JP2016010331A - Diagnostic method which is targeted to sequence specific to helicobacter suis, sequence concerned, and protein encoded by sequence concerned - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for determining the presence of Helicobacter suis which lives in a stomach similarly to Helicobacter pylori, and is supposed to cause gastritis, gastric ulcer, gastric cancer, lymphoma, or the like, and for which a measurement (detection)/diagnostic method is not yet established, and to provide a diagnostic method for Helicobacter suis infection, as well as to provide a drug or kit for use in the method concerned.SOLUTION: The invention relates to a method for determining the presence of Helicobacter suis in a sample comprising measuring at least one selected from the group consisting of (i) a nucleic acid having a base sequence or a part thereof described in a specific sequence, (ii) a protein or peptide having an amino acid sequence or a part thereof described in a specific sequence, and (iii) an antibody recognizing the protein or peptide of (ii), in the sample derived from a subject.

Description

  The present invention relates to a Helicobacter suis (hereinafter referred to as “H. Switzerland”) specific base sequence and amino acid sequence isolated from humans for which a pure culture method has not yet been established, and H targeting the sequence. . A method for determining the presence of Switzerland or The present invention relates to the field of Swiss infection diagnosis methods and drugs or kits for use in the methods.

  In 1983, Marshall (Marshall B, J,) et al. Succeeded in separating and cultivating Helicobacter pylori (hereinafter referred to as “H. pylori”) from chronic gastritis patients for the first time. Since this discovery, gastritis and some gastric ulcers have been found in H.264. It has been clarified that it is caused by Helicobacter pylori infection (Non-patent Document 1). H. H. pylori is believed to infect humans orally. In developing countries, the high infection rate of 70-80% or more already in childhood. On the other hand, infection rates are lower in developed countries than in developing countries. From the results of the epidemiological survey, in 1994, the World Health Organization (WHO) H. pylori has been recognized as a carcinogenic substance, and its relationship with gastric cancer has been attracting attention (Non-Patent Document 2). On the other hand, H. Although cases of gastritis and gastric ulcers were reported despite H. pylori negative, Various studies have also been conducted on the dynamics of related bacteria other than H. pylori. Helicobacter bacteria are classified into more than 30 types. Among them, in 1994, Helicobacter heilmannii (hereinafter referred to as “H. heilmani”) was detected as an infectious bacterium in the stomach of an animal (non-patented). Reference 3). Subsequently, gastric lymphocyte complex tissue (MALT) lymphoma (hereinafter referred to as “gastric MALT lymphoma”) and Research results showing an association with Hailmany were published. In addition to H. pylori, H. Heilmany became clear (Non-Patent Document 4).

  H. Hylemany is a zoonotic pathogen that infects the stomach of humans, dogs, cats, pigs, monkeys, etc. and induces gastric diseases. Based on the taxonomy based on the base sequence of the 16S ribosome gene, it is isolated from humans. Heilmany It was reclassified as Switzerland (Non-patent Document 5). J. et al. Syst. Evol. Microbiol. In the magazine, H. isolated from pigs. Since the genomic base sequences of Swiss HS1 and HS5 strains were announced, The name of bacteria was unified in Switzerland (Non-patent Document 6). Therefore, H. infects humans reported prior to 2008. Hylemany has reported on H. H. in subsequent reports. It is the same fungus as Switzerland (hereinafter unified in the present specification with the expression “H. Switzerland”).

  H. Switzerland has been confirmed to live in the stomach of many mammals, including humans, and animal-to-human transmission is suspected. H. Switzerland As with H. pylori, it causes gastritis, gastric ulcer, gastric cancer, lymphoma, etc. (Non-patent Document 5), suggesting the possibility of a major causative bacterium of gastric MALT lymphoma (Non-patent Document 7). H. Switzerland is a gram-negative spiral gonococcus with 5-7 rolls. It has 10-20 flagella in both poles and has high mobility (Non-Patent Document 5). H. H. pylori is positive for urease, whereas H. pylori is positive. In Switzerland, there are urease positive strains and negative strains. Especially, isolates from animals are reported to be urease negative, even though isolates from animals are positive for urease (Non-patent Documents). 8).

  In addition, H. coli isolated from humans. The Swiss isolation culture method has not yet been established and has been passaged in the stomach of experimental mice (Non-patent Document 8). H. There has been a report that growth has not been observed even though Swiss isolation culture was attempted (Non-patent Document 9). H. Although a screening method by PCR of a Heilmany-like strain (Helicobacter heilmannii'-like organisms: HHLO) has been published (Non-Patent Document 10), It was not unique to Switzerland, and there was a problem with detection sensitivity.

  Therefore, H.I. While it has been shown that the link between Swiss infection and gastric disease is very high, The culture method used for diagnosis of H. pylori and the urea breath test (UBT) using urease activity as an index cannot be applied, and an effective diagnostic method has not been established.

Marshal, B.M. J. et al. & Warren, J.A. R. Lancet, 1, 1273-1275, 1984. NIH Consensus Statement, 12 (1) 1-23, 1994. Stole, M.M. et al. , Scand. J. et al. Gastroenterol. , 29, 1061-1064, 1994. Matsui, H .; et al. Curr. Trends Microbiol. , 6, 27-34, 2010. Priestnall, S.M. L. et al. , J. et al. Clin. Microbiol. , 42, 2144-2151, 2004. Baele, M.M. et al. , Int. J. et al. Syst. Evol. Microbiol. , 58, 1350-1358, 2008. Mongner, A.M. et al. , Gastroenterology 118, 821-828, 2000. Matsui, et al. , Helicobacter, published 28 March 2014 PubMed doi: 10.1111 / hel. 12127. The 37th Kanto-Koshinetsu Medical Examination Society 2000.10.14-15, “A Trial to Isolate and Culture Helicobacter heilmannii” Chisholm, S.H. A. & Owen, R.A. J. et al. Diagn. Microbiol. Infect. Dis. , 46, 1-7, 2003.

  The present invention is based on H.P. for which no measurement (detection) / diagnostic method has been established yet. A method for determining the presence of Switzerland; The object is to provide a method for diagnosing Swiss infection, as well as a drug or kit for use in the method.

  We have described H.C. H. isolated from cynomolgus monkeys to enable Swiss measurement (detection) and diagnosis. H. isolated from Switzerland and humans Swiss genome sequence analysis and comparative genome analysis were performed. More specifically, these H.P. After preparing genomic DNA from the gastric mucosal suspension of Swiss infected mice and determining the whole genome base sequence, the determined whole genome sequence and the related Campylobacter (published on the EMBL / GenBank / DDBJ database ( The genome sequences of the 11 genomes of Campylobacter genus and 20 strains of Helicobacter genus were subjected to comparative genome analysis using RECOG (Research Environment for Comparative Genomics). Two Swiss-specific nucleotide sequences were identified. Then, the present inventors devised PCR primers targeting the two types of identified base sequences, and H. coli from DNA prepared from gastric biopsy. It was found that a Swiss specific base sequence can be measured (detected) by the PCR method.

  Furthermore, since one of the identified base sequences may encode a protein, the present inventors expressed the expression product of the same sequence in E. coli as a recombinant protein, By confirming by Western blotting, it was confirmed that it was expressed as a protein. In addition, the present inventors confirmed that this protein is different from the proteins reported so far, and newly named it F2R2 protein. In addition, the inventors have described H.C. The inventors have found that antibodies that recognize F2R2 protein as an antigen are present in blood samples derived from Swiss-infected mice, thereby completing the present invention.

