WO2022091793A1 - Développement d'un biomarqueur du cancer du pancréas à l'aide d'une protéine dérivée de selles - Google Patents

Développement d'un biomarqueur du cancer du pancréas à l'aide d'une protéine dérivée de selles Download PDF

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WO2022091793A1
WO2022091793A1 PCT/JP2021/037977 JP2021037977W WO2022091793A1 WO 2022091793 A1 WO2022091793 A1 WO 2022091793A1 JP 2021037977 W JP2021037977 W JP 2021037977W WO 2022091793 A1 WO2022091793 A1 WO 2022091793A1
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pancreatic cancer
protein
proteins
feces
testing
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Japanese (ja)
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伊織 元尾
收 小原
祐介 川島
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国立大学法人富山大学
公益財団法人かずさＤｎａ研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the present invention relates to a biomarker for pancreatic cancer testing composed of a protein derived from feces and a method for testing pancreatic cancer using the biomarker.
  • Pancreatic cancer is considered to be a representative of intractable tumors with a poor prognosis, but the main reason is that a method for early diagnosis has not been established. In addition, it is also experienced that the diagnosis of pancreatic cancer does not reach a definitive diagnosis before surgical resection because it is difficult to make a histological diagnosis or an image diagnosis for a small lesion.
  • Non-Patent Document 1 In recent years, with the improvement of mass spectrometers due to the progress of science and technology, comprehensive protein analysis technology has been rapidly advancing (Non-Patent Document 1). So far, there have been reports suggesting a relationship between specific proteins present in human stool and the onset or exacerbation of gastrointestinal diseases such as inflammatory bowel disease, but suggesting a relationship between proteins in feces and diseases. There are very few reports of comprehensive protein analysis (Non-Patent Documents 2 and 3). For the first time in 2016, a proteome analysis study using mouse feces was reported (Non-Patent Document 4), and since then, studies on the search for colorectal cancer markers targeting host-derived proteins in feces have begun to be reported (Non-Patent Document 4). 5).
  • cancer mainly gastrointestinal cancers such as gastric cancer and colorectal cancer
  • CA19-9 the positive rate of "CA19-9” is high, and it is used together with “CEA” for cancer diagnosis and treatment monitoring.
  • these tumor markers may increase in value even in inflammation and benign diseases, and are not reliable and are currently used as an auxiliary diagnosis.
  • pancreatic cancer biomarkers There are no reports of fecal-derived pancreatic cancer biomarkers yet.
  • the only cancer screening using feces in the health examination is colorectal cancer screening by fecal occult blood test (detection of hemoglobin in blood). It would be very useful if there was a pancreatic cancer biomarker that could non-invasively monitor the early detection and treatment of pancreatic cancer by a stool test, which is less physically burdensome than a blood test.
  • An object to be solved by the present invention is to provide a novel pancreatic cancer testing method using a fecal-derived protein as a biomarker for pancreatic cancer.
  • the inventors performed a comprehensive proteomics analysis in the feces of pancreatic cancer patients and healthy subjects, and identified 12 kinds of proteins as proteins that are significantly detected in pancreatic cancer patients, and completed the present invention. ..
  • the present invention includes the embodiments described below.
  • Item 1 A method for testing pancreatic cancer, which comprises measuring the protein in the feces of a subject, wherein the protein is a collagen ⁇ -1 (I) chain, protein S100-A8, All-trans-retinol dehydrogenase, and the like.
  • Peroxyledoxin 4 cysteine-rich secreted protein 3, protein-glutamin ⁇ -glutaminase transferase E, ⁇ -enolase, lactotransferase, signal peptidase complex catalytic subunit 11A, plasminogen activator inhibitor 2, eukaryotic translation initiation factor 6
  • Item 2 If the level of one or more of the proteins in the feces of the subject is higher than the level of the corresponding protein in the feces of a healthy subject, the subject has pancreatic cancer or the pancreas. Item 2. The test method according to Item 1, which indicates that there is a high possibility of contracting cancer.
  • Item 3. Item 2. The method according to Item 1 or 2, wherein the measurement includes measurement by mass spectrometry.
  • Item 4. Item 2. The method according to Item 1 or 2, wherein the measurement comprises measuring using an antibody against each one or two or more of the above proteins or polypeptides which are a part thereof.
  • the pancreatic cancer testing method includes a method for testing for the presence of pancreatic cancer, a method for testing the risk of developing pancreatic cancer, a method for testing the preventive effect of pancreatic cancer, and a method for testing the therapeutic effect of pancreatic cancer.
  • Item 1 including methods, methods for examining the possibility of recurrence after treatment for pancreatic cancer, methods for screening drugs that are effective in preventing pancreatic cancer, or methods for screening drugs that are effective in treating pancreatic cancer. The method according to any one of 4 to 4.
  • Collagen ⁇ -1 (I) chain protein S100-A8, All-trans-retinol dehydrogenase, peroxyredoxin 4, cysteine-rich secretory protein 3, protein-glutaminase ⁇ -glutaminasetransferase E, ⁇ -enolase, lactotransferase, signal peptidase complex
  • a pancreatic cancer detection kit containing antibodies against each.
  • Item 7 Collagen ⁇ -1 (I) chain, protein S100-A8, All-trans-retinol dehydrogenase, peroxyredoxin 4, cysteine-rich secreted protein 3, protein-glutaminase ⁇ -glutaminasetransferase E, ⁇ -enolase, lactotransferase, signal peptidase complex
  • a pancreatic cancer diagnostic agent containing an antibody against each.
  • Collagen ⁇ -1 (I) chain protein S100-A8, All-trans-retinol dehydrogenase, peroxyredoxin 4, cysteine-rich secretory protein 3, protein-glutaminase ⁇ for examination of pancreatic cancer using fecal samples
  • non-invasive pancreatic cancer can be diagnosed at an early stage using feces. Early diagnosis of pancreatic cancer also leads to early treatment of pancreatic cancer.
