WO2023004626A1 - Myeloma biomarker serpinf2 and use thereof - Google Patents

Myeloma biomarker serpinf2 and use thereof Download PDF

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WO2023004626A1
WO2023004626A1 PCT/CN2021/108930 CN2021108930W WO2023004626A1 WO 2023004626 A1 WO2023004626 A1 WO 2023004626A1 CN 2021108930 W CN2021108930 W CN 2021108930W WO 2023004626 A1 WO2023004626 A1 WO 2023004626A1
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serpinf2
myeloma
gene
protein
product
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PCT/CN2021/108930
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French (fr)
Chinese (zh)
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于晓波
许萌
陈文明
高文
杨世方
庞红艳
杜鹃
隋伟薇
蔡真
王立生
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北京沙东生物技术有限公司
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Priority to PCT/CN2021/108930 priority Critical patent/WO2023004626A1/en
Priority to CN202180100706.3A priority patent/CN117677715A/en
Publication of WO2023004626A1 publication Critical patent/WO2023004626A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the invention relates to the technical fields of molecular biology and biomedicine, in particular to a myeloma biomarker (SERPINF2) and its application.
  • SERPINF2 myeloma biomarker
  • Myeloma (also known as plasmacytoma) is a malignant tumor originating from plasma cells in the bone marrow. It is a relatively common malignant tumor. There are single and multiple points. Multiple myeloma, also known as multiple myeloma (MM for short), is a malignant transformation of plasma cells capable of synthesizing and secreting immunoglobulin. A large number of monoclonal malignant plasma cell hyperplasia easily involves soft tissues, and it may be extensive in the late stage. metastases, but rarely lung metastases. It is more common in the spine, accounting for 10% of primary spinal tumors, and is more common in the lumbar spine. It occurs mostly in men over 40 years old, and the ratio of men and women over 40 years old is about 2:1. The most common sites are the spine, ribs, skull, and sternum. In recent years, the incidence of MM is on the rise, and the age of onset is also decreasing.
  • R-ISS Revised International Staging System
  • Hematopoietic stem cell transplantation is the most effective method for the treatment of multiple myeloma. Due to the influence of bone marrow source, HLA type matching and high medical costs, its clinical application is limited. Chemotherapy is still the main treatment strategy, but with With the progress of drug treatment, patients are prone to drug resistance and intolerance. To address these problems, it is necessary to find a simple, non-invasive, sensitive, rapid, and specific diagnosis, treatment, and prognosis strategy.
  • an application of a product for detecting SERPINF2 gene or SERPINF2 protein in preparing a tool for diagnosis and/or prognosis of myeloma is provided.
  • products for detecting SERPINF2 gene or SERPINF2 protein include products for detecting the expression level of SERPINF2 gene or SERPINF2 protein.
  • a product that detects the SERPINF2 gene may include a nucleic acid capable of binding the SERPINF2 gene.
  • the product for detecting the SERPINF2 gene can exert its function based on known methods using nucleic acid molecules: for example, polymerase chain reaction (PCR), Southern blot hybridization, Northern blot hybridization, dot hybridization, fluorescence in situ hybridization can be used (FISH), DNA microarrays, high-throughput sequencing platforms, etc., especially PCR methods, such as real-time fluorescent quantitative PCR methods.
  • PCR polymerase chain reaction
  • FISH fluorescence in situ hybridization
  • DNA microarrays such as real-time fluorescent quantitative PCR methods.
  • analyzes can be performed qualitatively, quantitatively, or semi-quantitatively.
  • the nucleic acid contained in the product for detecting the SERPINF2 gene can be obtained by chemical synthesis, or by preparing a gene containing the desired nucleic acid from a biological material, and then amplifying it using primers designed to amplify the desired nucleic acid to get.
  • the nucleic acid may include primers that specifically amplify the SERPINF2 gene.
  • the nucleic acid may also include a probe that specifically recognizes the SERPINF2 gene.
  • the products for detecting the SERPINF2 gene can be reagents, kits, test strips, gene chips, etc., which can include nucleic acids capable of binding the SERPINF2 gene (such as primers for specifically amplifying the SERPINF2 gene and/or probes for specifically recognizing the SERPINF2 gene). needle); the product for detecting the SERPINF2 gene can also be a high-throughput sequencing platform, which can use nucleic acids capable of binding to the SERPINF2 gene (such as primers for specific amplification of the SERPINF2 gene and/or probes for specific recognition of the SERPINF2 gene) to target SERPINF2 genetic testing.
  • the product for detecting SERPINF2 protein may include a substance capable of binding SERPINF2 protein (such as an antibody or a fragment thereof).
  • products that detect the SERPINF2 protein can be based on known methods using the protein to exert its function: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blot, proteomics (such as antibody chips, mass spectrometry ( For example, data independent acquisition (Data Independent Acquision, DIA) mass spectrometry) and so on.
  • products for detecting SERPINF2 protein may include antibodies or fragments thereof that specifically bind to SERPINF2 protein.
  • Antibodies or fragments thereof of any structure, size, immunoglobulin class, origin, etc. can be used as long as it binds the target protein.
  • Antibodies or fragments thereof included in products for detecting SERPINF2 protein may be monoclonal or polyclonal.
  • An antibody fragment refers to a part of an antibody (partial fragment) or a peptide containing a part of an antibody that retains the antigen-binding activity of the antibody.
  • Antibody fragments may include F(ab')2, Fab', Fab, single-chain Fv (scFv), disulfide-bonded Fv (dsFv) or polymers thereof, dimerized V regions (diabodies), or containing CDR peptides.
  • a product for detecting a SERPINF2 protein may include an isolated nucleic acid encoding an antibody or an amino acid sequence encoding an antibody fragment, a vector comprising the nucleic acid, or a cell carrying the vector. Antibodies can be obtained by methods known to those skilled in the art, and available commercial products can also be used.