Therefore, the present invention relates to an H. coli by measuring (detecting) the nucleotide sequence set forth in SEQ ID NO: 1 (or FIG. 2) or SEQ ID NO: 2 (or FIG. 3) or the amino acid sequence set forth in SEQ ID NO: 3. The present invention relates to a method for determining the presence of a Swiss infection or a diagnostic method. The present invention also provides a nucleic acid that specifically binds to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a substance that specifically binds to the amino acid sequence of SEQ ID NO: 3 for use in the method ( Antibodies, aptamers, etc.) and H. Swiss detection drug or H.P. Diagnostic (testing) drugs for Swiss infections and H. Swiss detection kit or H.C. It relates to diagnostic (test) kits for Swiss infections. More specifically, the present invention relates to the following inventions:
(1) In a sample derived from a subject, (i) a nucleic acid having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof, (ii) a protein having the amino acid sequence described in SEQ ID NO: 3 or a part thereof Or (iii) measuring (detecting) at least one selected from the group consisting of an antibody recognizing a protein or peptide having the amino acid sequence of SEQ ID NO: 3 or a part thereof, or (iii) H. in the sample. How to determine the presence of Switzerland.
(2) The method according to (1), wherein the subject is an eczema gastritis patient.
(3) The method according to (1) or (2), wherein the sample is a gastric biopsy sample or stool, or blood or urine.
(4) H.I. A polynucleotide having the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof for use in a method for determining the presence of Switzerland.
(5) H.I. A nucleic acid construct that specifically binds to a polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof, for use in a method for determining the presence of Switzerland.
(6) H. A protein or peptide having the amino acid sequence of SEQ ID NO: 3 or a part thereof for use in a method for determining the presence of Switzerland.
(7) H. An antibody or an immunoreactive fragment thereof that recognizes a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof for use in a method for determining the presence of Switzerland.
(8) H. containing at least one selected from the following groups: Swiss detection drugs:
(A) the nucleic acid construct according to (5);
(B) the protein or peptide according to (6); and
(C) The antibody or immunoreactive fragment thereof according to (7).
(9) H. comprising the detection agent according to (8). Swiss infection detection kit.

(H. Switzerland)
In this specification, “H. Switzerland” means H.S. Helicobacter heilmannii'-like organisms (HHLO), non-H. H. pylori helicobacter species (NHPHS) or H. pylori. H. heilmannii sensu lato (hereinafter referred to as “H. heilimannii sensu lato”). It means a strain belonging to Hylemani type 1, Other strains classified as Heilimannii sensu lato, heilimannii sentric tricto, H. Does not include strains belonging to Hylemany type 2. A system diagram of the Helicobacter classification is shown in FIG. H. Switzerland is known to infect the stomachs of dogs, cats, pigs, monkeys, etc. in addition to humans. There are no particular limitations on the mammals that Switzerland infects. For example, H. The mammal to which Switzerland is infected may be a human or a monkey.

(H. Polynucleotide having Swiss-specific base sequence)
In the present specification, the “polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof” is a polynucleotide containing all of the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. It may be a polynucleotide containing a part thereof. Here, the “base sequence described in SEQ ID NO: 1” completely coincides with the base sequence described in SEQ ID NO: 1 (H. Swiss specific sequence of untranslated region obtained from H. Swiss TKY strain). 1 to 100 (or 1 to 80, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20) in the base sequence described in SEQ ID NO: 1 , 1 to 10, 1 to 5, and 1 to 3 sequences deleted or substituted, inserted or added (or the nucleotide sequence described in SEQ ID NO: 1 and 60%, 70%, 80 %, 90%, 95%, 98%, or 99% homology, or a sequence that hybridizes under stringent conditions with a sequence complementary to the base sequence described in SEQ ID NO: 1) For example, any one of the nucleotide sequences listed in FIG. H. Swiss H. Swiss specific base sequence of untranslated region obtained from SNTW101 strain), SEQ ID NO: 17 (H. Swiss specific base sequence of non-translated region obtained from H. Swiss SH8 strain), or sequence The base sequence described in No. 18 (H. Swiss specific base sequence of untranslated region obtained from H. Swiss SH10 strain) may be used.

  Similarly, the “base sequence described in SEQ ID NO: 2” refers to the base sequence described in SEQ ID NO: 2 (H. Swiss HS1 strain or H. Swiss HS-specific translation region obtained from H. Swiss HS5 strain). In addition to the sequence: F2R2 gene sequence), 1 to 100 (or 1 to 80, 1 to 60, 1 to 50, 1 to 40, 1 to 30) in the base sequence described in SEQ ID NO: 2 , 1-20, 1-10, 1-5, 1-3, a sequence in which a nucleic acid is deleted, substituted, inserted or added (or 60% of the nucleotide sequence described in SEQ ID NO: 2) Hybridizes under stringent conditions with a sequence having 70%, 80%, 90%, 95%, 98%, or 99% homology, or a sequence complementary to the base sequence described in SEQ ID NO: 2. Sequence), for example, any of the base sequences listed in FIG. Or SEQ ID NO: 19 (H. Swiss specific base sequence of translation region obtained from H. Swiss TKY strain), SEQ ID NO: 20 (H. Swiss specific base of translation region obtained from H. Swiss SNTW101 strain) Sequence), SEQ ID NO: 21 (H. Swiss specific base sequence of translation region obtained from H. Swiss SH8 strain) or SEQ ID NO: 22 (H. Swiss specific base of translation region obtained from H. Swiss SH10 strain) The base sequence described in (Sequence) may be used.

  In the present specification, a polynucleotide having a part of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is H.264. Must be Swiss-specific. Here, “H. Switzerland specific” means H.Swiss. This means that no polynucleotide having a highly homologous base sequence is found in any species other than Switzerland. This means that in any species other than Switzerland, a polynucleotide having 90% or more (or 95% or more, 98% or more, 99% or more, 100%) homology with the base sequence is not confirmed. The candidate partial sequence is H.264. Whether or not it is Swiss specific can be appropriately confirmed by searching a database such as GenBank and performing sequence comparison. Preferably, the polynucleotide having a part of the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 is 50 of the original polynucleotide (the polynucleotide comprising the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, respectively). %, More preferably 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the polynucleotide. It is.

  As used herein, “hybridize under stringent conditions” means to hybridize under hybridization conditions commonly used by those skilled in the art. For example, whether or not to hybridize can be determined by a method described in Molecular Cloning, Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). For example, the hybridization conditions were 42 ° C. in 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0,2 M NaH 2 PO 4, 20 mM EDTA · 2Na, pH 7.4). The condition may be such that the sample is hybridized with, and then washed with 0.5 × SSC at 42 ° C. In addition, the homology of the base sequence and amino acid sequence described in the present specification can be determined using a known program such as BLASTX, BLAST, FASTA, for example.

  In another aspect, the present invention relates to a nucleic acid construct that specifically binds to a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof. The nucleic acid construct of the present invention may be labeled as necessary. The nucleic acid construct of the present invention may be a PCR primer or a probe for measuring (detecting) the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. For example, the length of the nucleic acid construct of the present invention may be 10 to 400 bp, 15 to 200 bp, 20 to 100 bp. Preferably, the nucleic acid construct of the present invention has a sequence complementary to a polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof. The complementary sequence is complementary to the polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof as long as the nucleic acid construct retains the function of specifically binding to the polynucleotide. In a typical sequence, 1 to 3 nucleic acids may be substituted, deleted, added, or inserted.