  • proteins 1 to 12 The 12 types of intact proteins (hereinafter, may be simply referred to as "proteins 1 to 12") that can be used as biomarkers for the examination of pancreatic cancer of the present invention are as follows.
  • Protein 1 is a collagen ⁇ -1 (I) chain (gene names COL1A1, 139Da).
  • protein 1 is a human collagen ⁇ -1 (I) chain
  • protein 1 is a human collagen ⁇ -1 (I) chain
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 is included.
  • the identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the mouse collagen ⁇ -1 (I) chain is a protein or sequence consisting of Uniprot accession number P11087 (NCBI reference sequence NP_031768.2, SEQ ID NO: 2).
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of No. 2 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher. Is included.
  • the rat collagen ⁇ -1 (I) chain is a protein or sequence consisting of Uniprot accession number P02454 (NCBI reference sequence NP_445756.1, SEQ ID NO: 3).
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of No. 3 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 2 is protein S100-A8 (gene name S100A8, 11 kDa).
  • the human protein S100-A8 consists of Uniprot accession number P05109 (NCBI reference sequence NP_001306126.1, NP_001306127.1, NP_001306130.1, NP_002955.2, SEQ ID NO: 4).
  • a protein or a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 4 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • mouse protein S100-A8 contains 90% of the protein consisting of Uniprot accession number P27005 (NCBI reference sequence NP_038678.1, SEQ ID NO: 5) or the amino acid sequence of SEQ ID NO: 5.
  • a protein consisting of an amino acid sequence having the above-mentioned identity is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • rat protein S100-A8 contains 90% of the protein consisting of Uniprot accession number P50115 (NCBI reference sequence NP_446274.2, SEQ ID NO: 6) or the amino acid sequence of SEQ ID NO: 6.
  • a protein consisting of an amino acid sequence having the above-mentioned identity is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 3 is All-trans-retinol dehydrogenase (gene name ADH7, 41 kDa).
  • the human All-trans-retinol dehydrogenase is a protein or sequence consisting of Uniprot accession number P40394 (NCBI reference sequence NP_000664.2, NP_001159976.1, SEQ ID NO: 7).
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of No. 7 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • mouse All-trans-retinol dehydrogenase is a protein consisting of Uniprot accession number Q64437 (NCBI reference sequence P_033756.2, SEQ ID NO: 8), or the amino acid of SEQ ID NO: 8.
  • a protein consisting of an amino acid sequence having 90% or more identity with the sequence is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the rat All-trans-retinol dehydrogenase is a protein consisting of Uniprot accession number P41682 (NCBI reference sequence NP_599156.1, SEQ ID NO: 9), or the amino acid of SEQ ID NO: 9.
  • a protein consisting of an amino acid sequence having 90% or more identity with the sequence is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 4 is peroxiredoxin 4 (gene name PRDX4, 31 kDa).
  • human protein 4 is human peroxyredoxin 4
  • the human protein 4 is the protein consisting of Uniprot accession number Q13162 (NCBI reference sequence NP_006397.1, SEQ ID NO: 10), or the protein of SEQ ID NO: 10.
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the mouse peroxyredoxin 4 is a protein consisting of Uniprot accession number O08807 (NCBI reference sequence, NP_001300640.1, NP_058044.1, SEQ ID NO: 11), or SEQ ID NO: 11.
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • rat peroxyredoxin 4 contains 90% of the protein consisting of Uniprot accession number Q9Z0V5 (NCBI reference sequence NP_445964.1, SEQ ID NO: 12) or the amino acid sequence of SEQ ID NO: 12.
  • a protein consisting of an amino acid sequence having the above-mentioned identity is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 5 is cysteine-rich secreted protein 3 (gene name Crisp3, 28 kDa).
  • the human cysteine-rich secreted protein 3 includes a protein consisting of Uniprot accession number P54108 (NCBI reference sequence NP_001177915.1, NP_006052.2, SEQ ID NO: 13), or SEQ ID NO: 13.
  • the mouse cysteine-rich secreted protein 3 includes a protein consisting of Uniprot accession number O55135 (NCBI reference sequence NP_033769.1, SEQ ID NO: 14) or an amino acid sequence of SEQ ID NO: 14.
  • a protein consisting of an amino acid sequence having 90% or more identity is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the rat cysteine-rich secreted protein 3 includes a protein consisting of Uniprot accession number P12020 (NCBI reference sequence NP_074050.1, SEQ ID NO: 15) or an amino acid sequence of SEQ ID NO: 15.
  • a protein consisting of an amino acid sequence having 90% or more identity is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 6 is protein-glutamin ⁇ -glutaminase transferase E (gene name TGM3, 77 kDa).
  • the human protein-glutamine ⁇ -glutamyltransferase E is a protein consisting of Uniprot accession number Q08188 (NCBI reference sequence N NP_003236.3, SEQ ID NO: 16), or a SEQ ID NO: It contains a protein consisting of 16 amino acid sequences and an amino acid sequence having 90% or more identity. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • mouse protein-glutamine ⁇ -glutamyltransferase E is a protein consisting of Uniprot accession number Q08189 (NCBI reference sequence NP_033400.2, SEQ ID NO: 17), or SEQ ID NO: 17 Contains a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the rat protein-glutamine ⁇ -glutamyltransferase E is a protein consisting of Uniprot accession number D4A5U3 (NCBI reference sequence NP_001102429.1, SEQ ID NO: 18), or SEQ ID NO: 18 Contains a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 7 is ⁇ -enolase (gene name ENO1, 47kDa).
  • the human ⁇ -enolase includes the protein consisting of Uniprot accession number P04764 (NCBI reference sequence NP_001103378.1, NP_036686.2, SEQ ID NO: 19), or the amino acid sequence of SEQ ID NO: 19.