  • products for detecting SERPINF2 protein can be reagents, kits, test strips, gene chips, etc., which can contain substances (such as antibodies or fragments thereof) that can bind SERPINF2 protein; products for detecting SERPINF2 protein can also be instrument platforms, which It may include a measurement module (for measuring the content of the SERPINF2 protein in the test sample), and an analysis module (for analyzing the difference in the content of the SERPINF2 protein in the test sample and the reference sample).
  • a measurement module for measuring the content of the SERPINF2 protein in the test sample
  • an analysis module for analyzing the difference in the content of the SERPINF2 protein in the test sample and the reference sample.
  • the measurement module can be based on mass spectrometry, such as DIA-MS, wherein the DIA acquisition scheme consists of 32 fixed windows, and the acquisition range is 400-1200 mass-to-charge ratio (m/z).
  • mass spectrometry such as DIA-MS
  • DIA acquisition scheme consists of 32 fixed windows, and the acquisition range is 400-1200 mass-to-charge ratio (m/z).
  • myeloma includes single myeloma and multiple myeloma, especially multiple myeloma.
  • the sample used for the detection of SERPINF2 gene or SERPINF2 protein can use, for example, tissue samples or fluids obtained from biopsy subjects, for example, tissue, blood, plasma, serum, lymph fluid, urine, serous cavity fluid , spinal fluid, synovial fluid, aqueous humor, tears, saliva, etc. or their fractions or processed materials.
  • the sample used for the detection of SERPINF2 gene or SERPINF2 protein is the subject's blood (especially peripheral blood) or its fraction (such as serum), especially serum.
  • a tool for diagnosis and/or prognosis of myeloma which comprises a product for detecting SERPINF2 gene or SERPINF2 protein.
  • products for detecting SERPINF2 gene or SERPINF2 protein have the above-mentioned corresponding definitions of the present invention.
  • the above-mentioned tools may be reagents, kits, test strips, gene chips, high-throughput sequencing platforms, proteomic analysis products (such as antibody chips, DIA-MS), etc.
  • a use of a SERPINF2 gene or SERPINF2 protein inhibitor in the preparation of a medicament for treating myeloma is provided.
  • the inhibitor is capable of inhibiting the expression or activity of substances in the upstream or downstream pathways of SERPINF2.
  • a medicament for treating myeloma which comprises an inhibitor of SERPINF2 gene or SERPINF2 protein.
  • a method for diagnosing or prognosing myeloma which includes the step of detecting SERPINF2 gene or SERPINF2 protein.
  • the above method may include the following steps:
  • the subject can be diagnosed with myeloma or have a high risk of myeloma, or the subject can be determined to have a poor prognosis. It should be noted that the specific disease risk, severity, and prognosis need to be comprehensively evaluated by clinicians in combination with other test indicators of the subject.
  • myeloma includes single myeloma and multiple myeloma, especially multiple myeloma.
  • the sample may use, for example, a tissue sample or fluid obtained from a biopsy subject, e.g., tissue, blood, plasma, serum, lymph, urine, serous cavity fluid, spinal fluid, synovial fluid, aqueous humor, tear fluid , saliva, etc., or fractions thereof, or processed material.
  • a tissue sample or fluid obtained from a biopsy subject e.g., tissue, blood, plasma, serum, lymph, urine, serous cavity fluid, spinal fluid, synovial fluid, aqueous humor, tear fluid , saliva, etc., or fractions thereof, or processed material.
  • the sample is the subject's blood (especially peripheral blood) or a fraction thereof (eg serum), especially serum.
  • the subject is a mammal, especially a human.
  • a method for treating myeloma which comprises inhibiting SERPINF2 gene or SERPINF2 protein.
  • the method includes inhibiting the expression of the SERPINF2 gene, and/or inhibiting the activity of the SERPINF2 protein.
  • a method for screening tumor drugs which may include detecting the expression level of the SERPINF2 gene or SERPINF2 protein at a certain period after administering the test drug to the tumor cells or after the tumor model animal is administered the test drug. To assess the effect of a test drug on improving tumor outcomes.
  • the drug when the expression level of SERPINF2 gene or SERPINF2 protein decreases or returns to a normal level after administration of a test drug, the drug can be selected as a therapeutic drug for improving tumor prognosis.
  • the tumor is myeloma, especially multiple myeloma.
  • the present invention analyzes serum samples by DIA-MS to obtain differential proteins and selects potential diagnostic markers, prepares an antibody chip according to the selected diagnostic markers, and evaluates the stability and specificity of the antibody chip.
  • the myeloma marker screened by the invention can detect and screen myeloma patients with high sensitivity and high specificity.
  • the antibody chip prepared by the invention has high stability and high sensitivity, and can be used for clinical detection.
  • the myeloma serum markers screened by the invention and the antibody chip prepared can greatly improve the detection efficiency of myeloma, realize early diagnosis, and effectively reduce the detection cost under the high-throughput chip detection platform.
  • the present invention by detecting the expression level of the SERPINF2 gene or SERPINF2 protein in the subject, it can be judged whether the subject has myeloma, or whether the subject is at risk of suffering from myeloma, or whether the subject has myeloma or not. Whether the prognosis of the patients is good, so as to guide clinicians to provide effective preventive measures or treatment plans for the subjects, which is conducive to improving the survival rate of the subjects, and can also provide new drug targets for the development of drugs for the treatment of myeloma.
  • Figure 1 shows the detection results of the targets on the SERPINF2 protein by DIA-MS mass spectrometry.
  • Figure 2 shows the results of the validation of the antibody chip on the myeloma diagnostic markers.
  • Figure 3 shows the volcano plot of mass spectrometry and antibody chip detection results.
  • Figure 4 shows representative results of antibody chip detection.
  • SERPINF2 ⁇ -2 anti-plasmin (serpin peptidase inhibitor clade F member 2, SERPINF2), referred to as SERPINF2 in this paper.