  In the present specification, the “labeling” of a nucleic acid means a modification to enable the measurement (detection) of the presence of the nucleic acid, for example, a radioactive label (32P, 3H, 14C etc.), a fluorescent label (FAM And VIC, etc.), enzyme labeling (such as biotin), antigen labeling (such as digoxigenin (DIG)), and the like can be used.

(F2R2 protein or a partial peptide thereof)
In yet another aspect, the present invention relates to a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof. Here, the “amino acid sequence described in SEQ ID NO: 3” means a sequence that completely matches the amino acid sequence described in SEQ ID NO: 3, as well as one to several amino acids in the amino acid sequence described in SEQ ID NO: 3. Is deleted, substituted, inserted or added, or the amino acid sequence of SEQ ID NO: 3 and 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99 A sequence having at least% homology, or an amino acid sequence encoded by a polypeptide that hybridizes under stringent conditions with a polypeptide encoding the amino acid sequence set forth in SEQ ID NO: 3, It may be an amino acid sequence encoded by any of the listed nucleotide sequences. In the present specification, a peptide having a part of the amino acid sequence shown in SEQ ID NO: 3 is H.264. Must be Swiss-specific. Here, “H. Switzerland specific” means H.Swiss. This means that a polypeptide having high homology with the amino acid sequence is not confirmed in any species other than Switzerland. This means that no polypeptide having 90% or more (or 95%, 98% or more, 99% or more, 100%) homology with the amino acid sequence is found in any species other than Switzerland. The candidate partial sequence is H.264. Whether or not it is Swiss specific can be appropriately confirmed by searching a database such as GenBank and performing sequence comparison. Preferably, the peptide having a part of the amino acid sequence described in SEQ ID NO: 3 is a peptide having 50% or more amino acids of the original protein (protein having the amino acid sequence described in SEQ ID NO: 3), more preferably 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more of a peptide having an amino acid sequence.

  For example, the protein or peptide having the amino acid sequence shown in SEQ ID NO: 3 or a part thereof is preferably a protein or peptide recognized by an antibody (having antigenicity), and most preferably an F2R2 protein. Whether or not the candidate protein or peptide is recognized by the antibody is determined by, for example, bringing the sample containing the antibody or antibody already obtained into contact with the candidate protein or peptide and binding the candidate protein or peptide to the antibody. Can be determined by measuring (detecting) and determining that the candidate protein or peptide is recognized by the antibody when bound.

The protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 of the present invention or a part thereof may be labeled as necessary. As the label, a detectable label such as a radioactive label, an enzyme, a fluorescent label, a bioluminescent label, or a chemiluminescent label metal can be used. Examples of such labels include, but are not limited to, radiolabels such as ³² p3H, ¹²⁵ I, and ¹⁴ C; β-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, lactate oxidase, alcohol oxidase, Enzymes such as monoamine oxidase and horseradish peroxidase; coenzymes or prosthetic groups such as FAD, FMN, ATP, biotin and heme; fluorescein derivatives (fluorescein isothiocyanate (FITC), fluorescein ovalbamyl etc.), rhodamine derivatives (tetramethyl) Rhodamine, trimethylrhodamine (RITC), Texas red, rhodamine 110, etc.), Cy dyes (Cy3, Cy5, Cy5.5, Cy7), Cy-chrome, spectrum Fluorescent labeling such as benzene, spectrum orange, propidium iodide, allophycocyanin (APC), R-phycoerythrin (R-PE); bioluminescent labeling such as luciferase; or luminol, isoluminol, N- (4- Aminobutyl) -N-ethylisoluminose ester and other luminol derivatives, N-methylacridinium ester, N-methylacridinium acylsulfonamide ester and other acridinium derivatives, lucigenin, adamantyl dioxetane, indoxyl derivatives, ruthenium complexes And chemiluminescent labels such as gold colloids and the like.

(Antibodies or fragments thereof)
In another aspect, the present invention relates to an antibody or an immunoreactive fragment thereof that recognizes a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof. An “immunoreactive fragment of an antibody” means a part (partial fragment) of an antibody or a peptide containing a part of an antibody, which retains the binding action of the antibody to an antigen. Examples of such antibody fragments include F (ab ′) ₂ , Fab ′, Fab, single chain Fv (hereinafter referred to as “scFv”), disulfide bond Fv (hereinafter referred to as “dsFv”), A peptide including a polymer, a dimerized V region (hereinafter referred to as “Diabody”), or a CDR can be exemplified. Preferably, the antibody of the present invention or an immunoreactive fragment thereof specifically recognizes a protein or peptide having the amino acid sequence shown in SEQ ID NO: 3 or a part thereof.

  Throughout this specification, “specifically recognize” or “specifically binds” means that it binds to a target substance but does not bind to other substances. Whether or not the candidate substance binds to the target substance and / or does not bind to the other substance can be confirmed by any of Southern hybridization, PCR, Western blotting, and ELISA. May be. When confirming by Western blotting, for example, F2R2 protein is dissolved in 2% SDS, 10% glycerol, 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol solution, boiled to obtain anti-His antibody and test sample. This can be confirmed by performing Western blot analysis with an antibody. When confirming by Southern hybridization, PCR, or ELISA, it can confirm using the method mentioned later. Here, “does not bind” or “does not recognize” means that the binding of the test substance to a nucleic acid, protein or peptide that is considered not to be bound (or not recognized) in comparison with the binding to the target substance is sufficient. Includes cases that are low enough to be distinguished. For example, the cross-reaction rate with other substances compared to the target substance is 1% or less, 0.5% or less, 0.3% or less, 0.1% or less, 0.05% or less, 0.03 % Or less. The cross-reactivity can be calculated and obtained as {(actual value (concentration) relating to binding to other substance to be compared) / (concentration of added sample of other substance to be compared) × 100%}.

  The antibody of the present invention may be polyclonal or monoclonal as long as it can specifically recognize F2R2 protein, but is preferably a monoclonal antibody. Here, “monoclonal antibody” means an antibody derived from a single clone. The antibodies of the present invention include bispecific antibodies and single chain antibodies as long as they retain the binding activity to the antigen in addition to the complete antibody. Complete antibodies include non-human animal antibodies, non-human animal antibody amino acid sequences and human-derived antibody amino acid sequences, and human antibodies. Non-human animal antibodies include, for example, antibodies such as mice, rats, hamsters and rabbits, preferably animal antibodies capable of producing hybridomas, more preferably mouse antibodies. . Examples of antibodies having amino acid sequences of non-human animal antibodies and human-derived antibodies include human chimeric antibodies in which the antigen-binding domain Fv of animal-derived monoclonal antibodies is replaced with human antibodies, antigens of animal-derived antibody monoclonal antibodies A humanized antibody in which CDR, which is a sequence on the Fv domain directly involved in binding, is incorporated in the frame of a human antibody can be mentioned. Moreover, a human antibody is a human antibody that is an expression product of a completely human-derived antibody gene. The immunoglobulin class of the antibody of the present invention is not particularly limited, and may be any immunoglobulin class of IgG, IgM, IgA, IgE, IgD, and IgY, preferably IgG. In addition, the antibody of the present invention encompasses antibodies of any isotype.