  • a protein consisting of an amino acid sequence having 90% or more identity is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the mouse ⁇ -enolase is a protein consisting of Uniprot accession number P17182 (NCBI reference sequence NP_001020559.1, NP_075608.2, SEQ ID NO: 20), or the amino acid sequence of SEQ ID NO: 20 and 90.
  • a protein consisting of an amino acid sequence having an identity of% or more is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the rat ⁇ -enolase is a protein consisting of Uniprot accession number P04764 (NCBI reference sequence NP_001103378.1, NP_036686.2, SEQ ID NO: 21), or the amino acid sequence of SEQ ID NO: 21 and 90.
  • a protein consisting of an amino acid sequence having an identity of% or more is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 8 is lactotransferrin (gene name LTF, 78 kDa).
  • the human lactotransferase is a protein or sequence consisting of Uniprot accession number P02788 (NCBI reference sequence NP_001186078.1, NP_001308050.1, NP_001308051.1, NP_002334.2, SEQ ID NO: 22).
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of No. 22 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • mouse lactotransferrin has 90% or more identity with the protein consisting of Uniprot accession number P08071 (NCBI reference sequence NP_032548.2, SEQ ID NO: 23) or the amino acid sequence of SEQ ID NO: 23. Includes a protein consisting of an amino acid sequence having. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the rat lactotransferrin has 90% or more identity with the protein consisting of Uniprot accession number D3ZAB1 (NCBI reference sequence NP_001100334.1, SEQ ID NO: 24) or the amino acid sequence of SEQ ID NO: 24.
  • the identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 9 is a signal peptidase complex catalytic subunit 11A (gene names SEC11A, 21 kDa).
  • the human signal peptidase complex-catalyzed subunit 11A may contain Uniprot accession number P67812 (NCBI reference sequence NP_001258847.1, NP_001258848.1, NP_001258849.1, NP_001258850. 1, NP_001258851.1, NP_055115.1, SEQ ID NO: 25), or a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 25 is included.
  • the identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the mouse signal peptidase complex-catalyzed subunit 11A is a protein or sequence consisting of Uniprot accession number Q9R0P6 (NCBI reference sequence NP_064335.1, SEQ ID NO: 26).
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of No. 26 is included.
  • the identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the rat signal peptidase complex-catalyzed subunit 11A may be a protein or sequence consisting of Uniprot accession number P42667 (NCBI reference sequence NP_113911.2, SEQ ID NO: 27).
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of No. 27 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 10 is plasminogen activator inhibitor 2 (gene name SERPINB2, 47kDa).
  • the human plasminogen activator inhibitor 2 is a protein consisting of Uniprot accession number P05120 (NCBI reference sequence NP_001137290.1, NP_002566.1 SEQ ID NO: 28), or A protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 28 is included.
  • the identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the mouse plasminogen activator inhibitor 2 includes a protein consisting of Uniprot accession number P12388 (NCBI reference sequence NP_001167641.1, NP_035241.1, SEQ ID NO: 29). Alternatively, a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 29 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the rat plasminogen activator inhibitor 2 includes a protein consisting of Uniprot accession number P04764 (NCBI reference sequence NP_001103378.1, NP_036686.2, SEQ ID NO: 30).
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 30 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • Protein 11 is eukaryotic translation initiation factor 6 (gene names EIF6, 27 kDa).
  • EIF6, 27 kDa When protein 11 is human eukaryotic translation initiator 6, Uniprot accession number P56537 (NCBI reference sequence NP_001254739.1, NP_002203.1, NP_852131.1, NP_852133.1, A protein consisting of SEQ ID NO: 31) or a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 31 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the mouse eukaryotic translation initiation factor 6 is a protein consisting of Uniprot accession number O55135 (NCBI reference sequence NP_034709.1, SEQ ID NO: 32), or SEQ ID NO: 32. Contains a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the rat eukaryotic translation initiation factor 6 is a protein consisting of Uniprot accession number Q3KRD8 (NCBI reference sequence NP_001032429.1, SEQ ID NO: 33), or SEQ ID NO: 33.
  • Protein 12 is malate dehydrogenase 1 (gene name MDH1, 36 kDa).
  • the human apple acid dehydrogenase 1 is a protein consisting of Uniprot accession number P40925 (NCBI reference sequence NP_001186040.1, NP_001186041.1, NP_001303303.1, NP_005908.1, SEQ ID NO: 34).
  • a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 34 is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the mouse apple acid dehydrogenase 1 is a protein consisting of Uniprot accession number P14152 (NCBI reference sequence NP_001303604.1, NP_032644.3, SEQ ID NO: 35), or an amino acid of SEQ ID NO: 35.
  • a protein consisting of an amino acid sequence having 90% or more identity with the sequence is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the rat apple acid dehydrogenase 1 is 90% with the protein consisting of Uniprot accession number O88989 (NCBI reference sequence NP_150238.1, SEQ ID NO: 36) or the amino acid sequence of SEQ ID NO: 36.
  • a protein consisting of an amino acid sequence having the above-mentioned identity is included. The identity is preferably 95% or higher, more preferably 98% or higher, and even more preferably 99% or higher.
  • the "test" for pancreatic cancer includes detection of pancreatic cancer, determination of pancreatic cancer, determination of a pancreatic cancer patient (responder) for whom a therapeutic agent is effective (companion diagnostic method), and pancreatic cancer. Judgment of preventive effect, judgment of therapeutic effect of pancreatic cancer, auxiliary test for diagnosis of pancreatic cancer (especially early diagnosis), and auxiliary test for treatment of pancreatic cancer (especially early treatment) Inspection is included. Detection of pancreatic cancer includes detecting the "presence or absence" of pancreatic cancer.
  • Determination of pancreatic cancer includes not only determining the presence or absence of pancreatic cancer, but also prophylactically determining the possibility of developing pancreatic cancer and predicting the prognosis of pancreatic cancer after treatment. This includes determining the therapeutic effect of a therapeutic agent for pancreatic cancer.
