JP2005151854A - Marker for diagnosing human subarachnoid hemorrhage and use of the marker - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polypeptide marker and a gene marker for diagnosing subarachnoid hemorrhage for use in detecting and diagnosing subarachnoid hemorrhage lesions and capable of specifically detecting the lesions, to provide an antibody specifically bindable to the polypeptide, to provide a probe for detecting the expression of the gene for diagnosing subarachnoid hemorrhage, and to provide respective methods for detecting and diagnosing the lesions using the marker. <P>SOLUTION: A polypeptide having an amino acid sequence represented by sequence No.1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 in the sequence table and a gene encoding the polypeptide are used as the objective polypeptide marker and the objective gene marker for diagnosing subarachnoid hemorrhage capable of specifically detecting the subarachnoid hemorrhage lesions. The antibody for detecting the polypeptide marker, the DNA probe for detecting the gene marker, and the like are also included. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to detection and diagnosis of human subarachnoid hemorrhage lesions, and in particular, a polypeptide marker for diagnosing human subarachnoid hemorrhage and a gene for diagnosing human subarachnoid hemorrhage The present invention also relates to a marker and a method for detecting and diagnosing human subarachnoid hemorrhage using the marker.

  Subarachnoid hemorrhage is a symptom of bleeding into the subarachnoid space, which is mainly caused by a ruptured cerebral aneurysm. An aneurysm is a state in which an artery is dilated with reduced resistance to internal pressure. The causes of aneurysms are generally (1) arteriosclerosis, (2) trauma, (3) nonspecific arteritis, (4) Idiopathic saccular medial necrosis, (5) syphilis, (6) bacterial infection, (7) dilatation after stenosis and the like. In recent years, it has been reported that many are caused by arteriosclerosis. Symptoms of lesions of subarachnoid hemorrhage are meningeal irritation symptoms and cerebral hypertension symptoms caused by blood spreading in the subarachnoid space. In the latter case, headache, nausea, sore throat and even fundus bleeding appear. In bleeding due to a ruptured cerebral aneurysm, disturbance of consciousness appears. The occurrence of cerebral aneurysms is known to have a relatively strong genetic background from epidemiological studies (Neurosurgery, 46 (6) 1301-1306, 2000).

  For the diagnosis of cerebral aneurysms, blood and biochemical diagnostic methods have not been conventionally used practically, and radiological diagnostic methods such as MRI and CTscan are performed.

Recently, several biochemical diagnostic methods have been disclosed in connection with the diagnosis of cerebral aneurysms. For example, by identifying genetic polymorphisms part of intron of the human elastin gene, which determine the presence of risk of developing cerebral aneurysms (JP 2002-238577 JP), Na ⁺ -HCO ₃ ^- co Diagnosis of brain disorders such as cerebral infarction and subarachnoid hemorrhage using antibody of transporter protein (JP 2000-23685 A, WO 00/37637), human brain type prostaglandin D synthesis in body fluid sample One that detects intracranial hemorrhage such as subarachnoid hemorrhage using the enzyme (hPGDS) concentration as an index (WO98 / 49999), one that measures creatine kinase isozyme BB and detects vascular disorder disease (WO98 / 40749), Vascular disorders such as aneurysms using monoclonal antibodies against human smooth muscle myosin and its active fragments (WO97 / 29784) and the like are disclosed.

As described above, several methods have been recently disclosed for the diagnosis of subarachnoid hemorrhage lesions, but their detection is not necessarily applied to subarachnoid hemorrhage lesions such as cerebral aneurysms. Therefore, it is not a specific detection and diagnosis method. Therefore, for the specific diagnosis of a lesion of arteriosclerosis, further development of a marker capable of specifically detecting the lesion is desired.
JP 2002-238777 A. Japanese Unexamined Patent Publication No. 2000-236885. WO00 / 37637. WO 98/49599. WO 98/40749. WO 97/29784. Neurosurgery, 46 (6) 1301-1306,2000.

  An object of the present invention is to use a polypeptide marker for diagnosing subarachnoid hemorrhage and a genetic marker for diagnosing subarachnoid hemorrhage, which can be used for detection and diagnosis of subarachnoid hemorrhage lesions, Provided are an antibody that specifically binds to a polypeptide for diagnosing subarachnoid hemorrhage, a probe for detecting expression of the gene for diagnosing subarachnoid hemorrhage, and a method for detecting and diagnosing subarachnoid hemorrhage lesions using the marker There is to do.