  • expression level refers to the measurable amount of the SERPINF2 gene product in a sample, wherein the gene product can be a transcription product or a translation product.
  • expression levels are relative to nucleic acid gene products (eg, mRNA or cDNA) or polypeptide gene products (eg, SERPINF2 protein).
  • SERPINF2 gene includes the polynucleotide of the SERPINF2 gene itself and any functional equivalent of the SERPINF2 gene, for example, it has more than 70% (for example, more than 80%, More than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%) homology, and the DNA sequence encoding the same functional protein.
  • diagnosing myeloma includes not only judging whether a subject has myeloma, but also judging whether a subject is at risk of suffering from myeloma.
  • prognosis refers to the process or result of a myeloma patient after myeloma is suppressed or alleviated by surgical treatment or the like.
  • the prognosis may be the vital state at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 years or more after suppression or remission of myeloma by surgical treatment.
  • Prognosis can be predicted by checking biomarkers namely SERPINF2 gene or SERPINF2 protein.
  • Prognosis prediction can be performed by determining whether a patient has a good prognosis or a poor prognosis, or the probability of a good prognosis or a poor prognosis, based on the presence or absence, or increase or decrease, of biomarkers.
  • good prognosis means that after the myeloma is suppressed or remitted for the patient by surgical treatment etc. long) is not critical.
  • good prognosis may mean survival, metastasis-free, and recurrence-free for such a long period of time.
  • good prognosis may mean at least 3 years or especially at least 5 years of existence, preferably no metastasis or recurrence. The most preferred state of good prognosis is long-term disease-free survival.
  • “good prognosis” may also include a state in which a disease such as metastasis can be found, but nausea is low and does not seriously affect viability.
  • “poor prognosis” means that a patient develops a fatal condition within a short period (for example, 1, 2, 3, 4, 5 years or less) after myeloma is suppressed or remitted by surgical treatment or the like.
  • “poor prognosis” refers to death, metastasis or recurrence within such a short period of time.
  • “poor prognosis” may mean recurrence, metastasis or death within at least 3 years, especially at least 5 years.
  • Predicting prognosis refers to predicting the course or outcome of a patient's condition and does not mean that the course or outcome of a patient's condition can be predicted with 100% accuracy. Predicting prognosis means determining whether the likelihood of a certain course or outcome increases, and does not imply determining the likelihood of a certain course or outcome occurring by comparison with the situation where the certain course or outcome did not occur. As per the present invention, a particular process or outcome is more likely to be observed in patients with elevated or decreased levels of the SERPINF2 gene or SERPINF2 protein of the present invention than in patients who do not exhibit this feature.
  • the above samples were detected by mass spectrometry using DIA-MS technology, and the differential proteins of the N and MM samples were obtained and potential diagnostic markers were selected.
  • Example 1 The potential diagnostic markers obtained in Example 1 were verified using a special antibody chip.
  • SERPINF2 antibody was purchased from R&D Company, product number: MAB1470-SP.
  • Negative control 100 ⁇ g/mL BSA and 1 ⁇ PBS.
  • Positive control 10 ⁇ g/mL Alexx-55-goat anti-human IgG; 100 ⁇ g/mL Biotin-IgG.
  • Sample diluent 1 ⁇ PBS (137mMNaCl, 2.7mMKCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 );
  • Tween 0.05% PBST (0.05% Tween, 1 ⁇ PBS), Tween was purchased from American Amresco company;
  • 5% milk blocking solution (5% milk, 1 ⁇ PBST), skimmed milk powder was purchased from BD Company in the United States;
  • Biotin labeling reagent NHS-PEG4-Biotin
  • fluorescent dye Streptavidin, R-Phycoerythrin Conjugate (SAPE)
  • BSA Albumin from bovine serum
  • Sigma-Aldrich Company of the United States
  • Biochip scanner (4300A), purchased from U.S. Molecular Devices instrument company;
  • the incubation plate (3/5) was purchased from PEPperPRINT, Germany;
  • Temperature control mixer (MixMate) and desktop centrifuge (Centrifuge 5810R), purchased from Germany Eppendorf AG;
  • PCR-384M2-C microplates were purchased from Axygen, USA.
  • Chip detection dry the chip, scan it with a fluorescent chip scanner and extract the fluorescent signal data.
  • Embodiment 3 The clinical detection accuracy of antibody chip
  • Figure 4 is a representative result of antibody chip detection, wherein, picture A is the detection result of the antibody chip of healthy people, and picture B is the test result of myeloma patients.
  • the inventors found that the signal of the point in picture B is generally stronger than that of picture A . It is preliminarily shown that there are more up-regulated proteins in the serum of myeloma patients than in healthy people.

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Abstract

The present invention relates to the technical fields of molecular biology and biomedicine, and specifically relates to a myeloma biomarker and the use thereof. According to the above-mentioned use of the present invention, whether a subject has myeloma, or whether the subject is at a risk of having myeloma, or whether the prognosis of the subject is good can be judged by means of detecting an expression level of an SERPINF2 gene or an SERPINF2 protein in a subject, thereby guiding a clinician to provide an effective prevention means or treatment regimen for the subject, which is beneficial for improving the survival rate of the subject.

Description

一种骨髓瘤生物标志物SERPINF2及其应用A myeloma biomarker SERPINF2 and its application 技术领域technical field
本发明涉及分子生物学与生物医药技术领域，具体涉及一种骨髓瘤生物标志物(SERPINF2)及其应用。The invention relates to the technical fields of molecular biology and biomedicine, in particular to a myeloma biomarker (SERPINF2) and its application.