(Detection drug)
The present invention relates to an H. coli containing the nucleic acid construct, the protein or peptide, or the antibody or immunoreactive fragment thereof. Swiss detection drug. In other words, the present invention relates to H.264. The nucleic acid construct, the protein or peptide, or the antibody or immunoreactive fragment thereof for use in Swiss detection. Alternatively, the present invention relates to H.264. It relates to the use of said nucleic acid construct, said protein or peptide, or said antibody or immunoreactive fragment thereof for the manufacture of a Swiss detection agent.

(kit)
In another embodiment, the present invention provides a nucleic acid construct that specifically binds to a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof, or the amino acid sequence set forth in SEQ ID NO: 3 or a sequence thereof A protein or peptide having a part thereof, or an antibody or an immunoreactive fragment thereof recognizing a protein or peptide having the amino acid sequence shown in SEQ ID NO: 3 or a part thereof. Swiss infection determination kit, or H.P. The present invention relates to a diagnostic kit for Swiss infections. The kit of the present invention comprises a nucleic acid construct that specifically binds to a polynucleotide having the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof, or the amino acid sequence shown in SEQ ID NO: 3 or a part thereof. In addition to an antibody or an immunoreactive fragment thereof that recognizes a protein or peptide having the amino acid sequence described in SEQ ID NO: 3 or a part thereof, or an immunoreactive fragment thereof, preferably from a solid phase, a hapten, and an insoluble carrier A carrier selected from the group consisting of:

  A measurement (detection) kit containing a nucleic acid construct that specifically binds to a polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof can be based on a known method using a nucleic acid. . Examples of such a method include a method using hybridization; a method using PCR; and a method known as an Invader (registered trademark) method (for example, Kwiatkowski, RW, et al., Mol. Diagn. (1999). ) 4: 353-364).

  For example, the kit of the present invention comprises a solid phase or hapten on which a nucleic acid construct that specifically binds to a labeled polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof is immobilized. It can be a kit. When the kit of the present invention contains a hapten, it may further contain a solid phase on which a substance that specifically binds to the hapten is immobilized.

  Alternatively, the kit of the present invention may include a primer (or primer pair) that specifically binds to a polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof.

  Alternatively, the kit of the present invention comprises an allele comprising a sequence complementary to a polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof and a sequence (flap) complementary to a part of a quenching probe Fluorescent label comprising a specific probe, an invader probe comprising a sequence complementary to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof, a triple strand specific DNA-degrading enzyme, and a quenching probe A universal probe can be provided. The flap is preferably different between each allele-specific probe. As the fluorescent label, a probe known to those skilled in the art can be appropriately selected, and it is preferable that the fluorescent label is different among the fluorescent labeled universal probes. For example, FAM and VIC can be used as fluorescent labels.

  A protein or peptide having the amino acid sequence shown in SEQ ID NO: 3 or a part thereof, or an antibody or an immunoreactive fragment thereof that recognizes a protein or peptide having the amino acid sequence shown in SEQ ID NO: 3 or a part thereof The measurement (detection) kit can be based on a known method using antibody molecules. Examples of such methods include labeled immunoassay method; immunoblotting method; immunochromatography method; chromatography method; turbidimetric method (TIA method); specific wax method (NIA method); colorimetric method; latex agglutination method (LIA) Method); particle counting method (CIA method); chemiluminescence measurement method (CLIA method, CLEIA method); precipitation reaction method; surface plasmon resonance method (SPR method); resonant mirror detector method (RMD method); comparative interference method, etc. Can be mentioned. Whether or not the kit of the present invention can perform a desired measurement can be detected by performing each measurement method by a method well known to those skilled in the art using the sample or the sample at the concentration. It can be confirmed by measuring whether or not.

  For example, the kit of the present invention comprises (i) a protein or peptide having the amino acid sequence shown in SEQ ID NO: 3 or a part thereof, or a protein or peptide having the amino acid sequence shown in SEQ ID NO: 3 or a part thereof ( A kit for immunochemical measurement comprising a solid phase or hapten to which an antibody or an immunoreactive fragment thereof to be specifically bound is immobilized, and (ii) a labeled second antibody. When the kit of the present invention contains a hapten, it may further contain a solid phase on which a substance that specifically binds to the hapten is immobilized.

  Alternatively, the kit of the present invention comprises (i) a solid phase on which a labeled first antibody is immobilized, and (ii) a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof, or A kit for immunochemical measurement comprising a hapten to which a second antibody or an immunoreactive fragment thereof that binds (specifically) a protein or peptide having the amino acid sequence shown in SEQ ID NO: 3 or a part thereof is immobilized. it can. The kit may further contain a labeled substance that specifically binds to the hapten.

  In the kit of the present invention, the solid phase is not particularly limited as long as it can be used for immunochemical measurement. For example, nitrocellulose, sepharose, nylon, vinylon, polyester, acrylic, polyolefin, polyurethane, rayon, polynosic, cupra, Lyocell, acetate, polyvinylidene difluoride, silicone rubber, latex, polystyrene, polypropylene, polyethylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, fluorinated resin, ABS resin, AS resin, acrylic resin, polymer alloy, Plates, tubes, chips (eg, protein chips, lab chips, etc.), beads, membranes, absorptions containing glass fibers, carbon fibers, glass, gelatin, polyamino acids and / or magnetically sensitive materials And / or the like can be mentioned particles, preferably, the plate and magnetic beads. In the present specification, the insoluble carrier means a suspendable insoluble solid phase such as beads, and examples thereof include latex beads and magnetic beads.

  The kit of the present invention may contain a coloring reagent, a reaction stopping reagent, a standard antigen reagent, a sample pretreatment reagent, a blocking reagent, and the like, if necessary. In addition, when the kit of the present invention contains a labeled antibody, it may further contain a substrate that reacts with the label. Furthermore, the kit of the present invention may appropriately include a package insert, instructions, a container for storing the kit, and the like.

  H. according to the present invention. By using a Swiss specific base sequence and a PCR diagnostic method targeting the base sequence, the conventional H.P. H. pylori not detected by H. pylori diagnosis A quick and reliable diagnosis of Swiss infection is possible.