  The present inventor has detected the onset of a cerebral aneurysm capable of specifically detecting a subarachnoid hemorrhage lesion and found a diagnostic marker that enables diagnosis of the subarachnoid hemorrhage. As a result of searching for proteins expressed in patient sera, 12 clones showing positive reaction with many patient sera from proteins expressed by introducing cDNA obtained from cerebral aneurysm patient sera into E. coli The clone was detected, and the onset of a cerebral aneurysm was detected, and it was found that it became a diagnostic marker that enables diagnosis of subarachnoid hemorrhage, and the present invention was completed. The present invention includes the polypeptide marker for diagnosis of subarachnoid hemorrhage of the present invention, and an antibody that specifically binds to the polypeptide marker for detecting the polypeptide marker. The amino acid sequence of the polypeptide for diagnosis of subarachnoid hemorrhage of the present invention is shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 in the sequence listing.

That is, the polypeptide for diagnosing subarachnoid hemorrhage of the present invention comprises a polypeptide encoded by the following gene.
(1) Cerebral aneurysm marker-1 (SEQ ID NO: 1 in the sequence listing)
Clone name: 3A1
Accession No .: NM # 015874
Gene name: H-2K binding factor-2
Gene description: A transcription factor that binds to the NFκB site of the MHC class I gene.
Literature: Proc. Natl. Acad. Sci. USA 100 (14), 8490-8495, 2003
(2) Cerebral aneurysm marker-2 (SEQ ID NO: 3 in the sequence listing)
Clone name: 6T1
Accession No .: XM # 039236
Gene name: molecule interacting with Rab13
Gene description: A protein that has a calponin domain and seems to be related to Rab13, but its functional details are unknown.
Literature: Unpublished (3) Cerebral aneurysm marker-3 (SEQ ID NO: 5 in the sequence listing)
Clone name: 11C1
Accession No .: NM # 004446
Gene name: Glutamyl-prolyl-tRNA synthetase
Literature: Genomics 19 (2), 280-290, 1994
(4) Cerebral aneurysm marker-4 (SEQ ID NO: 7 in the sequence listing)
Clone name: 11D1
Accession No .: NM # 002337
Gene name: Low density lipoprotein-related protein-associated protein 1
Gene description: Contributes to the stabilization of the LDL receptor. There are reports that it is related to intracellular signal transduction.
Reference: J. Biol. Chem. 278 (20), 17986-17992, 2003
(5) Cerebral aneurysm marker-5 (SEQ ID NO: 9 in the sequence listing)
Clone name: 1112
Accession No .: NM # 014497
Gene name: NP220 nuclear protein
Gene description: One of nuclear proteins. It binds to double-stranded DNA and is involved in the regulation of transcription.
Reference: J. Cell. Biochem. 84 (3), 556-566, 2002
(6) Cerebral aneurysm marker-6 (SEQ ID NO: 11 in the sequence listing)
Clone name: 11P1
Accession No .: BC014861
Gene name: Actin, beta
Gene Description: Cytoskeleton constituent protein.
Literature: Proc. Natl. Acad. Sci. USA 99 (26), 16899-16903, 2002
(7) Cerebral aneurysm marker-7 (SEQ ID NO: 13 in the sequence listing)
Clone name: 14C1
Accession No .: NM # 003563
Gene name: Speckle-type POZ protein
Gene description: Protein with self-antigenicity. It has a POZ domain and has DNA or actin binding properties to regulate protein interactions.
Reference: FEBS Lett. 418 (1-2), 23-25, 1997
(8) Cerebral aneurysm marker-8 (SEQ ID NO: 15 in the sequence listing)
Clone number 14N
Accession No .: BC004544.
Gene name: hypothetical protein BC004544
Gene description: Unknown function.
Literature: Submitted (14-MAR-2001) National Institutes of Health Mammalian Institute
(9) Cerebral aneurysm marker-9 (SEQ ID NO: 17 in the sequence listing)
Clone name: 16A
Accession No .: NM # 014832.
Gene name: TBC1 domain family, member 4
Gene description: Unknown function.
Literature: Genomics 79 (2), 154-161, 2002
(10) Cerebral aneurysm marker-10 (SEQ ID NO: 19 in the sequence listing)
Clone name: 17L
Accession No .: BC043617
Gene name: DNA (cytosine-5-)-methyltransferase 3 alpha
Gene description: Documents that perform DNA methylation required during embryo development: Proc. Natl. Acad. Sci. USA 99 (26), 16899-16903, 2002
(11) Cerebral aneurysm marker-11 (SEQ ID NO: 21 in the sequence listing)
Clone name: 41D3
Accession No .: NM # 005531
Gene name: interferon, gamma-inducible protein 16
Gene Description: Shares 200 residues with other interferon gamma-inducible proteins presumed to be involved in myeloid differentiation.
Reference: Oncogene 22 (31), 4831-4840, 2003
(12) Cerebral aneurysm marker-12 (SEQ ID NO: 23 in the sequence listing)
Clone name: 44Q2
Accession No .: NM # 001810
Gene name: centromere protein B, 80kDa
Gene Description: A protein that has the function of collecting centromere structures in the nucleus at the middle and mitotic stages of the cell cycle. Known as autoantigen in patients with anti-centromere antibodies.
Atherosclerotic lesion marker literature: Mol. Cell. Biol. 22 (7), 2229-2241, 2002
Furthermore, the present invention relates to a subarachnoid hemorrhage gene marker comprising a base sequence encoding the polypeptide for diagnosing subarachnoid hemorrhage of the present invention, and a subarachnoid hemorrhage having a DNA sequence that hybridizes to the base sequence under stringent conditions. It includes a diagnostic marker gene detection probe. The base sequence of the marker gene for diagnosis of subarachnoid hemorrhage of the present invention is shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 in the sequence listing.

