 2001-07-02 Kenneth Nilsson Ny peptid, diagnostiskt reagens och kit för detektion av rickettsioser

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CN117721227A (zh) * 2023-12-22 2024-03-19 内蒙古医科大学 一种蜱传立克次体三重荧光定量pcr检测试剂盒

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