A system diagram of the genus Helicobacter is shown. H. of untranslated region (approximately 590 base pairs) Shows the Swiss specific base sequence. Translation region (approximately 550 base pairs) Swiss specific base sequence (F2R2 gene region) is shown. H. PCR primers targeting two Swiss-specific sequences; It is the photograph which performed the agarose gel electrophoresis of the PCR product performed using the primer which targeted the vacA gene of H. pylori. Samples used in each lane are as follows. Lane M: 100-base pair DNA marker; Lane 1: DNA from uninfected mice (100 ng); Lane 2: H. Switzerland DNA from TKY infected mice (100 ng); Lane 3: H. Switzerland DNA from SNTW101 infected mice (100 ng); Swiss DNA (100 ng) from SH8 infected mice; Swiss DNA (100 ng) from SH10 infected mice; Switzerland HS5 DNA (10 ng); Heilmy s. s. DNA from ASB1.4 (10 ng); DNA from Ferris ATCC 49179 (10 ng); DNA from bacriformis M50 (10 ng); lane 10: H.I. DNA from Bizozerony R1051 (10 ng); Lane 11: H.I. Synogatrix DNA from JKM4 (10 ng); Lane 12: H.I. Mustele DNA from NCTC12032 (10 ng); Lane 13: H.E. DNA from Salmonis R1053 (10 ng); Lane 14: H.I. H. pylori from SS1 (10 ng); lane 15: H. pylori. H. pylori DNA from TN2GF4 (10 ng); lane 16: H. pylori H. pylori DNA from NCTC11637 (10 ng); Lane 17: H. pylori. H. pylori DNA from ATCC 43579 (10 ng); Lane 18: H. pylori. DNA from H. pylori RC-1 (10 ng). Moreover, the arrow on the right side of the photograph shows the following bands. “418 bp”: band amplified by VAC3624 / VAC4041R primer pair (H. pylori vacA gene); “348 bp”: band amplified by HH-F5 / HH-R4 primer pair (H. Swiss untranslated region) "220 bp": Band amplified by HH-F2 / HH-R2 primer pair (H. Swiss F2R2 gene). HH. PCR primers targeting two Swiss-specific sequences; It is the photograph which performed the agarose gel electrophoresis of the PCR product performed using the primer which targeted the vacA gene of H. pylori. Samples used in each lane are as follows. Lane M: 100-base pair DNA marker; H. pylori DNA from SS1 (10 ng); lane 2: H. pylori Switzerland DNA from TKY infected mice (100 ng); lanes 3, 4, 5 and 16: DNA from stomach biopsy of UBT negative patients (100 ng); lanes 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15: DNA from gastric biopsy of UBT positive patients (100 ng). Moreover, the arrow on the right side of the photograph shows the following bands. “418 bp”: band amplified by VAC3624 / VAC4041R primer pair (H. pylori vacA gene); “220 bp”: band amplified by HH-F2 / HH-R2 primer pair (H. Swiss F2R2 gene). It is a photograph of the electrophoresis analysis of the protein encoded by the F2R2 sequence. Samples used in each lane are as follows. Lane M: Molecular weight marker; Lane 1, 2: Transformant 1; Lane 3, 4: Transformant 2; Lane 5, 6: Transformant 3; Lane 1, 3, 5: No expression induction by IPTG; 2 4, 6: Expression induced by IPTG. The arrow indicates the band of F2R2 protein. H. It is a photograph of Western blotting analysis using Swiss infected mouse serum. Samples used in each lane are as follows. Lane M: molecular weight marker; lane 1, 2, secreted protein; lane 3, 4, 5, 6: cell protein; lane 1, 3, 5: no expression induced by IPTG; 2, 4, 6: expression induced by IPTG Yes. The arrow indicates the band of F2R2 protein.

  The polynucleotide, nucleic acid construct, protein or peptide, and antibody or immunoreactive fragment thereof of the present invention can be appropriately prepared by methods well known to those skilled in the art. Moreover, the detection agent and detection kit of the present invention can be appropriately produced by methods well known in the art.

  H. in the present invention. The method for determining the presence of Switzerland is as follows: (i) a polynucleotide having a base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof; (ii) an amino acid sequence described in SEQ ID NO: 3 in a sample derived from a subject Or (iii) measuring (detecting) at least one selected from the group consisting of a protein or peptide having a part thereof and (iii) an antibody recognizing the amino acid sequence described in SEQ ID NO: 3 or a protein or peptide having a part thereof ) To prepare.

(I) measuring (detecting) a polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof; A method for determining the presence of Switzerland by measuring (detecting) a polynucleotide. Detection in Switzerland is a method using hybridization; a method using PCR; and a method known as the Invader (registered trademark) method (for example, Kwiatkowski, RW, et al., Mol. Diagn. (1999) 4: 353-364).

Examples of methods utilizing hybridization include Southern hybridization, Northern hybridization, dot hybridization, fluorescence in situ hybridization (FISH), DNA microarray, ASO method and the like. For example, H. in the present invention. A method for determining the presence of Switzerland may comprise the following steps:
(A) contacting a sample derived from a subject with a nucleic acid construct that binds to at least one of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof;
(B) measuring (detecting) binding of the nucleic acid construct to a polynucleotide in the sample; and
(C) A sample in which a polynucleotide to which the nucleic acid construct binds is measured (detected) A sample in which Switzerland was determined to be present and the polynucleotide to which the nucleic acid construct binds was not measured (detected) By determining Switzerland as a non-existing sample, Step to determine the presence of Switzerland.

In addition, examples of methods using PCR include ARMS (Amplification Reference Mutation System) method, RT-PCR (Reverse transcriptase-PCR) method, and nested PCR method. For example, H. in the present invention. A method for determining the presence of Switzerland may comprise the following steps:
(A) Amplifying a nucleic acid having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof:
(B) detecting the amplification amount of the nucleic acid, and measuring (detecting) the presence of a nucleic acid having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof; and
(C) A sample in which the amplification of the nucleic acid was measured (detected) A sample in which Switzerland was determined to be present and the amplification of the nucleic acid was not measured (detected) By determining Switzerland as a non-existing sample, Step to determine the presence of Switzerland.

  Amplified nucleic acid is obtained by dot blot hybridization method, surface plasmon resonance method (SPR method), PCR-RFLP method, in situ RT-PCR method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method, Examples thereof include AMPFLP (Amplifiable Fragment Length Polymorphism) method, MVR-PCR method, and PCR-SSCP (Single Strand Conformation Polymorphism) method.

When performed using a method known as the Invader (registered trademark) method, for example, it comprises measuring (detecting) a polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention or a part thereof. H. A method for determining the presence of Switzerland may comprise the following steps:
(A) contacting the sample with the nucleic acid described in the following (i) and (ii) to form a triplex in DNA complementary to the allele probe:
(I) an allele-specific probe comprising a sequence complementary to a part of the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 and a sequence (flap) complementary to a part of the quenching probe, and / or SEQ ID NO: 1 or an allele-specific probe comprising a sequence complementary to a part of the base sequence described in SEQ ID NO: 2 and a sequence complementary to a part of the quenching probe (flap),
(Ii) an invader probe comprising a sequence complementary to a part of the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2;
(B) contacting the nucleic acid sample obtained in (a) with a triplex-specific DNA-degrading enzyme to release a flap from the triplexed nucleic acid;
(C) contacting the liberated flap with a fluorescently labeled universal probe each comprising a sequence complementary to the flap and a quenching probe;
(D) contacting the nucleic acid sample obtained in (c) with a triplex-specific DNA-degrading enzyme to release a fluorescent label and causing fluorescence to develop;
(E) a step of measuring (detecting) the polypeptide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 by detecting the colored fluorescence; A sample that was determined as a sample in which Switzerland was present and in which no fluorescence was detected was designated H.264. By determining Switzerland as a non-existing sample, Step to determine the presence of Switzerland.

(Ii) measuring (detecting) a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof; Method for determining the presence of Switzerland H. by measuring (detecting) proteins or peptides. Swiss detection uses labeled immunoassay (EIA method), simplified EIA method, enzyme-linked immunosorbent assay (ELISA method), radioimmunoassay method (RIA method), fluorescent immunoassay method (FIA method), etc. Measurement method; immunoblotting method such as Western blotting method; immunochromatography method such as gold colloid aggregation method; chromatography method such as ion exchange chromatography method and affinity chromatography method; turbidimetric method (TIA method); specific wax method (NIA method); Latex aggregation method (LIA method); particle counting method (CIA method); chemiluminescence measurement method (CLIA method, CLEIA method); precipitation reaction method; surface plasmon resonance method (SPR method); resonant mirror detector method ( RMD method); can be performed by comparative interference method or the like.