  The present invention also provides a method for detecting and diagnosing subarachnoid hemorrhage caused by the onset of cerebral aneurysm using the polypeptide marker for diagnosing subarachnoid hemorrhage and the gene marker for diagnosing subarachnoid hemorrhage according to the present invention, and It includes kits used for diagnostic methods.

In the present invention, the background to the completion of the invention will be described. The present inventor is able to detect a subarachnoid hemorrhage lesion without specifically detecting a subarachnoid hemorrhage lesion. Sera of patients with subarachnoid hemorrhage lesions admitted to the hospital were screened with the consent of the person or family. The obtained cDNA clone was incorporated into the plasmid pBlueScriptII and sequenced. Thereafter, the plasmid pGEX was recombined and introduced into Escherichia coli, and IPTG was added to express a large amount of protein to prepare a protein extract. Using it, the reaction with many patient sera was examined by Western method. As a result, it was found that 12 clones, which are markers of the present invention, showed a positive reaction with sera of a plurality of cerebral aneurysm patients and were useful as markers for predicting cerebral aneurysm morbidity or cerebral aneurysm rupture. Then, using the antigenicity of these clones and gene probes, we developed a detection and diagnosis method for subarachnoid hemorrhage lesions, and furthermore, it is possible to create a diagnostic kit using the gene and protein array used in the detection and diagnosis method It was.

  Specifically, the present invention specifically relates to the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 in the sequence listing, or a partial amino acid sequence thereof. A polypeptide marker for diagnosis of human subarachnoid hemorrhage (Claim 1), a human marker for diagnosis of subarachnoid hemorrhage consisting of a base sequence encoding the polypeptide of Claim 1 (Claim 2), The human subarachnoid hemorrhage according to claim 2, which has the DNA sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 in the sequence listing. A diagnostic gene marker (Claim 3) or an antibody derived from the polypeptide of Claim 1 and specifically binding to the polypeptide (Claim 4), or an antibody comprising a monoclonal antibody The antibody according to claim 4 (Claim 5), the antibody according to claim 4, wherein the antibody is a polyclonal antibody, (Claim 6), or Claim 2 or A probe for detecting a human subarachnoid hemorrhage marker gene having a DNA sequence that hybridizes under stringent conditions with the base sequence according to claim 3 (claim 7), or all or one of the antisense strands of the base sequence according to claim 3. A human subarachnoid hemorrhage marker gene detection probe according to claim 7 (Claim 8) or at least one DNA of Claim 7 or 8 immobilized thereon It consists of a microarray for detecting bleeding marker genes or a DNA chip (claim 9).