背景技术Background technique
骨髓瘤(又称浆细胞瘤)是起源于骨髓中浆细胞的恶性肿瘤，是一种较常见的恶性肿瘤。有单发性和多发性之分。多发性骨髓瘤又称细胞骨髓瘤(Multiple myeloma，简称MM)，是由具有合成和分泌免疫球蛋白的浆细胞发生恶变，大量单克隆的恶性浆细胞增生引起易累及软组织，晚期可有广泛性转移，但少有肺转移。较多见于脊，占脊柱原发肿瘤的10％，以腰椎部多见。多发于40岁以上男性，好发年龄在40岁以上男性与女性之比约2∶1。好发部位依次为脊椎、肋骨、颅骨、胸骨等。近年来MM的发病率呈上升趋势，发病年龄也日益下降。Myeloma (also known as plasmacytoma) is a malignant tumor originating from plasma cells in the bone marrow. It is a relatively common malignant tumor. There are single and multiple points. Multiple myeloma, also known as multiple myeloma (MM for short), is a malignant transformation of plasma cells capable of synthesizing and secreting immunoglobulin. A large number of monoclonal malignant plasma cell hyperplasia easily involves soft tissues, and it may be extensive in the late stage. metastases, but rarely lung metastases. It is more common in the spine, accounting for 10% of primary spinal tumors, and is more common in the lumbar spine. It occurs mostly in men over 40 years old, and the ratio of men and women over 40 years old is about 2:1. The most common sites are the spine, ribs, skull, and sternum. In recent years, the incidence of MM is on the rise, and the age of onset is also decreasing.
多发性骨髓瘤起病徐缓，早期无明显症状，其临床表现多样，常会造成误诊和漏诊，最终导致病情延误。多发性骨髓瘤的诊断一般需要进行骨髓活检、影像学检查和血液检查等，并结合临床表现来进行鉴别诊断。针对多发性骨髓瘤修订的国际分期系统(R-ISS)纳入了血浆生物标志物(乳酸脱氢酶、β2-微球蛋白和白蛋白)和已知预后意义的细胞遗传学异常来预测疾病行为，但不同患者异质性比较大。造血干细胞移植是治疗多发性骨髓瘤最有效的方法，由于受骨髓来源、HLA配型以及高医疗费用等因素的影响，限制了其在临床上的应用，化疗仍然是主要的治疗策略，但随着药物治疗的进程患者容易产生耐药和不耐受等现象。针对这些问题，寻找一种简便无创、灵敏快速、特异性强的诊断、治疗和预后策略是很有必要的。Multiple myeloma has a slow onset and no obvious symptoms in the early stage. Its clinical manifestations are diverse, which often leads to misdiagnosis and missed diagnosis, eventually leading to delays in treatment. The diagnosis of multiple myeloma generally requires bone marrow biopsy, imaging examination, and blood examination, etc., combined with clinical manifestations for differential diagnosis. The Revised International Staging System (R-ISS) for multiple myeloma incorporates plasma biomarkers (lactate dehydrogenase, β2-microglobulin, and albumin) and cytogenetic abnormalities of known prognostic significance to predict disease behavior , but the heterogeneity of different patients is relatively large. Hematopoietic stem cell transplantation is the most effective method for the treatment of multiple myeloma. Due to the influence of bone marrow source, HLA type matching and high medical costs, its clinical application is limited. Chemotherapy is still the main treatment strategy, but with With the progress of drug treatment, patients are prone to drug resistance and intolerance. To address these problems, it is necessary to find a simple, non-invasive, sensitive, rapid, and specific diagnosis, treatment, and prognosis strategy.
发明内容Contents of the invention
在本发明的第一方面，提供一种检测SERPINF2基因或SERPINF2蛋白的产品在制备用于骨髓瘤的诊断和/或预后的工具中的应用。In the first aspect of the present invention, an application of a product for detecting SERPINF2 gene or SERPINF2 protein in preparing a tool for diagnosis and/or prognosis of myeloma is provided.
具体地，检测SERPINF2基因或SERPINF2蛋白的产品包括检测SERPINF2基因或SERPINF2蛋白的表达水平的产品。Specifically, products for detecting SERPINF2 gene or SERPINF2 protein include products for detecting the expression level of SERPINF2 gene or SERPINF2 protein.
具体地，检测SERPINF2基因的产品可以包括能够结合SERPINF2基因的核酸。Specifically, a product that detects the SERPINF2 gene may include a nucleic acid capable of binding the SERPINF2 gene.
具体地，检测SERPINF2基因的产品可基于使用核酸分子的已知方法来发挥其功能：例如，可以采用聚合酶链式反应(PCR)、Southern印迹杂交、Northern印迹杂交、点杂交、荧光原位杂交(FISH)、DNA微阵列、高通量测序平台等，特别是PCR方法，例如实时荧光定量PCR法。使用该产品可以定性地、定量地、或半定量地实施分析。Specifically, the product for detecting the SERPINF2 gene can exert its function based on known methods using nucleic acid molecules: for example, polymerase chain reaction (PCR), Southern blot hybridization, Northern blot hybridization, dot hybridization, fluorescence in situ hybridization can be used (FISH), DNA microarrays, high-throughput sequencing platforms, etc., especially PCR methods, such as real-time fluorescent quantitative PCR methods. Using this product, analyzes can be performed qualitatively, quantitatively, or semi-quantitatively.
具体地，检测SERPINF2基因的产品中所含的核酸可以通过化学合成来获得，或通过从生物材料制备含有所需核酸的基因，然后使用设计用于扩增所需核酸的引物对其进行扩增来获得。Specifically, the nucleic acid contained in the product for detecting the SERPINF2 gene can be obtained by chemical synthesis, or by preparing a gene containing the desired nucleic acid from a biological material, and then amplifying it using primers designed to amplify the desired nucleic acid to get.
具体地，该核酸可以包括特异性扩增SERPINF2基因的引物。Specifically, the nucleic acid may include primers that specifically amplify the SERPINF2 gene.
具体地，该核酸还可包括特异性识别SERPINF2基因的探针。Specifically, the nucleic acid may also include a probe that specifically recognizes the SERPINF2 gene.