Thus, the method of the present invention may comprise the following steps:
(A) contacting a sample derived from a subject with an antibody that recognizes at least one amino acid sequence of SEQ ID NO: 3 or a part thereof, or an immunoreactive fragment thereof (first antibody);
(B) If necessary, an antibody or an immunoreactive fragment thereof (second antibody) that recognizes the amino acid sequence of SEQ ID NO: 3 or a part thereof, which is different from the antibody or the immunoreactive fragment thereof. Contacting the sample obtained by step (a);
(C) measuring (detecting) binding of the protein or peptide having the amino acid sequence of SEQ ID NO: 3 or a part thereof (using a label) to the antibody or immunoreactive fragment thereof;
(C) A sample in which the binding of a protein or peptide to the antibody or immunoreactive fragment thereof is measured (detected). A sample that was determined as a sample in which Switzerland was present and in which the binding of the protein or peptide to the antibody or immunoreactive fragment thereof was not measured (detected) By determining Switzerland as a non-existing sample, Step to determine the presence of Switzerland.
In the above method, the first antibody and / or the second antibody may be labeled as necessary.

(Iii) measuring (detecting) an antibody that recognizes a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof; Method for determining the presence of Switzerland H. by measuring (detecting) antibodies recognizing F2R2. Swiss detection uses labeled immunoassay (EIA method), simplified EIA method, enzyme-linked immunosorbent assay (ELISA method), radioimmunoassay method (RIA method), fluorescent immunoassay method (FIA method), etc. Measurement method; immunoblotting method such as Western blotting method; immunochromatography method such as gold colloid aggregation method; chromatography method such as ion exchange chromatography method and affinity chromatography method; turbidimetric method (TIA method); specific wax method (NIA method); Latex aggregation method (LIA method); particle counting method (CIA method); chemiluminescence measurement method (CLIA method, CLEIA method); precipitation reaction method; surface plasmon resonance method (SPR method); resonant mirror detector method ( RMD method); can be performed by comparative interference method or the like.

More specifically, the method of the present invention may comprise the following steps:
(A) contacting a sample derived from a subject with a protein or peptide having at least one amino acid sequence set forth in SEQ ID NO: 3 or a part thereof;
(B) detecting the binding of the antibody in the subject-derived sample to a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof;
(C) A sample in which the binding of the antibody to a protein or peptide having the amino acid sequence of SEQ ID NO: 3 or a part thereof is measured (detected) A sample which was determined as a sample in which Switzerland was present and in which binding of the antibody to a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof was not measured (detected) By determining Switzerland as a non-existing sample, Step to determine the presence of Switzerland.

  Measurement (detection) of the binding of an antibody in a subject-derived sample to a protein or peptide having the amino acid sequence shown in SEQ ID NO: 3 or a part thereof is performed by, for example, labeling a sample with a labeled anti-IgG antibody or anti-Fc antibody. It can be carried out by contacting and measuring using the label.

  Labeling of an antibody or a fragment thereof can be performed by a general method in the art. For example, when fluorescently labeling a protein or peptide, after washing the protein or peptide with a phosphate buffer, a dye adjusted with DMSO, buffer, etc. is added, mixed, and then allowed to bind at room temperature for 10 minutes. be able to. In addition, as a commercially available labeling kit, a biotin labeling kit (Biotin Labeling Kit-NH2, Biotin Labeling Kit-SH: Dojin Chemical Laboratory Co., Ltd.), an alkaline phosphatase labeling kit (Alkaline Phosphatase Labeling Kit-NH2, Alkaline Phosphatase Kit) SH: Dojindo Laboratories Co., Ltd.), peroxidase labeling kit (Peroxidase Labeling Kit-NH2, Peroxidase Labeling Kit-NH2: Dojindo Laboratories Co., Ltd.), phycobiliprotein labeling kit (Allophycocyanin Labeling Kit-NH2, Allophycocyan) Labeling Kit-SH, B-Phycoerythrin Labeling Kit-NH2, B-Phycoerythrin Labeling Kit-SH, R-Phycoerythrin Labeling Kit-NH2, R-Phycoerythrin Label Lab. Labeling Kit-NH2, HiLyte Fluor (registered trademark) 555 Labeling Kit-NH2, HiLyte Fluor (registered trademark) 647 Labeling Kit-NH2: Dojin Chemical Laboratory Co., Ltd., DyLight 547, DyLight 647 (Technochemical Co., Ltd.) Trademark) Ale a Fluor (TM) antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen), can be labeled using EZ-Label Protein Labeling Kit (Funakoshi Corporation), and the like. In addition, the labeled antibody or a fragment thereof can be detected by using a device suitable for labeling as appropriate.

  H. Because Switzerland widely infects mammals, the H.S. The subject in the method for determining Swiss presence can be a mammal such as a human, rabbit, guinea pig, rat, mouse, cow, sheep, goat, pig, and horse, preferably a human, more preferably A human goose bump gastritis patient. Further, the method of the present invention can be performed qualitatively, quantitatively or semi-quantitatively.

  As described in the specification. As a sample used in the method for determining the presence of Switzerland, for example, a tissue sample collected from a subject as a biopsy or a liquid collected from the subject can be used. The sample is not particularly limited as long as it is a target of the method of the present invention. For example, tissue, blood, plasma, serum, lymph, urine, feces, serous fluid, spinal fluid, joint fluid, aqueous humor, tear fluid , Saliva or fractions or processed products thereof. As described in the specification. When nucleic acid is a detection target in the method for determining the presence in Switzerland, the sample is preferably a tissue (particularly stomach tissue (gastric biopsy sample)) or stool. As described in the specification. In the method of determining the presence of Switzerland, when an antibody is a detection target, blood, plasma, serum, and lymph are preferable as a sample.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. It should be noted that all documents cited throughout this application are incorporated herein by reference in their entirety.

Example 1 Determination of base sequence in Switzerland H. for base sequence determination Swiss preparation H. isolated from cynomolgus monkeys Swiss TKY strain and H. pneumoniae gastric biopsy isolated from female gonorrhea gastritis patients. Swiss SNTW101 strain, and H. isolated from male patients with gastric MALT lymphoma for reference. Swiss SH8 strain and H. pneumoniae isolated from male patients with chronic gastritis. Swiss SH10 strains were passaged in the stomach of different female C57BL / 6J mice. Passage is to suspend the gastric mucosa of infected mice with phosphate buffered saline (BSG, pH 7.4) containing 0.01% gelatin and orally infect uninfected C57BL / 6J mice at 4 weeks of age. It went by. The passage was from one infected mouse to three non-infected mice, and the passage was repeated at intervals of 3-6 months.

2. H. from mouse gastric mucosa suspension Preparation of Swiss genomic DNA H. pylori from suspensions of gastric mucosa of infected mice. In addition to Switzerland, it has been found that H. Genomic DNA was prepared after extracting Switzerland, and the entire base sequence of the prepared DNA was determined using a next-generation DNA sequencer (SOLiD, Life Technology). Specifically, H.C. The mouse gastric mucosa infected with Switzerland is scraped with a glass slide and homogenized with two glass slides in ice-cold phosphate buffer (PBS; pH 7.4) containing 0.05% (vol / vol) Tween20. I let you. Cell clumps were removed by filtering the mucosal homogenate with a cell strainer (40 μm, nylon; BD Falcon, Farranklin Lakes, NJ, USA). In order to avoid non-specific adsorption, the filtered ones were treated with non-immune rabbit IgG-coated magnetic beads (Sepa-Max Hp; Wako Pure Chemical Industries). Non-adsorbed supernatant was collected from rabbit anti-H. Treatment with H. pylori antibody-coated magnetic beads (Sepa-Max Hp; Wako Pure Chemical Industries, Ltd.) separated Helicobacter bacteria. The bacterial cells obtained were suspended in distilled water, heat-treated (95 ° C., 5 minutes) and centrifuged (10,000 g, 5 minutes), and then the supernatant was treated with RNase A (10 μg / mL, 37 ° C., 1 H.) by phenol / chloroform extraction and ethanol precipitation. Swiss genomic DNA was prepared. DNA purity was confirmed by quantitative real-time PCR with CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) using iQ SYBR Green Supermix (Bio-Rad) and the following primer pairs:

  The sequences of each primer are shown in Table 2 below.