  • Substance screening methods include methods for screening substances useful for "detection,””determination,” and “treatment” of pancreatic cancer.
  • treatment means cure or amelioration of a disease or symptom, or suppression of a symptom, and includes “prevention”.
  • Prevention means preventing the onset of a disease or symptom.
  • polypeptide refers to a molecule formed by binding two or more and less than 100 amino acids.
  • protein refers to a molecule formed by binding 100 or more amino acids.
  • measuring one or more of the proteins in a subject's feces refers to measuring the presence (presence) or level of the protein, and the "level” of the protein. “” refers to the concentration, amount, or signal intensity of protein.
  • proteins 1-12 are proteins consisting of the amino acid sequences of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34. Alternatively, it refers to a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence (identity is preferably 95% or more, more preferably 98% or more, still more preferably 99% or more), respectively, and a mouse. In the case of the protein of SEQ ID NO: 2, 5, 8, 11, 14, 15, 20, 23, 26, 31, 32, 35, a protein consisting of the amino acid sequence or an amino acid having 90% or more identity with the amino acid sequence.
  • a protein consisting of a sequence (identity is preferably 95% or more, more preferably 98% or more, even more preferably 99% or more), and in the case of a rat protein, SEQ ID NOs: 3, 6, 9, 12 respectively. , 15, 18, 21, 24, 27, 30, 33, 36 or a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence (identity is preferably 95% or more, More preferably 98% or more, and even more preferably 99% or more).
  • a method for testing pancreatic cancer which comprises measuring one or more selected from the group consisting of the above proteins 1 to 12 in the feces of a subject.
  • the measurement of proteins 1 to 12 can also be performed by measuring the concentration, amount, or signal intensity of the intact protein of proteins 1 to 12, or the full length of the partial sequence of proteins 1 to 12, the concentration, amount, or signal intensity. It can also be measured by measuring.
  • the level (concentration, amount, or signal intensity) of the polypeptide containing some of the amino acid residues of these 12 proteins also has a high level (concentration, amount, or signal intensity).
  • concentration, amount, or signal intensity As long as it reflects the level (concentration, amount, or signal intensity) of intact proteins 1-12, it can also be a marker for pancreatic cancer as long as it is a marker that can distinguish between healthy individuals and pancreatic cancer patients.
  • a part of the amino acid residues means 10 or more consecutive amino acids among the amino acid residues of each protein, preferably 15 or more consecutive amino acids, and more preferably 20 consecutive amino acids.
  • proteins 1 to 12 correlate with the presence or absence of pancreatic cancer. Specifically, the expression levels of proteins 1 to 12 in feces are all significantly higher in pancreatic cancer patients than in healthy subjects.
  • proteins 1 to 12 alone or in combination of two or more as biomarkers for pancreatic cancer testing, the disease can be tested with high accuracy in vitro.
  • the sensitivity (presence accuracy rate) and specificity (disease-free accuracy rate) of the test can be further increased.
  • the subject may be a healthy person or a person suffering from pancreatic cancer.
  • Subjects include those suspected of having pancreatic cancer. "Persons suspected of having pancreatic cancer” include those who are subjectively suspected by the subject (not limited to those who have some subjective symptoms, but who simply wish to undergo preventive screening. ), Or a person who has been determined or diagnosed as pancreatic cancer based on some objective basis (for example, as a result of conventionally known pancreatic cancer tests and / or examinations, pancreatic cancer is rational. It may be a person who is judged by the doctor to have a possibility of illness).
  • the subject is a mammal, preferably a human, a mouse, a rat, a rabbit, a goat, a sheep, a horse, a guinea pig, a hamster, etc., more preferably a human, a mouse, a rat, and further preferably a human. ..
  • the proteins 1 to 12 of the present invention are measured using a fecal sample derived from a subject as a test sample.
  • the stool sample may be centrifuged to remove substances other than the proteins 1 to 12 of the present invention, or the protein-containing fraction may be trypsinized, if necessary.
  • proteins 1 to 12 of the present invention for example, fecal samples are subjected to various molecular mass spectrometry methods (eg, gel electrophoresis, etc.) and various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase).
  • molecular mass spectrometry methods eg, gel electrophoresis, etc.
  • separation and purification methods eg, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase.
  • ionization method eg, electron impact ionization method, field dispersion method, secondary ionization method, high-speed atomic collision method, matrix-assisted laser desorption ionization method, electrospray ionization method, etc.
  • mass spectrometers eg, double convergent mass spectrometer, quadrupole type analyzer, flight time type mass spectrometer, Fourier conversion mass spectrometer, ion cyclotron mass spectrometer, immunomass spectrometer, stable isotope inside Used in a method of combining a standardized mass spectrometer, an MS-MS mass spectrometer using stable isotope-labeled fragment ions as an internal standard, an immunomicroscope, a mass spectrometer, etc.), and matches the molecular weight of proteins 1 to 12. This can be done by detecting fragment ions, spots, or peaks of the band or partial polypeptide thereof, but is
  • the preferred method for measuring proteins 1 to 12 in the test method of the present invention is mass spectrometry. Therefore, according to one aspect of the present invention, one or more of the proteins consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 36 in the feces of the subject are measured by mass spectrometry. Methods for testing for pancreatic cancer are provided.
  • both undegraded proteins and similar polypeptides are biomarkers proteins 1-12 of the invention (particularly proteins consisting of the amino acid sequences represented by SEQ ID NOs: 1-36) or portions thereof. It is completely separated from the polypeptide, and the exact molecular weight of the polypeptide can be quantified quickly and with high accuracy (specificity, sensitivity).
  • proteins 1 to 12 of the present invention are obtained from information on molecular weight.
  • the presence or level of can be identified.
  • programs are well known and that one of ordinary skill in the art can easily construct or modify such programs using known information processing techniques.