具体地，检测SERPINF2基因的产品可以为试剂、试剂盒、试纸、基因芯片等，其可以包含能够结合SERPINF2基因的核酸(例如特异性扩增SERPINF2基因的引物和/或特异性识别SERPINF2基因的探针)；检测SERPINF2基因的产品也可以为高通量测序平台，其可以使用能够结合SERPINF2基因的核酸(例如特异性扩增SERPINF2基因的引物和/或特异性识别SERPINF2基因的探针)针对SERPINF2基因进行检测。Specifically, the products for detecting the SERPINF2 gene can be reagents, kits, test strips, gene chips, etc., which can include nucleic acids capable of binding the SERPINF2 gene (such as primers for specifically amplifying the SERPINF2 gene and/or probes for specifically recognizing the SERPINF2 gene). needle); the product for detecting the SERPINF2 gene can also be a high-throughput sequencing platform, which can use nucleic acids capable of binding to the SERPINF2 gene (such as primers for specific amplification of the SERPINF2 gene and/or probes for specific recognition of the SERPINF2 gene) to target SERPINF2 genetic testing.
具体地，检测SERPINF2蛋白的产品可以包括能够结合SERPINF2蛋白的物质(例如抗体或其片段)。Specifically, the product for detecting SERPINF2 protein may include a substance capable of binding SERPINF2 protein (such as an antibody or a fragment thereof).
具体地，检测SERPINF2蛋白的产品可基于使用蛋白的已知方法来发挥 其功能：例如，可以采用ELISA、放射免疫测定法、免疫组织化学法、Western印迹、蛋白质组学(例如抗体芯片、质谱(例如数据非依赖采集(Data Independent Acquision，DIA)质谱)等。Specifically, products that detect the SERPINF2 protein can be based on known methods using the protein to exert its function: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blot, proteomics (such as antibody chips, mass spectrometry ( For example, data independent acquisition (Data Independent Acquision, DIA) mass spectrometry) and so on.
具体地，检测SERPINF2蛋白的产品可以包括特异性结合SERPINF2蛋白的抗体或其片段。可以使用任何结构、尺寸、免疫球蛋白类别、起源等的抗体或其片段，只要它结合靶蛋白质即可。检测SERPINF2蛋白的产品中所包括的抗体或其片段可以是单克隆的或多克隆的。抗体片段指保留抗体对抗原的结合活性的抗体一部分(部分片段)或含有抗体一部分的肽。抗体片段可以包括F(ab′)2、Fab′、Fab、单链Fv(scFv)、二硫化物键合的Fv(dsFv)或其聚合物、二聚化V区(双抗体)、或含有CDR的肽。检测SERPINF2蛋白的产品可以包括编码抗体或编码抗体片段的氨基酸序列的分离的核酸、包含该核酸的载体或携带该载体的细胞。抗体可以通过本领域技术人员公知的方法来获得，也可采用可用的市售产品。Specifically, products for detecting SERPINF2 protein may include antibodies or fragments thereof that specifically bind to SERPINF2 protein. Antibodies or fragments thereof of any structure, size, immunoglobulin class, origin, etc. can be used as long as it binds the target protein. Antibodies or fragments thereof included in products for detecting SERPINF2 protein may be monoclonal or polyclonal. An antibody fragment refers to a part of an antibody (partial fragment) or a peptide containing a part of an antibody that retains the antigen-binding activity of the antibody. Antibody fragments may include F(ab')2, Fab', Fab, single-chain Fv (scFv), disulfide-bonded Fv (dsFv) or polymers thereof, dimerized V regions (diabodies), or containing CDR peptides. A product for detecting a SERPINF2 protein may include an isolated nucleic acid encoding an antibody or an amino acid sequence encoding an antibody fragment, a vector comprising the nucleic acid, or a cell carrying the vector. Antibodies can be obtained by methods known to those skilled in the art, and available commercial products can also be used.
具体地，检测SERPINF2蛋白的产品可以为试剂、试剂盒、试纸、基因芯片等，其可以包含能够结合SERPINF2蛋白的物质(例如抗体或其片段)；检测SERPINF2蛋白的产品也可以为仪器平台，其可以包含测量模块(用于测量待测样本中SERPINF2蛋白的含量)，还可以包含分析模块(用于分析待测样本与参比样本中SERPINF2蛋白的含量差异)。Specifically, products for detecting SERPINF2 protein can be reagents, kits, test strips, gene chips, etc., which can contain substances (such as antibodies or fragments thereof) that can bind SERPINF2 protein; products for detecting SERPINF2 protein can also be instrument platforms, which It may include a measurement module (for measuring the content of the SERPINF2 protein in the test sample), and an analysis module (for analyzing the difference in the content of the SERPINF2 protein in the test sample and the reference sample).
具体地，该测量模块可基于质谱，例如DIA-MS，其中DIA采集方案由32个固定窗口组成，采集范围为400-1200质荷比(m/z)。Specifically, the measurement module can be based on mass spectrometry, such as DIA-MS, wherein the DIA acquisition scheme consists of 32 fixed windows, and the acquisition range is 400-1200 mass-to-charge ratio (m/z).