  The present invention also uses the antibody according to any one of claims 4 to 6 to detect the expression of a marker polypeptide for diagnosis of human subarachnoid hemorrhage in a test sample. And Western blot method, ELISA method, protein array method, radioimmunoassay method, fluorescent antibody method, or SEREX method for detecting the expression of the diagnostic polypeptide (Claim 10) and marker polypeptide for diagnosis of human subarachnoid hemorrhage The method for detecting and diagnosing arteriosclerosis according to claim 10 (claim 11) or the serum from a subject according to claim 10 is reacted with the marker polypeptide for diagnosis of human subarachnoid hemorrhage according to claim 1. A method for detecting and diagnosing a human subarachnoid hemorrhagic lesion characterized by detecting the expression of a serum antibody in a subject (claim 12), or claim 7 or 8. A method for detecting and diagnosing a human subarachnoid hemorrhage lesion characterized by detecting expression of a human subarachnoid hemorrhage marker gene in a test cell using a diagnostic probe, or a human subarachnoid hemorrhage marker gene 14. Detection of human subarachnoid hemorrhage lesion according to claim 13 and detection of human subarachnoid hemorrhage marker gene expression, and detection of human subarachnoid hemorrhage marker gene The method for detecting and diagnosing human subarachnoid hemorrhage lesion according to claim 13, wherein the human subarachnoid hemorrhage marker gene detection microarray or DNA chip according to claim 9 is used. Detection of human subarachnoid hemorrhage marker gene expression according to any of claims 13 to 14 using quantitative or semi-quantitative PCR The method for detecting and diagnosing a human subarachnoid hemorrhage lesion characterized by the fact that the use of quantitative or semi-quantitative PCR is RT-PCR (Claim 16). A method for detecting and diagnosing arteriosclerosis of the present invention (Claim 17), and amplifying a gene in a test cell using at least one pair of a predetermined sense primer and antisense primer, and the amplified gene Or a method for detecting and diagnosing a human subarachnoid hemorrhage lesion according to claim 13 (claim 18), or a method for detecting and diagnosing a human subarachnoid hemorrhage lesion according to claim 7, The probe for detecting a human subarachnoid hemorrhage marker gene, the microarray or DN for detecting a human subarachnoid hemorrhage marker gene according to claim 9, wherein the probe is immobilized. A kit for detecting and diagnosing a human subarachnoid hemorrhage lesion comprising the A chip and at least one of the antibodies according to any one of claims 4 to 6 (claim 19).

  Conventionally, radiological diagnostic methods such as MRI and CTscan have been used to diagnose arteriosclerotic lesions of subarachnoid hemorrhage lesions. In order to lead to early detection of subarachnoid hemorrhage lesions, The method by physical inspection is important. By using the polypeptide marker or gene marker of the present invention, it becomes possible to detect and diagnose specific lesions of subarachnoid hemorrhage, leading to early detection of lesions of subarachnoid hemorrhage. For detection of the polypeptide marker or gene marker of the present invention, an antibody that binds to the marker polypeptide of the present invention or a DNA probe that hybridizes to the marker gene of the present invention is prepared in the form of a chip or an array. Thus, it is possible to detect and diagnose a lesion of subarachnoid hemorrhage by a simple operation, and it can be expected to greatly contribute to early detection of subarachnoid hemorrhage.

  The present invention detects and diagnoses a subarachnoid hemorrhage lesion by detecting the expression of a marker polypeptide for diagnosing subarachnoid hemorrhage or a marker gene for diagnosing subarachnoid hemorrhage that is specifically expressed in a lesion of subarachnoid hemorrhage Consists of. In the present invention, polypeptides serving as polypeptide markers for diagnosis of subarachnoid hemorrhage are shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 in the sequence listing. A polypeptide having an amino acid sequence. In the present invention, the gene serving as a gene marker for diagnosing subarachnoid hemorrhage is a base represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 in the sequence listing. A gene having a sequence.

  Polypeptides shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 in the sequence listing are any of the polypeptides shown in the cerebral aneurysm markers 1-12. Is also a known polypeptide. The gene is also a known gene. The amino acid sequence and DNA sequence information of the marker polypeptide for diagnosing subarachnoid hemorrhage and the marker gene by detecting the aneurysm of the present invention are the accession numbers: NM — 015874, XM — 039236, NM — 004446, NM — 002337, NM — 014497, in the NCBI gene database. , BC0144861, NM_003563, BC004544, NM_0143482, BC043617, NM_005531, and NM_001810.