  • the measurement of proteins 1 to 12 of the present invention can also be performed using an antibody against a partial polypeptide of each protein 1 to 12. Therefore, according to one aspect of the present invention, one or more selected from the group consisting of proteins 1 to 12 in the feces of a subject is subjected to an antibody against the proteins 1 to 12 or a partial polypeptide thereof. Methods for testing pancreatic cancer, including measuring with, are provided. Such an antibody against the protein of the present invention or a partial polypeptide thereof can be used as a test agent or a diagnostic agent for pancreatic cancer.
  • Antibodies to proteins 1 to 12 or partial polypeptides thereof of the present invention are prepared, and the presence or level of proteins 1 to 12 or partial polypeptides thereof is determined by various immunological methods such as ELISA, immunochromatography, immunoassay, and Western blotting. It can also be a measuring method.
  • Antibodies to proteins 1 to 12 of the present invention or partial polypeptides thereof can be prepared, for example, by immunizing an animal with a sequence of proteins 1 to 12 of the present invention or a partial polypeptide thereof as an epitope antigen.
  • the polypeptide can be isolated and purified from a biological sample derived from a patient expressing the polypeptide, or it can be prepared by a well-known genetic engineering method such as amplification of a cDNA fragment encoding the polypeptide. Furthermore, it can be prepared in large quantities by an organic synthesis method.
  • the antibody against the proteins 1 to 12 or a partial polypeptide thereof of the present invention can specifically bind to the proteins 1 to 12 or a partial polypeptide thereof via the antigen-binding site of the antibody, and the proteins 1 to 12 or the partial polypeptide thereof can be specifically bound to the antibody.
  • Epitope antigens which are 12 or a partial polypeptide thereof, can be made by conventional techniques using one or more immunogens.
  • the epitope antigen is a polypeptide consisting of at least 7, at least 8, at least 10, at least 15, and at least 20 consecutive amino acid residues in the amino acid sequence of each polypeptide represented by SEQ ID NOs: 1 to 36. It consists of a fragment or the full length of each polypeptide.
  • the antibody against the proteins 1 to 12 or a partial polypeptide thereof of the present invention may be either a polyclonal antibody or a monoclonal antibody, and can be produced by a well-known immunological method.
  • the polyclonal antibody can be produced by immunizing an animal such as a bird (for example, a chicken) or a mammal (for example, a rabbit, a goat, a horse, a sheep, a rat, etc.) with the polypeptide according to the present invention.
  • Monoclonal antibodies can be obtained by techniques comprising producing hybridoma cell lines in mice by conventional techniques that produce monoclonal antibodies specific for each polypeptide.
  • the test method of the present invention using an antibody is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample is detected by chemical or physical means. Any measuring method may be used as long as it is a measuring method for calculating this from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method, sandwich method and the like are preferably used. Upon measurement, the antibody or antigen may be bound to a labeling agent such as a radioisotope, enzyme, fluorescent substance, or luminescent substance. Furthermore, a biotin-avidin system can also be used for binding an antibody or antigen to a labeling agent. These individual immunoassays can be applied to the quantification method of the present invention by the ordinary skill of those skilled in the art.
  • pancreatic cancer testing method using an antibody is useful in that the polypeptide can be detected easily and with high accuracy without the need for a mass spectrometer.
  • the expression level (expression concentration, expression level, signal intensity) of proteins 1 to 12 in feces collected from pancreatic cancer patients is based on the expression level of the corresponding polypeptide in feces collected from healthy subjects. Is also expensive.
  • test method of the present invention can be carried out by collecting fecal samples from a patient in chronological order and examining the expression of proteins 1 to 12 of the present invention in each sample only once, or a plurality of changes with time. It can also be done by examining.
  • the sample collection interval is not particularly limited when the test is performed over time, but the sample collection interval can be, for example, every 1 to several days, 1 to 4 weeks, or 1 to 6 months.
  • the method for testing pancreatic cancer of the present invention is a method for testing the presence of pancreatic cancer, which is one or more of proteins 1 to 12 in the feces of a subject. This includes comparing the level or measured value with the reference value and determining that the subject is likely to have pancreatic cancer if it is equal to or higher than the reference value.
  • the reference value is, for example, a cutoff value that distinguishes between a pancreatic cancer patient and a healthy person, or a mean value, a median value, or a value larger than those of the corresponding proteins 1 to 12 in a pancreatic cancer patient.
  • the method for testing pancreatic cancer of the present invention is a method for testing the presence of pancreatic cancer, which is one or more of proteins 1 to 12 in the feces of a subject. This includes comparing the level or measured value with the reference value and determining that the subject is unlikely to have pancreatic cancer if it is below the reference value.
  • the reference value is, for example, a cutoff value which is a threshold for distinguishing between a pancreatic cancer patient and a healthy person, or a mean value, a median value, or a value smaller than those of the corresponding proteins 1 to 12 in the healthy person.
  • the method for testing pancreatic cancer of the present invention is a method for testing the presence of pancreatic cancer, which is one or more of proteins 1 to 12 in the feces of a subject.
  • pancreatic cancer which is one or more of proteins 1 to 12 in the feces of a subject.
  • Level or measured value is compared with the standard value, and if it is equal to or higher than the standard value, it is determined that the subject is likely to have pancreatic cancer, and if it is lower than the standard value. Including determining that the subject is unlikely to have pancreatic cancer.
  • the reference value is, for example, a cutoff value that distinguishes between a pancreatic cancer patient and a healthy person, or a mean value, a median value, or a value larger than those of the corresponding proteins 1 to 12 in a pancreatic cancer patient.
  • the method for testing pancreatic cancer of the present invention is a method for testing the risk of developing pancreatic cancer, and one or more of proteins 1 to 12 in the feces of a subject. Includes comparing the level or measured value of the patient with the reference value and determining that the subject is more likely to develop pancreatic cancer if it is equal to or higher than the reference value.
  • the reference value is, for example, a cutoff value that distinguishes between a pancreatic cancer patient and a healthy person, or a mean value, a median value, or a value larger than those of the corresponding proteins 1 to 12 in a pancreatic cancer patient.