  CFX96 real-time PCR was performed with the following protocol: organization and thermal initiation enzyme activation program (95 ° C., 3 minutes), amplification and quantification program (95 ° C., 10 minutes, and 60 ° C., 15 minutes, single fluorescence measurement ) 50 times and melting curve program (65-95 ° C., 0.5 ° C./s heating rate, and continuous fluorescence measurement). Standard serial dilutions were used in each experimental plate, and semi-quantification of 16S RNA and β-actin gene normalization were based on average values. The amount of the target gene was prepared using the chromosomal DNA of the isolated Streptococcus pyogenes GAS472 strain. The yield of bacterial genomic DNA (including H. Switzerland) is Swiss TKY strain or H. 1 μg from one mouse infected with the Swiss SNTW101 strain; Swiss DNA purity was 30-60%.

3. Genome sequencing Whole genome sequencing was performed by cycled ligation sequencing using a SOLiD sequencer (Life Technologies, Carlsbad, CA, USA). Approximately 3-5 μg of purified bacterial genomic DNA was transferred to a Duty Cycle 20%, Intensity 5 using a Covaris S2 system (Covaris Inc., Woburn, Mass., USA) in a Covaris microtube containing 120 μL of 10 mmol / L TE buffer. Cycles per Burst 200, by crushing at 5 ° C. for 60 seconds, cutting into 100-150 bp. The obtained DNA fragment was placed in a fragment library to which a barcode of SOLiD was assigned using a SOLid fragment library construction kit (Life Technologies). The DNA fragment library was amplified by emulsion PCR (ePCR) at a library concentration of 0.5 pmol / L. The 3 ′ end of the template enrichment beads was modified and deposited on a SOLiD sequencer slide. The slide was loaded on a SOLiD4 sequencer, and information of 20 billion bases (20 Gb) or more was obtained with a paired end of 50 + 35 bases.

4). Array assembly The color data obtained from the SOLiD sequencer is converted into the SOLiD de novo accessory tool v. Assembled using 2.0 (Denovo 2). It uses Velvet as an assembly engine, internally combined with pre- and post-processes including error correction, gap filling, and translation from color data to base data. In the assembly with the default parameter set, only very short contigs were obtained (H. Swiss TKY strain 2.12 Mb, H. Swiss SNTW101 strain 1.62 Mb). So, by trying several assembly parameters, one sequence was selected that produced the longest total contig size. The parameter set used for this sequence was hsize = 23, cov cutoff = 0.3, maxcov = 1000, and min_pair_count = 3.

Example 2 Search for Swiss specific sequences H. Comparative analysis of Swiss genomic sequences Numerous contig sequences obtained by assembling analysis and H. coli registered in the EMBL / GenBank / DDBJ database. Swiss HS1 strain, The base sequences of Swiss HS5 strains (both strains isolated from pigs) were aligned using FASTA. Registered in the EMBL / GenBank / DDBJ database. The Swiss contig base sequence is as follows.
HS1 strain GenBank accession No. ADGY00000000
HS5 strain GenBank accession No. ADHO00000000

  In addition, BLASTX search was performed on EMBL / GenBank / DDBJ database for many contig sequences obtained by assembly analysis. As a result, in the case of 89.9%, H.H. Switzerland, other Helicobacter species, and Campylobacter species were top hit sequences. Therefore, RECOG (Research Environment for Comparative Genomics; ///Mbgd.genome.ad.jp/RECOG/), comparative genome analysis was performed.

  As a result, H.C. Two H. pneumoniae common in Switzerland and not present in related genera Campylobacter, Helicobacter and Enterobacteria. A Swiss-specific base sequence was found (FIG. 2: SEQ ID NO: 1 and FIG. 3: SEQ ID NO: 2). One of these sequences (FIG. 2: SEQ ID NO: 1) was a sequence that does not encode an open reading frame, while the other sequence (FIG. 3, SEQ ID NO: 2) contains a gene encoding a protein, This sequence is H.264. The sequence was not present in the chromosomes of other Helicobacter species other than Switzerland. A sequence close to this sequence was searched in the database. There were no sequences derived from sources other than Switzerland that gave more than 50% homology. The closest sequence is 44% identical to the sequence of the carros gene of Azospirillum brasilense, but this fungus is a bacterium in the soil that helps the plant growth by carbon fixation, and the carR gene is a gene of the gene related to carbon fixation. It was a regulatory gene of expression. H. Since Switzerland is thought to have no carbon fixation function, the protein found by this experiment is considered to be a protein different from carR. Therefore, the present inventors named the gene encoded by this base sequence F2R2.

Example 3 H.P. Detection of Switzerland PCR primers targeting the two sequences obtained in Example 2 above were constructed. 5 Swiss strains By performing PCR using DNA prepared from Helicobacter pylori 5 and other Helicobacter 7 strains as a template, these two target sequences are transformed into H. pylori. Confirmed to be Swiss-specific.

1. Preparation of PCR template DNA Using the DNeasy Blood & Tissue kit (Qiagen, Dusseldorf, Germany), DNA was extracted from the strains listed in Table 6 below and from gastric biopsy samples of 14 goose dermatitis patients according to the manufacturer's protocol. Thus, template DNA for PCR was prepared. Among the strains listed in Table 6, those whose preparation method is “cultivation” are strains that can be cultured. Therefore, template samples were prepared by culturing by the methods described below. In addition, among the strains described in Table 6, those whose preparation method is “mouse gastric mucosa” are strains that cannot be cultured, so that template samples were obtained from the stomach mucosa of infected mice according to the method described in Example 1. Was prepared.

The culture method of the culturable strain is as follows.
H. Ferris ATCC 49179 strain, H.P. Mustele NCTC 12032, H.C. H. pylori SS1 strain, H. pylori. H. pylori TN2GF4 strain, H. pylori. H. pylori NCTC11637 strain, H. pylori H. pylori ATCC 43579 strain and H. pylori The H. pylori RC-1 strain was inoculated on Brucella (Oxoid Ltd., Hampshire, UK) agar plates containing 7% (vol / vol) inactivated calf serum (FCS), 5% O ₂ , 10% CO _2. In a mixed gas of 85% N ₂ , the cells were cultured for 3 days at 37 ° C. and 100% humidity. The resulting colonies were picked and cultured in a liquid medium for another 3 days with shaking.
H. Swiss HS5 strain (isolated from pig) Hylemany s. s. ASB1.4 strain (isolated from cat) Bakry Formis M50, H. Bizozerony R1051 strain, H.P. Synogatrix JKM4 strain and H. Salmonis R1053 strain was cultured according to previously reported culture conditions (Baele M et al., Int J Syst Evol Microbiol (2008) 58: 1350-8; Vermote M et al., Vet Res (2011) 42:51; Joosten M et al. , Vet Res (2013) 44:65; Baele M et al., Int J Syst Evol Microbiol (2008) 58: 357-64; Baele M et al., Baele M et al., J Clin Microbiol (2004) 42: 1115-22; Van den. See Bulk K, et al., Int J Syst Evol Microbiol (2006) 56: 1559-64).