  In the present invention, in order to detect the expression of the marker gene for diagnosis of subarachnoid hemorrhage of the present invention, a known gene expression detection method can be used. For example, the Northern blotting method can be used to detect the expression of the marker gene for diagnosis of subarachnoid hemorrhage of the present invention. In addition, in order to detect and identify a subarachnoid hemorrhage lesion using the gene marker of the present invention, a probe having a DNA sequence that hybridizes under stringent conditions with the DNA sequence of the gene marker for subarachnoid hemorrhage diagnosis of the present invention Can be used. In order to detect lesions of subarachnoid hemorrhage using the probe, it can be appropriately performed using a known method. For example, a DNA probe having an appropriate length is prepared from the DNA sequence of the gene marker shown in the sequence listing, and a label such as a fluorescent label is appropriately added, and this is hybridized with a specimen, thereby subarachnoid hemorrhage. The expression of the marker gene is detected. As the DNA probe, a probe for detecting a marker gene for diagnosis of subarachnoid hemorrhage consisting of all or part of the antisense strand of the base sequence of the gene marker of the present invention shown in the sequence table can be used. In addition, the probe can be used in the form of a microarray or DNA chip for detecting a marker gene having at least one or more immobilized thereon.

  In the preparation of the above DNA probe, the conditions of “hybridize under stringent conditions with the DNA sequence of the marker gene for diagnosis of subarachnoid hemorrhage” in the base sequence of the present invention include, for example, hybridization at 42 ° C. , And 1 × SSC (0.15M NaCl, 0.015M sodium citrate), and a washing solution at 42 ° C. with a buffer containing 0.1% SDS (Sodium dodecyl sulfate). And a washing treatment at 65 ° C. with a buffer containing 0.1 × SSC and 0.1% SDS can be more preferably mentioned. In addition to the above temperature conditions, there are various factors that influence the stringency of hybridization. Those skilled in the art can combine various elements to obtain a string equivalent to the above-described hybridization stringency. It is possible to realize a genie.

  In the present invention, the polypeptide marker for diagnosis of subarachnoid hemorrhage of the present invention can be detected using an antibody that is induced by the polypeptide and specifically binds to the polypeptide. Examples of the antibody include a monoclonal antibody and a polyclonal antibody. The antibody can be prepared by a conventional method using the polypeptide marker of the present invention as an antigen. In order to detect the expression of a marker polypeptide for diagnosis of subarachnoid hemorrhage in a test sample using the antibody of the present invention, it can be performed using an immunological assay using a known antibody. Examples of the immunological measurement method include a Western blot method, an ELISA method, a radioimmunoassay method, and a fluorescent antibody method. Moreover, SEREX (Serological iderecombinant expression cloning) method which is a serological antigen identification method by recombinant expression cloning can also be used.

  In the present invention, the marker gene detection probe for diagnosis of subarachnoid hemorrhage of the present invention and / or the antibody for detection of marker polypeptide for diagnosis of subarachnoid hemorrhage of the present invention and / or the marker antibody detection of diagnosis of subarachnoid hemorrhage of the present invention Using the antigen polypeptide, the expression of a marker gene for diagnosis of subarachnoid hemorrhage and / or marker polypeptide for diagnosis of subarachnoid hemorrhage and / or marker antibody for diagnosis of subarachnoid hemorrhage is detected in a test sample, and lesions of subarachnoid hemorrhage Can be diagnosed. In detecting a marker gene for diagnosis of subarachnoid hemorrhage in a test cell, quantitative or semi-quantitative PCR can be used to amplify the gene in the test sample. RT-PCR (reverse transcription PCR) can be used as the PCR. When performing the PCR, a primer comprising a sense primer and an antisense primer for amplifying the marker gene for diagnosis of subarachnoid hemorrhage of the present invention is used. The primer can be appropriately constructed based on the nucleotide sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 in the sequence listing.

  In the present invention, to detect and diagnose lesions of subarachnoid hemorrhage, the gene in the cells of the test sample was amplified using at least one or more pairs of the above-described sense primer and antisense primer, and amplified. The gene is detected using the marker gene detection probe for diagnosis of subarachnoid hemorrhage of the present invention.