  • the method for testing pancreatic cancer of the present invention is a method for testing the risk of developing pancreatic cancer, and one or more of proteins 1 to 12 in the feces of a subject. This includes comparing the level or measured value of the patient with the reference value and determining that the subject is unlikely to develop pancreatic cancer if the value is lower than the reference value.
  • the reference value is, for example, a cutoff value which is a threshold for distinguishing between a pancreatic cancer patient and a healthy person, or a mean value, a median value, or a value smaller than those of the corresponding proteins 1 to 12 in the healthy person.
  • the pancreatic cancer testing method of the present invention is a method for testing the risk of developing pancreatic cancer, and one or two of the proteins 1 to 12 in the feces of a subject.
  • the subject is judged to be more likely to develop pancreatic cancer when it is equal to or higher than the reference value, and when it is lower than the reference value. Includes determining that the subject is unlikely to develop pancreatic cancer.
  • the reference value is, for example, a cutoff value that distinguishes between a pancreatic cancer patient and a healthy person, or a mean value, a median value, or a value larger than those of the corresponding proteins 1 to 12 in a pancreatic cancer patient.
  • the method for testing pancreatic cancer of the present invention is a method for testing the preventive effect of pancreatic cancer, and among the proteins 1 to 12 in the feces of the subject after receiving the treatment. This includes comparing the level or measured value of one or more types with the reference value, and determining that the preventive effect of the measure is high for pancreatic cancer when the value is lower than the reference value.
  • the reference value is, for example, the level or measured value of one or more of the proteins 1 to 12 in the feces of the same subject before receiving the treatment, and the threshold value for distinguishing between pancreatic cancer patients and healthy subjects.
  • Measures include surgery for pancreatic cancer, radiation therapy, administration of therapeutic agents, diet therapy, exercise therapy, lifestyle improvement guidance, etc.
  • the subject may be given one or more of the measures listed above.
  • the method for testing pancreatic cancer of the present invention is a method for testing the preventive effect of pancreatic cancer, a method for testing the presence of pancreatic cancer, and a test after receiving treatment.
  • the level or measured value of one or more of the proteins 1 to 12 in the feces of a person is compared with the reference value, and if it is the same as or higher than the reference value, pancreatic cancer due to the measure is taken. It includes determining that the preventive effect is low.
  • the reference value is, for example, the level or measured value of one or more of the proteins 1 to 12 in the feces of the same subject before receiving the treatment, and the cutoff value for distinguishing between pancreatic cancer patients and healthy subjects. , Or the average, median, or greater than those of the corresponding proteins 1-12 in patients with pancreatic cancer.
  • Measures include surgery for pancreatic cancer, radiation therapy, administration of therapeutic agents, diet therapy, exercise therapy, lifestyle improvement guidance, etc.
  • the subject may be given one or more of the measures listed above.
  • the pancreatic cancer testing method of the present invention is a method for testing the preventive effect of pancreatic cancer, and among the proteins 1 to 12 in the feces of the subject after receiving the treatment.
  • the level or measured value of one or more of the above is compared with the standard value, and if it is lower than the standard value, it is judged that the preventive effect of pancreatic cancer by the measure is high, and it is the same as or lower than the standard value. If it is high, it includes determining that the preventive effect of the measures for pancreatic cancer is low.
  • the reference value is, for example, the level or measured value of one or more of the proteins 1 to 12 in the feces of the same subject before receiving the treatment, and the threshold value for distinguishing between pancreatic cancer patients and healthy subjects.
  • Measures include surgery for pancreatic cancer, radiation therapy, administration of therapeutic agents, diet therapy, exercise therapy, lifestyle improvement guidance, etc.
  • the subject may be given one or more of the measures listed above.
  • the method for testing pancreatic cancer of the present invention is a method for testing the therapeutic effect of pancreatic cancer, and among the proteins 1 to 12 in the feces of the subject after receiving the treatment. It includes comparing the level or measured value of one or more types with the reference value, and determining that the therapeutic effect of pancreatic cancer by the measure is high when the level is lower than the reference value.
  • the therapeutic effect of pancreatic cancer means that the pancreatic cancer is improving.
  • the reference value is, for example, the level or measured value of one or more of the proteins 1 to 12 in the feces of the same subject before receiving the treatment, and the threshold value for distinguishing between pancreatic cancer patients and healthy subjects.
  • Measures include surgery for pancreatic cancer, radiation therapy, administration of therapeutic agents, diet therapy, exercise therapy, lifestyle improvement guidance, etc.
  • the subject may be given one or more of the measures listed above.
  • the method for testing pancreatic cancer of the present invention is a method for testing the therapeutic effect of pancreatic cancer, a method for testing the presence of pancreatic cancer, and a test after receiving treatment.
  • the level or measured value of one or more of the proteins 1 to 12 in the feces of a person is compared with the reference value, and if it is the same as or higher than the reference value, pancreatic cancer due to the measure is taken. Includes determining that the therapeutic effect is low.
  • the reference value is, for example, the level or measured value of one or more of the proteins 1 to 12 in the feces of the same subject before receiving the treatment, and the cutoff value for distinguishing between pancreatic cancer patients and healthy subjects. , Or the average, median, or greater than those of the corresponding proteins 1-12 in patients with pancreatic cancer.
  • Measures include surgery for pancreatic cancer, radiation therapy, administration of therapeutic agents, diet therapy, exercise therapy, lifestyle improvement guidance, etc.
  • the subject may be given one or more of the measures listed above.
  • the method for testing pancreatic cancer of the present invention is a method for testing the therapeutic effect of pancreatic cancer, and among the proteins 1 to 12 in the feces of the subject after receiving the treatment.
  • the level or measured value of one or more of the above is compared with the standard value, and if it is lower than the standard value, it is judged that the therapeutic effect of pancreatic cancer by the measure is high, and it is the same as or lower than the standard value. If it is high, it includes determining that the therapeutic effect of pancreatic cancer by the measure is low.