2. PCR reaction The 1 × PCR reaction solution (total volume 25 μL) had the following composition.
200 μM dNTP mix 1.5 mM MgCl ₂
0.2 μM primer pair 10 ng DNA prepared from cultured strain,
Or DNA prepared from 100 ng mouse gastric mucosa
1 unit Tfi DNA polymerase (Invitrogen, Carlsbad, CA, USA)
Primer pairs were VAC3624F / VAC4041R (for detecting vacA gene of H. pylori), HH-F5 / HH-R4 (for detecting H. Swiss untranslated region: Fig. 2 and SEQ ID NO: 1), F2R22F / F2R22R (H For detection of Swiss F2R2 gene: the combination of Fig. 3 and SEQ ID NO: 2) was used. The sequence of each primer is shown in Table 7.

  The PCR reaction was performed using Mastercycler ep (Eppendorf AG, Hamburg, Germany) according to the following procedure. After denaturation and hot start at 94 ° C. for 1 minute, the amplification program was repeated 35 times at 94 ° C. for 15 seconds, 57 ° C. for 30 seconds, and 72 ° C. for 1 minute. Then, it was made to react at 72 degreeC for 10 minutes as final extension. 1 μL of 6 × loading buffer was added to 5 μL of the obtained PCR reaction solution, separated by 2.0% (wt / vol) agarose gel electrophoresis, and the resulting DNA band was detected by staining with exidium bromide.

  The results are shown in FIG. The two H.C. found in Example 2. Swiss specific sequences are Amplified in all Swiss strains (TKY, SNTW101, SH8, and SH10). It was not amplified in H. pylori and other Helicobacter 7 strains. On the other hand, H. The vacA gene of H. pylori is H. pylori. It was amplified only with the H. pylori sample. Thus, the two H.C. Swiss-specific sequences are A common sequence in Switzerland; Confirmed to be specific to Switzerland.

Example 4 Diagnosis of human gastric biopsy by PCR Preparation of human gastric biopsy sample Human gastric biopsy samples of 14 goosebumps gastritis patients were suspended in physiological saline and then used for PCR using DNeasy Blood & Tissue kit (Qiagen; Dusseldorf, Germany) according to the attached instructions. Template DNA was prepared.

2. H.P. Swiss infection diagnosis Reaction solution (total volume 25 μL) was prepared as follows.
1 × PCR reaction solution 200 μM dNTP mix 1.5 mM MgCl 2
0.2 μM primer pair (using VAC3624F / VAC4041R and HH-F2 / HH-R2)
DNA prepared from 10 ng cultured strain,
Or DNA prepared from 100 ng mouse gastric mucosa,
Or DNA prepared from 100 ng human gastric biopsy
1 unit Tfi DNA polymerase (Invitrogen, Carlsbad, CA, USA)

  The PCR reaction was performed using Mastercycler ep (Eppendorf AG, Hamburg, Germany) according to the following procedure. After denaturation and hot start at 94 ° C. for 1 minute, the amplification program was repeated 35 times at 94 ° C. for 15 seconds, 57 ° C. for 30 seconds, and 72 ° C. for 1 minute. Then, it was made to react at 72 degreeC for 10 minutes as final extension. 1 μL of 6 × loading buffer was added to 5 μL of the obtained PCR reaction solution, separated by 2.0% (wt / vol) agarose gel electrophoresis, and the resulting DNA band was detected by staining with exidium bromide.

  The results are shown in FIG. From the gastric biopsy of 14 goose bump gastritis patients, H. pylori or H. pylori. We were able to detect a Swiss infection. All UBT positive patients are H.264. H. pylori infection was observed, and one UBT negative patient (lane 16) was treated with H. pylori. A Swiss infection was confirmed. Thus, the method of the present invention can be used in H.B. It was shown that it can detect Swiss infections.

(Example 5) Confirmation of expression of F2R2 gene Since one of the Swiss-specific sequences (FIG. 3, SEQ ID NO: 2) contains a gene encoding a protein, it was confirmed whether or not this F2R2 gene actually produces a protein as an expression product. .
The F2R2 sequence was cloned into pETite N-His and transformed with E. coli BL21 (DE3). Three transformants were selected and cultured in a Lennox medium containing 1 mM (medium concentration) of isopropyl β-D-1-thiogalactopyroside (IPTG) with shaking overnight at 37 ° C. to prepare recombinant F2R2 protein. Proteins were analyzed by 12% polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. Proteins secreted from the microbial cells during culture and microbial proteins (20 μg each) were separately analyzed by SDS-PAGE.

  The results are shown in FIG. In any Escherichia coli, F2R2 was expressed as an intracellular protein by IPTG stimulation. Therefore, it was confirmed that F2R2 is indeed a gene expressed at the protein level.

Example 6 Detection of anti-F2R2 antibody in the blood of Swiss-infected mice The secreted protein and bacterial protein (20 μg) of transformant 1 in FIG. 6 were separately separated by SDS-PAGE, transferred to a PVDF membrane, and diluted 200-fold. . Reaction was performed with antiserum prepared from a 1-year-old mouse in Switzerland or control serum prepared from a non-infected mouse, and further reacted with a secondary antibody (Peroxidase-conjugated affinity antigen-mouse IgG) diluted 10,000 times. Finally, a chemiluminescence detection reagent was used to detect a protein band specifically recognized by the antibody.

  The results are shown in FIG. H. In the serum of Swiss-infected mice, there was an antibody that reacts with a protein (F2R2 protein) in IPTG-induced cells. That is, H.I. In the blood (serum) of Swiss infected animals, It was confirmed that an antibody recognizing F2R2, a Swiss-specific protein, was included. Therefore, by measuring / detecting anti-F2R2 antibody in the sample using the blood (serum) of the subject as a sample, It has been shown that it is possible to test / diagnose / determine Swiss infections.

Claims (9)

  In a sample derived from a subject, (i) a nucleic acid having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof, (ii) a protein or peptide having the amino acid sequence described in SEQ ID NO: 3 or a part thereof, And (iii) measuring at least one selected from the group consisting of antibodies that recognize proteins or peptides having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof, in the sample. How to determine the presence of Switzerland.   The method according to claim 1, wherein the subject is a goosebumps gastritis patient.   The method according to claim 1 or 2, wherein the sample is a gastric biopsy sample or stool, or blood or urine.   H. A polynucleotide having the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof for use in a method for determining the presence of Switzerland.   H. A nucleic acid construct that specifically binds to a polynucleotide having the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof, for use in a method for determining the presence of Switzerland.   H. A protein or peptide having the amino acid sequence of SEQ ID NO: 3 or a part thereof for use in a method for determining the presence of Switzerland.   H. An antibody or an immunoreactive fragment thereof that recognizes a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 3 or a part thereof for use in a method for determining the presence of Switzerland. H. containing at least one selected from the following group: Swiss detection drugs:
(A) the nucleic acid construct according to claim 5;
(B) the protein or peptide of claim 6; and
(C) The antibody or immunoreactive fragment thereof according to claim 7. H. comprising the detection agent according to claim 8. Swiss detection kit.