具体地，检测SERPINF2蛋白的产品为仪器平台时，在检测前待测样本经过预处理，预处理可包括：将待测样本用裂解缓冲液稀释，二硫化物还原，烷基化处理，酶解，酸化，脱盐；具体地，预处理可包括：将待测样本用尿素溶液稀释，用二硫苏糖醇(Dithiothreitol，DTT)在37℃水浴进行二硫化物还原，然后在25℃下用500mmol/L的碘乙酰胺(Iodoacetamide，IAA)避光烷基化处理，用胰蛋白酶37℃下酶解，将酶解的肽用三氟乙酸溶液 (Trifluoroacetic acid，TFA，pH＝2-3)进行酸化，然后用C18脱盐柱进行脱盐，然后将脱盐的肽在真空下干燥后溶于含有0.1％甲酸和2％乙腈的缓冲液中，用分析柱分离得到定量的肽，用于DIA-MS分析。Specifically, when the product for detecting SERPINF2 protein is an instrument platform, the sample to be tested is pretreated before detection, and the pretreatment may include: diluting the sample to be tested with lysis buffer, disulfide reduction, alkylation, enzymatic hydrolysis , acidification, desalination; specifically, the pretreatment may include: dilute the sample to be tested with urea solution, use dithiothreitol (Dithiothreitol, DTT) to carry out disulfide reduction in a 37°C water bath, and then use 500mmol /L of iodoacetamide (Iodoacetamide, IAA) was alkylated in the dark, enzymolyzed with trypsin at 37°C, and the enzymatically hydrolyzed peptides were treated with trifluoroacetic acid solution (Trifluoroacetic acid, TFA, pH=2-3) Acidification, followed by desalting with a C18 desalting column, and then the desalted peptides were dried under vacuum and dissolved in a buffer containing 0.1% formic acid and 2% acetonitrile, separated by an analytical column to obtain quantitative peptides for DIA-MS analysis .
具体地，骨髓瘤包括单发性骨髓瘤和多发性骨髓瘤，特别是多发性骨髓瘤。Specifically, myeloma includes single myeloma and multiple myeloma, especially multiple myeloma.
具体地，用于SERPINF2基因或SERPINF2蛋白的检测的样本，可以使用例如自活检受试者获得的组织样品或流体，例如，组织、血液、血浆、血清、淋巴液、尿液、浆膜腔液、脊髓液、滑液、房水、泪液、唾液等或其级分或经过处理的材料。Specifically, the sample used for the detection of SERPINF2 gene or SERPINF2 protein can use, for example, tissue samples or fluids obtained from biopsy subjects, for example, tissue, blood, plasma, serum, lymph fluid, urine, serous cavity fluid , spinal fluid, synovial fluid, aqueous humor, tears, saliva, etc. or their fractions or processed materials.
在本发明的一个实施方式中，用于SERPINF2基因或SERPINF2蛋白的检测的样本为受试者的血液(特别是外周血)或其级分(例如血清)，特别是血清。In one embodiment of the present invention, the sample used for the detection of SERPINF2 gene or SERPINF2 protein is the subject's blood (especially peripheral blood) or its fraction (such as serum), especially serum.
在本发明的第二方面，提供一种用于骨髓瘤的诊断和/或预后的工具，其包含检测SERPINF2基因或SERPINF2蛋白的产品。In the second aspect of the present invention, a tool for diagnosis and/or prognosis of myeloma is provided, which comprises a product for detecting SERPINF2 gene or SERPINF2 protein.
具体地，检测SERPINF2基因或SERPINF2蛋白的产品具有本发明上述相应定义。Specifically, products for detecting SERPINF2 gene or SERPINF2 protein have the above-mentioned corresponding definitions of the present invention.
具体地，上述工具可以为试剂、试剂盒、试纸、基因芯片、高通量测序平台、蛋白质组学分析产品(例如抗体芯片、DIA-MS)等。Specifically, the above-mentioned tools may be reagents, kits, test strips, gene chips, high-throughput sequencing platforms, proteomic analysis products (such as antibody chips, DIA-MS), etc.
在本发明的第三方面，提供一种SERPINF2基因或SERPINF2蛋白的抑制剂在制备治疗骨髓瘤的药物中的应用。In the third aspect of the present invention, a use of a SERPINF2 gene or SERPINF2 protein inhibitor in the preparation of a medicament for treating myeloma is provided.
具体地，该抑制剂能够抑制SERPINF2上游或下游途径的物质的表达或活性。Specifically, the inhibitor is capable of inhibiting the expression or activity of substances in the upstream or downstream pathways of SERPINF2.
在本发明的第四方面，提供一种治疗骨髓瘤的药物，其包含SERPINF2基因或SERPINF2蛋白的抑制剂。In the fourth aspect of the present invention, a medicament for treating myeloma is provided, which comprises an inhibitor of SERPINF2 gene or SERPINF2 protein.
在本发明的第五方面，提供一种诊断或预后骨髓瘤的方法，其包括检测SERPINF2基因或SERPINF2蛋白的步骤。In the fifth aspect of the present invention, a method for diagnosing or prognosing myeloma is provided, which includes the step of detecting SERPINF2 gene or SERPINF2 protein.
具体地，上述方法可包括如下步骤：Specifically, the above method may include the following steps:
(1)获取受试者样本；(1) Obtain subject samples;
(2)检测受试者样本中的SERPINF2基因或SERPINF2蛋白的表达水平；(2) Detect the expression level of SERPINF2 gene or SERPINF2 protein in the subject sample;
(3)将测得的SERPINF2基因或SERPINF2蛋白的表达水平与受试者的患病与否关联起来。(3) Correlating the measured expression level of the SERPINF2 gene or SERPINF2 protein with the subject's illness.
特别是，与正常对照相比，SERPINF2基因或SERPINF2蛋白的表达水平升高，则该受试者可被诊断为骨髓瘤或患骨髓瘤风险高，或该受试者被确定为预后不良。需要说明的是，具体疾病风险、严重程度、预后情况，还需临床医生结合该受试者的其他检测指标综合评估。In particular, if the expression level of SERPINF2 gene or SERPINF2 protein is increased compared with the normal control, the subject can be diagnosed with myeloma or have a high risk of myeloma, or the subject can be determined to have a poor prognosis. It should be noted that the specific disease risk, severity, and prognosis need to be comprehensively evaluated by clinicians in combination with other test indicators of the subject.
具体地，骨髓瘤包括单发性骨髓瘤和多发性骨髓瘤，特别是多发性骨髓瘤。Specifically, myeloma includes single myeloma and multiple myeloma, especially multiple myeloma.