  • the reference value is, for example, the level or measured value of one or more of the proteins 1 to 12 in the feces of the same subject before receiving the treatment, and the threshold value for distinguishing between pancreatic cancer patients and healthy subjects.
  • Measures include surgery for pancreatic cancer, radiation therapy, administration of therapeutic agents, diet therapy, exercise therapy, lifestyle improvement guidance, etc.
  • the subject may be given one or more of the measures listed above.
  • the method for testing pancreatic cancer of the present invention is a method for testing the possibility of recurrence after treatment for pancreatic cancer, and the protein 1 in the feces of the subject after receiving the treatment. Comparing the level or measured value of one or more of 12 to the reference value, if it is the same as or higher than the reference value, it is highly possible that pancreatic cancer has recurred after the treatment. It includes determining and / or determining that pancreatic cancer is unlikely to recur after treatment if it is below the reference value.
  • the reference value is, for example, the level or measured value of one or more of the proteins 1 to 12 in the feces of the same subject before receiving the treatment, and the cutoff value for distinguishing between pancreatic cancer patients and healthy subjects. , Or the average, median, or greater than those of the corresponding proteins 1-12 in patients with pancreatic cancer.
  • Measures include surgery for pancreatic cancer, radiation therapy, administration of therapeutic agents, diet therapy, exercise therapy, lifestyle improvement guidance, etc.
  • the subject may be given one or more of the measures listed above.
  • the method for testing pancreatic cancer of the present invention is a method for screening a drug effective for the prevention of pancreatic cancer, and among the proteins 1 to 12 in the feces of a subject after administration of the drug. It includes comparing the level or measured value of one or more of the above with the reference value, and determining that the drug is likely to be effective in the prevention of pancreatic cancer when it is lower than the reference value.
  • the reference value is, for example, the level or measured value of one or more of proteins 1 to 12 in the feces of the same subject before drug administration, and the threshold value for distinguishing between pancreatic cancer patients and healthy subjects.
  • the method for testing pancreatic cancer of the present invention is a method for screening a drug effective for the prevention of pancreatic cancer, and among the proteins 1 to 12 in the feces of a subject after administration of the drug.
  • the level or measured value of one or more of the above is compared with the standard value, and if it is the same as or higher than the standard value, it is judged that the drug is unlikely to be effective in preventing pancreatic cancer. Including doing.
  • the reference value is, for example, the level or measured value of one or more proteins 1 to 12 in the feces of the same subject before administration of the drug, the cutoff value for distinguishing between pancreatic cancer patients and healthy subjects, and the reference value. Or the mean, median, or greater than the corresponding proteins 1-12 in patients with pancreatic cancer.
  • the method for testing pancreatic cancer of the present invention is a method for screening a drug effective for the prevention of pancreatic cancer, and the protein 1 to 12 in the feces of a subject after administration of the drug.
  • the level or measured value of one or more of them is compared with the standard value, and if it is lower than the standard value, it is judged that the drug is highly likely to be effective in preventing pancreatic cancer, and the standard value is determined. Includes determining that the drug is unlikely to be effective in preventing pancreatic cancer if it is equal to or higher than.
  • the reference value is, for example, the level or measured value of one or more of the same subject's fecal proteins 1 to 12 before administration of the drug, and the cutoff which is a threshold for distinguishing between pancreatic cancer patients and healthy subjects.
  • the method for testing pancreatic cancer of the present invention is a method for screening a drug effective for treating pancreatic cancer, and among the proteins 1 to 12 in the feces of a subject after administration of the drug. It includes comparing the level or measured value of one or more of the above with the reference value, and determining that the drug is likely to be effective for the treatment of pancreatic cancer when it is lower than the reference value.
  • the reference value is, for example, the level or measured value of one or more of proteins 1 to 12 in the feces of the same subject before drug administration, and the threshold value for distinguishing between pancreatic cancer patients and healthy subjects.
  • the method for testing pancreatic cancer of the present invention is a method for screening a drug effective for treating pancreatic cancer, and among the proteins 1 to 12 in the feces of a subject after administration of the drug.
  • the level or measured value of one or more of the above is compared with the reference value, and if it is the same as or higher than the reference value, it is judged that the drug is unlikely to be effective in treating pancreatic cancer. Including doing.
  • the reference value is, for example, the level or measured value of one or more proteins 1 to 12 in the feces of the same subject before administration of the drug, the cutoff value for distinguishing between pancreatic cancer patients and healthy subjects, and the reference value. Or the mean, median, or greater than the corresponding proteins 1-12 in patients with pancreatic cancer.
  • the method for testing pancreatic cancer of the present invention is a method for screening a drug effective for treating pancreatic cancer, and the protein 1 to 12 in the feces of a subject after administration of the drug.
  • the level or measured value of one or more of them is compared with the standard value, and if it is lower than the standard value, it is judged that the drug is likely to be effective for the treatment of pancreatic cancer, and the standard value is determined. Includes determining that the drug is unlikely to be effective in treating pancreatic cancer if it is equal to or higher than.
  • the reference value is, for example, the level or measured value of one or more of the same subject's fecal proteins 1 to 12 before administration of the drug, and the cutoff which is a threshold for distinguishing between pancreatic cancer patients and healthy subjects.
  • the proteins 1 to 12 of the present invention are not only markers for diagnosing or detecting pancreatic cancer, but also markers for examining the risk of developing pancreatic cancer and determining the preventive effect of pancreatic cancer. Markers, markers for determining the therapeutic effect of pancreatic cancer, markers for testing the possibility of recurrence after pancreatic cancer treatment, markers for screening drugs that are effective in preventing pancreatic cancer, pancreatic cancer It can also be a marker for screening therapeutically effective drugs.