  The expression of a marker polypeptide for diagnosis of subarachnoid hemorrhage in a test sample cell can be detected by using a fluorescent antibody method using the antibody of the present invention, and a lesion of subarachnoid hemorrhage can be detected visually. In order to label a sample cell expressing a marker polypeptide for diagnosing subarachnoid hemorrhage by the fluorescent antibody method, an antibody that specifically binds to the polypeptide marker for diagnosing subarachnoid hemorrhage of the present invention is fluorescently labeled, and this is labeled with an antigen. Either bind to the expressed sample cells and label cells expressing the polypeptide marker for subarachnoid hemorrhage diagnosis (direct fluorescent antibody method) or unlabeled cells on antigen-expressing cells After binding the specific antibody of the invention, a labeled secondary antibody (anti-immunoglobulin antibody) is bound to label the polypeptide for diagnosis of subarachnoid hemorrhage (indirect fluorescent antibody method), and the labeled subarachnoid hemorrhage Diagnosis is performed by detecting cells expressing the diagnostic polypeptide.

  Probe for detecting marker gene for diagnosis of subarachnoid hemorrhage used for detection and diagnosis of subarachnoid hemorrhage lesion of the present invention, microarray or DNA chip for detecting marker gene for diagnosis of subarachnoid hemorrhage to which the probe is fixed, and subarachnoid hemorrhage of the present invention Antibodies for detecting diagnostic marker polypeptides can be commercialized as kits for detecting and diagnosing subarachnoid hemorrhagic lesions equipped with them.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Experimental procedure-SEREX method, Western method, ELISA method]
(Sample preparation)
(1) Targeting patients with carotid artery stenosis who visited hospitals and research cooperation facilities, normal subjects with matched gender and age (± 5 years) were selected as controls from outpatients. At the time of outpatient visit or at the time of hospitalization for neurosurgery, 1) Explaining the purpose of the study and the voluntary nature of research cooperation to the family and obtaining informed consent, 2) Family history, past history, drinking alcohol, difficulty in smoking, etc. Inquire about lifestyle and working mode. 3) Collect blood to separate and store serum and blood cell components. In addition, 4) Describe the clinical severity, treatment course, blood test findings, etc. based on the doctor's medical record. The patient blood to be analyzed is cryopreserved until the start of the study at minus 80 degrees after serum separation.
(2) A commercially available λZAP II phage vector (STRATAGENE) incorporating a human umbilical vein endothelium (HUVEC) -derived cDNA library is used as a screening target with serum.

(SEREX method experimental procedure)
(3) Screening by the SEREX method is performed according to the method of Sahin et al.
1) The phage vector of (2) above is infected with E. coli (XL1-Blue) and cultured on a φ15 cmNZY agar plate medium.
2) After confirming the appearance of plaques, place a nitrocellulose membrane treated with IPTG (isopropyl thiogalactoside) on the medium to express and transfer the phage-derived protein on the membrane.
3) The patient's serum diluted 2000 times with 0.5% BSA and the membrane are incubated overnight to react the expressed protein with the antibody in the serum.
4) After washing, alkaline phosphatase-labeled goat anti-human IgG antibody is reacted with the membrane as a secondary antibody.
5) Identify serum IgG-recognizing clones using NBT and BCIP as coloring reagents.
6) Perform positive and secondary screening (φ10 cm plate) for positive clones and eliminate false positive clones.
7) The selected phage is cloned into pBlueScript using the ExAssist helper phage system (Stratagene, La Jolla, CA).
8) Purify the plasmid using Rapid plasmid miniprep system (Marigen).
9) The nucleotide sequence is determined by ABI PRISM 3700 DNA Analyzer (Applied Biosystems) using outsourcing or DNA Sequencing kit Big Dye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems). The homology search of clones that can be obtained by collating the obtained base sequence with the base sequence on the public database is performed. At this point, clones with different reading frames are excluded, and the remaining clones are used as antigen protein candidates to proceed to the protein purification process.