具体地，样本可以使用例如自活检受试者获得的组织样品或流体，例如，组织、血液、血浆、血清、淋巴液、尿液、浆膜腔液、脊髓液、滑液、房水、泪液、唾液等或其级分或经过处理的材料。在本发明的一个实施方式中，样本为受试者的血液(特别是外周血)或其级分(例如血清)，特别是血清。In particular, the sample may use, for example, a tissue sample or fluid obtained from a biopsy subject, e.g., tissue, blood, plasma, serum, lymph, urine, serous cavity fluid, spinal fluid, synovial fluid, aqueous humor, tear fluid , saliva, etc., or fractions thereof, or processed material. In one embodiment of the present invention, the sample is the subject's blood (especially peripheral blood) or a fraction thereof (eg serum), especially serum.
具体地，受试者为哺乳动物，特别是人类。In particular, the subject is a mammal, especially a human.
在本发明的第六方面，提供一种治疗骨髓瘤的方法，其包括抑制SERPINF2基因或SERPINF2蛋白。In the sixth aspect of the present invention, there is provided a method for treating myeloma, which comprises inhibiting SERPINF2 gene or SERPINF2 protein.
具体地，该方法包括抑制SERPINF2基因的表达，和/或，抑制SERPINF2蛋白的活性。Specifically, the method includes inhibiting the expression of the SERPINF2 gene, and/or inhibiting the activity of the SERPINF2 protein.
在本发明的第七方面，提供一种肿瘤药物的筛选方法，其可以包括对肿瘤细胞施用测试药物后或在肿瘤模型动物施用测试药物后的某个时期检测SERPINF2基因或SERPINF2蛋白的表达水平来评估测试药物对改善肿瘤预后的效果。In the seventh aspect of the present invention, a method for screening tumor drugs is provided, which may include detecting the expression level of the SERPINF2 gene or SERPINF2 protein at a certain period after administering the test drug to the tumor cells or after the tumor model animal is administered the test drug. To assess the effect of a test drug on improving tumor outcomes.
具体地，当SERPINF2基因或SERPINF2蛋白的表达水平在施用测试药 物后降低或恢复正常水平时，可选择该药物作为改善肿瘤预后的治疗药物。Specifically, when the expression level of SERPINF2 gene or SERPINF2 protein decreases or returns to a normal level after administration of a test drug, the drug can be selected as a therapeutic drug for improving tumor prognosis.
具体地，肿瘤为骨髓瘤，特别是多发性骨髓瘤。In particular, the tumor is myeloma, especially multiple myeloma.
本发明通过DIA-MS分析血清样本得到差异蛋白并挑选出潜在的诊断标志物，根据挑选出的诊断标志物制备抗体芯片，并评价该抗体芯片的稳定性和特异性。本发明筛选的骨髓瘤标志物能够高灵敏度、高特异性检测筛查骨髓瘤患者。本发明制备的抗体芯片稳定性高、灵敏度高，能够用于临床检测。本发明筛选的骨髓瘤血清标志物及制备的抗体芯片可大大提高骨髓瘤的检测效率，实现早期诊断，同时在高通量芯片的检测平台下，有效降低了检测成本。根据本发明上述应用，通过检测受试者中SERPINF2基因或SERPINF2蛋白的表达水平，可以判断受试者是否患有骨髓瘤、或者判断受试者是否存在患有骨髓瘤的风险、或判断受试者的预后是否良好，从而指导临床医师为受试者提供有效的预防手段或者治疗方案，有利于提供受试者生存率，也可以为骨髓瘤治疗药物的开发提供新的药物靶点。The present invention analyzes serum samples by DIA-MS to obtain differential proteins and selects potential diagnostic markers, prepares an antibody chip according to the selected diagnostic markers, and evaluates the stability and specificity of the antibody chip. The myeloma marker screened by the invention can detect and screen myeloma patients with high sensitivity and high specificity. The antibody chip prepared by the invention has high stability and high sensitivity, and can be used for clinical detection. The myeloma serum markers screened by the invention and the antibody chip prepared can greatly improve the detection efficiency of myeloma, realize early diagnosis, and effectively reduce the detection cost under the high-throughput chip detection platform. According to the above application of the present invention, by detecting the expression level of the SERPINF2 gene or SERPINF2 protein in the subject, it can be judged whether the subject has myeloma, or whether the subject is at risk of suffering from myeloma, or whether the subject has myeloma or not. Whether the prognosis of the patients is good, so as to guide clinicians to provide effective preventive measures or treatment plans for the subjects, which is conducive to improving the survival rate of the subjects, and can also provide new drug targets for the development of drugs for the treatment of myeloma.
附图说明Description of drawings
图1所示为DIA-MS质谱对SERPINF2蛋白上靶标的检测结果。Figure 1 shows the detection results of the targets on the SERPINF2 protein by DIA-MS mass spectrometry.
图2所示为抗体芯片对骨髓瘤诊断标志物进行验证的结果。Figure 2 shows the results of the validation of the antibody chip on the myeloma diagnostic markers.
图3所示为质谱和抗体芯片检测结果的火山图。Figure 3 shows the volcano plot of mass spectrometry and antibody chip detection results.
图4所示为抗体芯片检测的代表性结果。Figure 4 shows representative results of antibody chip detection.
具体实施方式Detailed ways
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.
α-2抗纤溶酶(serpin peptidase inhibitor clade F member 2，SERPINF2)，在本文中简称为SERPINF2。α-2 anti-plasmin (serpin peptidase inhibitor clade F member 2, SERPINF2), referred to as SERPINF2 in this paper.
在本发明中，“表达水平”是指样品中SERPINF2基因产物的可测量的 量，其中基因产物可以是转录产物或翻译产物。因此，表达水平与核酸基因产物(如mRNA或cDNA)或多肽基因产物(例如SERPINF2蛋白)有关。In the present invention, "expression level" refers to the measurable amount of the SERPINF2 gene product in a sample, wherein the gene product can be a transcription product or a translation product. Thus, expression levels are relative to nucleic acid gene products (eg, mRNA or cDNA) or polypeptide gene products (eg, SERPINF2 protein).