  • the proteins 1-12 of the present invention can be used for screening drug discovery target molecules for the treatment of pancreatic cancer and / or can be used as a companion diagnostic for selecting patients or adjusting the dosage of therapeutic agents. can.
  • the method for inspecting pancreatic cancer using proteins 1 to 12 of the present invention determines or diagnoses pancreatic cancer instead of the conventional method for diagnosing pancreatic cancer or in combination with the conventional method for diagnosing pancreatic cancer. Applicable to assistance.
  • test method using the proteins 1 to 12 of the present invention uses the feces of the patient as a sample, it can be carried out as a screening test for a health diagnosis in daily medical care. If a patient with strong suspicion of pancreatic cancer can be accurately detected by a medical examination, it can be linked to endoscopic ultrasonography as a secondary examination for pancreatic cancer scrutiny, and early diagnosis of pancreatic cancer can be made. , Useful for treatment.
  • a pancreatic cancer test or diagnostic kit containing an antibody against each of one or more of proteins 1 to 12 is provided.
  • the test or diagnostic kit may further include a container for containing antibodies against one or more of the proteins 1 to 12 individually or as a mixture, respectively.
  • Antibodies to proteins 1-12 or partial polypeptides thereof may be attached or bound to a solid phase carrier or may be in free form.
  • Antibodies to proteins 1-12 or partial polypeptides thereof may optionally be labeled with labels such as fluoresceins, enzymes, radioisotopes, etc., or secondary antibodies may be labeled as such. May be combined.
  • test or diagnostic kits may further include labeled secondary antibodies, carriers, washings, sample diluents, enzyme substrates, reaction arrests, purified standard marker polypeptides, instructions for use and the like. ..
  • the sample tested by the above test or diagnostic kit is, for example, feces collected from the subject.
  • a pancreatic cancer test kit using an antibody is useful in that it can easily and accurately detect each protein 1 to 12 or a partial polypeptide thereof without the need for a mass spectrometer.
  • the pancreatic cancer test or diagnostic kit of the present invention has the above-mentioned pancreatic cancer test method of the present invention, for example, a test for the presence of pancreatic cancer, a test for the risk of developing pancreatic cancer, and a preventive effect on pancreatic cancer. It can be used for testing, testing the therapeutic effect of pancreatic cancer, screening for drugs effective in preventing pancreatic cancer, and the like.
  • Example 1 1 mL of phosphate buffered saline (PBS-PI) containing a protease inhibitor was added to 50 mg of mouse feces, and the feces were gently crushed with a tip and gently stirred by pipetting until the feces were dissolved. Then, centrifugation was performed at 4 ° C. at 15000 G for 10 minutes, only the supernatant was recovered, and low molecular weight components were removed by trichloroacetic acid precipitation. 100 mM Tris-HCl pH 8.5-0.5% sodium dodecanoate was added to the precipitate containing the protein component, and the protein was redissolved by an ultrasonic crusher.
  • PBS-PI phosphate buffered saline
  • the total protein content was measured by BCA assay to adjust the protein concentration to 1 mg / mL.
  • 2 ⁇ L of 110 mM dithiothreitol was added, and the mixture was allowed to stand at 50 ° C. for 30 minutes to reduce the disulfide bond of the protein.
  • 2 uL of 360 mM iodoacetamide was added, and the mixture was allowed to stand at room temperature for 30 minutes to alkylate the cysteine side chain.
  • 2 ⁇ L of 780 mM cysteine was added and allowed to stand at room temperature for 10 minutes to stop the reaction of iodoacetamide.
  • the recovered sample was desalted by a C18 spin column, the eluate was completely dried on a centrifugal evaporator and then redissolved in 20 ⁇ L of 3% acetonitrile-0.1% formic acid.
  • the total amount of peptide was measured by BCA assay, the peptide concentration was adjusted to 0.25 mg / mL, and 2 ⁇ L was analyzed by liquid chromatography-mass spectrometer (LC / MS).
  • the LC / MS LC uses the UltiMate 3000 RSLC nano System manufactured by Thermo Fisher Scientific, the flow velocity is 100 nL / min, and the column is 75 umID x 400 mm packed with C18 2.6um core shell particles (manufactured by Osaka Soda Co., Ltd.). used.
  • MS an Orbitrap Exploris 480 Mass Spectrometer manufactured by Thermo Fisher Scientific was used.
  • the MS data obtained by LC / MS analysis was collated with the spectral library of human proteins using Scaffold DIA manufactured by Proteome Software to identify the proteins. The identification threshold was set so that the false positive rate of peptide-level and protein-level identification was 1% or less, respectively. Quantitative values were calculated from the MS / MS chromatograph area values for the identified proteins, and protein variability in each sample was analyzed.
  • the expression levels of the 12 proteins were shown to be significantly higher in pancreatic cancer patients (T01 to T10) than in healthy subjects (H01 to H10).
  • Table 1 shows the average expression level of each protein in pancreatic cancer patients (T01 to T10), the average expression level in healthy subjects (H01 to H10), and the p-value. The two groups were tested with the Mann-Whitney U Test.

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Abstract

L'invention concerne une méthode d'essai du cancer du pancréas consistant à mesurer une protéine dans les selles d'un sujet, la protéine étant un ou deux éléments choisis dans le groupe constitué par le collagène chaîne alpha-1(I), la protéine S100-A8, l'all-trans-rétinol déshydrogénase, la péroxyrédoxine 4, la protéine sécrétoire riche en cystéine 3, la gamma-glutamyltransférase E glutamine-protéine, l'alpha-énolase, la lactotransferrine, une sous-unité catalytique complexe de la peptidase signal 11A, un inhibiteur de l'activateur du plasminogène 2, un facteur 6 d'initiation de la traduction eucaryote et la malate déshydrogénase 1.
PCT/JP2021/037977 2020-10-27 2021-10-13 Développement d'un biomarqueur du cancer du pancréas à l'aide d'une protéine dérivée de selles WO2022091793A1 (fr)

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