(ELISA method experimental procedure)
(4) Preparation of ELISA method as secondary screening 1) Recombination of pBlueScript containing an antigen protein candidate insert into a protein expression and purification vector pGEX-4T (Amersham Bioscience). A restriction enzyme suitable for recombination into pGEX from pBlueScript, pGEX plasmid (4T1-3) is selected from the obtained base sequence information, and restriction enzyme treatment is performed.
2) After polyacrylamide gel electrophoresis, the target band is excised using the purification kit GeneElute Minus EtBr Spin Columns (SIGMA), and the restriction enzyme-treated insert and pGEX are recovered.
3) Ligation of the insert and pGEX using Ligation kit ver.2 (Takara) produces a plasmid containing the insert.
4) If an appropriate restriction enzyme does not exist depending on the clone, an insert is prepared by PCR. A primer having a restriction enzyme recognition site is prepared in advance by outsourcing, and reverse transcription PCR is performed using RNA extracted from an aneurysm tissue as a template to prepare cDNA, and further PCR is performed to obtain a full-length insert. Similarly, ligation using restriction enzyme and ligation kit is performed as follows. 5) Transformation of pGEX obtained in E. coli competent cell BL21 (DE3) RIL codon plus (STRATAGENE) improved for eukaryotic protein expression 6) Culture in ampicillin-containing LB medium and induce expression of insert DNA-derived protein with IPTG.
7) Centrifugal recovery of E. coli and sonication and separation into soluble and insoluble fractions 8) Solubilization with urea when the target protein enters the insoluble fraction 9) Obtained soluble protein The GSTrapFF (Amersham Bioscience) is used to purify only the GST fusion protein 10) Protein extraction from the column is performed by thrombin cleavage.

(Western method experimental procedure)
(5) Secondary screening by Western method 1) The transformed BL21 (DE3) RIL codon plus obtained above is cultured overnight in 2 ml of LB ampicillin.
2) Transfer to 20 ml of LB ampicillin and incubate for 1 hour and add to a final IPTG concentration of 1 mM and control for another 3 hours.
3) After centrifuging the culture solution, E. coli is recovered, dissolved in a sample buffer, and used as a Western method sample.
4) After electrophoresis of the sample on a 12% polyacrylamide gel, transfer the protein to the nitrocellulose membrane using a flat plate transfer device.
5) After blocking with 1% skim milk, add 5000 times patient serum as primary antibody and incubate overnight.
6) After washing with TBST, add 10,000-fold diluted HRP-labeled goat anti-human IgG antibody and react for 20 minutes. 7) After washing with TBST, develop color using immunostar (WAKO).
8) Expose to film.
9) A band present in the IPTG induction but not in the control is defined as a positive band.

(ELISA method experimental procedure)
(6) Secondary screening by ELISA method 1) The purified protein obtained by the SEREX method is used as the solid phase of the ELISA method. Place purified protein at a concentration of 50 μg / ml in a 96-well ELISA plate and store at 4 ° C. overnight for immobilization.
2) The goal is to collect 5 mg of purified protein per clone by the protein purification described above.
3) After washing with PBS, blocking with 0.5% skim milk, patient serum and control serum are diluted 5000 times and reacted with the immobilized protein.
4) After washing with PBS, add HRP-labeled goat anti-human IgG antibody 5) Add substrate and develop color and measure OD with a plate reader 6) Perform three experiments for each protein and serum, and use the average as the serum antibody value .

[Detection of peptide markers by Western blotting]
Sera from patients with cerebral aneurysms admitted to hospitals and cooperating facilities were used for screening with the consent of the patient or family. The obtained cDNA clone was incorporated into the plasmid pBlueScriptII and sequenced. Thereafter, the plasmid pGEX was recombined and introduced into Escherichia coli, and IPTG was added to express a large amount of protein to prepare a protein extract. Using it, the reaction with many patient sera was examined by Western method.

  For analysis by the Western method, a cDNA library derived from human umbilical vein endothelial cells using λZAPII as a vector was used. The integrated cDNA was expressed by IPTG treatment, and the one that reacted with an antibody present in a patient suffering from cerebral aneurysm was isolated. The isolated cDNA clone was further expressed in the pGEX plasmid in large quantities, and then the reaction of patients suffering from cerebral aneurysms was analyzed by the Western method, and clones showing a positive reaction were selected.

Pictures of blotting of clones detected by the Western method are shown in FIGS. 1a, 1b, 1c, 1d, FIGS. 2a, 2b, 2c, 2d, and FIGS. 3a, 3b, 3c, 3d.
[Confirmation of peptide marker gene by SEREX method]
Sequencing of the detected clone gene was performed using the SEREX method. As a result of collating the obtained DNA sequence with the nucleotide sequence on the publicly available gene database, the genes of the obtained clones are DNA sequences encoding the polypeptides described in the cerebral aneurysm markers 1 to 12, respectively. Matched.

1a to 1d are photographs of blotting of cerebral aneurysm markers of clones in which the expression of the detected polypeptide for diagnosis of arteriosclerosis was detected by the Western method in Examples of the present invention. 2a to 2d are photographs of blotting of cerebral aneurysm markers 1 to 4 of clones in which the expression of the detected polypeptide for diagnosis of arteriosclerosis was detected by the Western method in the Examples of the present invention. 3a to 3d are photographs of blotting of cerebral aneurysm markers 5 to 8 of clones in which expression of the detected polypeptide for diagnosis of arteriosclerosis was detected by the Western method in Examples of the present invention.

Claims (19)

Polypeptide for diagnosing human subarachnoid hemorrhage having the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 of the sequence listing, or a partial amino acid sequence thereof marker. A genetic marker for diagnosis of human subarachnoid hemorrhage, comprising a base sequence encoding the polypeptide according to claim 1. The base sequence has a DNA sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 in the sequence listing. Genetic marker for diagnosis of human subarachnoid hemorrhage. An antibody, which is derived using the polypeptide according to claim 1 and specifically binds to the polypeptide. The antibody according to claim 4, wherein the antibody is a monoclonal antibody. The antibody according to claim 4, wherein the antibody is a polyclonal antibody. A probe for detecting a human subarachnoid hemorrhage marker gene having a DNA sequence that hybridizes with the base sequence according to claim 2 or 3 under stringent conditions. The probe for detecting a human subarachnoid hemorrhage marker gene according to claim 7, comprising all or part of the antisense strand of the base sequence according to claim 3. A microarray or DNA chip for detecting a human subarachnoid hemorrhage marker gene, wherein at least one of the DNAs according to claim 7 or 8 is immobilized. A method for detecting and diagnosing a human subarachnoid hemorrhagic lesion, which comprises detecting the expression of a marker polypeptide for diagnosing human subarachnoid hemorrhage in a test sample using the antibody according to any one of claims 4 to 6. The detection of the expression of a marker polypeptide for diagnosing human subarachnoid hemorrhage is performed using Western blotting, ELISA, protein array, radioimmunoassay, fluorescent antibody, or SEREX. The method for detecting and diagnosing arteriosclerosis as described. A method for detecting and diagnosing a human subarachnoid hemorrhagic lesion, characterized by reacting serum from a subject with the marker polypeptide for diagnosis of human subarachnoid hemorrhage according to claim 1 and detecting the expression of serum antibody in the subject . A method for detecting and diagnosing a human subarachnoid hemorrhage lesion, which comprises detecting the expression of a human subarachnoid hemorrhage marker gene in a test cell using the diagnostic probe according to claim 7 or 8. The method for detecting and diagnosing a human subarachnoid hemorrhage lesion according to claim 13, wherein the expression of the human subarachnoid hemorrhage marker gene is detected using a Northern blotting method. Detection of human subarachnoid hemorrhage marker gene expression using the microarray or DNA chip for detecting human subarachnoid hemorrhage marker gene according to claim 9, And diagnostic methods. A method for detecting and diagnosing a human subarachnoid hemorrhage lesion, wherein the detection of the expression of the human subarachnoid hemorrhage marker gene according to any one of claims 13 to 14 includes the use of quantitative or semi-quantitative PCR. . The method for detecting and diagnosing arteriosclerosis according to claim 16, wherein the use of quantitative or semi-quantitative PCR is RT-PCR. A gene in a test cell is amplified using at least one pair of primers of a predetermined sense primer and antisense primer, and the amplified gene is detected using the probe for detecting an arteriosclerosis marker gene according to claim 7 or 8. The method for detecting and diagnosing a human subarachnoid hemorrhagic lesion according to claim 13. The probe for detecting a human subarachnoid hemorrhage marker gene according to claim 7 or 8, the microarray or DNA chip for detecting a human subarachnoid hemorrhage marker gene according to claim 9, and the probe according to any one of claims 4 to 6 A kit for detecting and diagnosing a human subarachnoid hemorrhagic lesion, comprising at least one of the above antibodies.

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