在本发明中，“SERPINF2基因”包括SERPINF2基因本身以及SERPINF2基因的任何功能等同物的多核苷酸，例如与目前国际公共核酸序列数据库GeneBank中SERPINF2基因DNA序列具有70％以上(例如80％以上，90％以上，95％以上，96％以上，97％以上，98％以上，99％以上，99.5％以上)同源性，且编码相同功能蛋白质的DNA序列。In the present invention, "SERPINF2 gene" includes the polynucleotide of the SERPINF2 gene itself and any functional equivalent of the SERPINF2 gene, for example, it has more than 70% (for example, more than 80%, More than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%) homology, and the DNA sequence encoding the same functional protein.
在本发明中，“诊断骨髓瘤”既包括判断受试者是否已经患有骨髓瘤，也包括判断受试者是否存在罹患骨髓瘤的风险。In the present invention, "diagnosing myeloma" includes not only judging whether a subject has myeloma, but also judging whether a subject is at risk of suffering from myeloma.
在本发明中，“预后”是指骨髓瘤患者在通过手术治疗等抑制或缓解骨髓瘤后的过程或结果。在本说明书中，预后可以是通过手术治疗抑制或缓解骨髓瘤后1、2、3、4、5、6、7、8、9、10、15、20年或更久时的生机状态。预后可以通过检查生物标志物即SERPINF2基因或SERPINF2蛋白来预测。预后预测可以这样进行：根据生物标志物的有或无，或者升高或降低，确定患者的预后是良好还是不良，或者确定良好预后或不良预后的概率。In the present invention, "prognosis" refers to the process or result of a myeloma patient after myeloma is suppressed or alleviated by surgical treatment or the like. In this specification, the prognosis may be the vital state at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 years or more after suppression or remission of myeloma by surgical treatment. Prognosis can be predicted by checking biomarkers namely SERPINF2 gene or SERPINF2 protein. Prognosis prediction can be performed by determining whether a patient has a good prognosis or a poor prognosis, or the probability of a good prognosis or a poor prognosis, based on the presence or absence, or increase or decrease, of biomarkers.
在本发明中，“预后良好”是指在通过手术治疗等为患者抑制或缓解骨髓瘤之后，患者长时期(例如3、5、6、7、8、9、10、15、20年或更长)没有危急状况。或者，“预后良好”可以意指这样长时间内存活、无转移、无复发。例如，“预后良好”可以意指至少3年或特别是至少5年存在，优选没有转移或复发。预后良好最优选的状态是长期无疾病的存活。在本发明中，“预后良好”还可以包括这样的状态：可以发现疾病如转移，但是恶心低且不严重地影响生存能力。In the present invention, "good prognosis" means that after the myeloma is suppressed or remitted for the patient by surgical treatment etc. long) is not critical. Alternatively, "good prognosis" may mean survival, metastasis-free, and recurrence-free for such a long period of time. For example, "good prognosis" may mean at least 3 years or especially at least 5 years of existence, preferably no metastasis or recurrence. The most preferred state of good prognosis is long-term disease-free survival. In the present invention, "good prognosis" may also include a state in which a disease such as metastasis can be found, but nausea is low and does not seriously affect viability.
在本发明中，“预后不良”是指患者在通过手术治疗等抑制或缓解骨髓瘤后的短时期(例如1、2、3、4、5年或更短)内发生致命状况。或者，“预后不良”是指在这样的短时期内死亡、转移或复发。例如，“预后不良”可以意指至少3年特别是至少5年内复发、转移或死亡。In the present invention, "poor prognosis" means that a patient develops a fatal condition within a short period (for example, 1, 2, 3, 4, 5 years or less) after myeloma is suppressed or remitted by surgical treatment or the like. Alternatively, "poor prognosis" refers to death, metastasis or recurrence within such a short period of time. For example, "poor prognosis" may mean recurrence, metastasis or death within at least 3 years, especially at least 5 years.
预测预后是指预测患者状况的过程或结果，并不意味着能以100％的准确度预测患者状况的过程或结果。预测预后是指确定某些过程或结果的可能性是否增加，而并不意味着通过与某些过程或结果不发生的情况比较来确定发生某些过程或结果的可能性。如本发明而言，本发明中SERPINF2基因或SERPINF2蛋白的水平升高或降低的患者中，与不显示该特征的患者相比，更有可能观察到特定过程或结果。Predicting prognosis refers to predicting the course or outcome of a patient's condition and does not mean that the course or outcome of a patient's condition can be predicted with 100% accuracy. Predicting prognosis means determining whether the likelihood of a certain course or outcome increases, and does not imply determining the likelihood of a certain course or outcome occurring by comparison with the situation where the certain course or outcome did not occur. As per the present invention, a particular process or outcome is more likely to be observed in patients with elevated or decreased levels of the SERPINF2 gene or SERPINF2 protein of the present invention than in patients who do not exhibit this feature.
本文所引用的各种出版物、专利和公开的专利说明书，其公开内容通过引用整体并入本文。The disclosures of the various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.
实施例1：骨髓瘤诊断标志物的筛选Example 1: Screening of myeloma diagnostic markers
1、样本收集1. Sample collection
样本收集于首都医科大学附属北京朝阳医院，在朝阳医院招募了22名健康志愿者(N)，志愿者平均年龄为57.95±3.40岁，男女比例为8：3，作为对照组；选择临床诊断确诊骨髓瘤的患者，骨髓瘤患者(MM)平均年龄为58.16±9.14岁，男女比例8：3，所有患者均获得知情同意书。Samples were collected in Beijing Chaoyang Hospital Affiliated to Capital Medical University. 22 healthy volunteers (N) were recruited in Chaoyang Hospital. The average age of the volunteers was 57.95±3.40 years old, and the ratio of male to female was 8:3. They were used as the control group; the clinical diagnosis was confirmed. Myeloma patients, the average age of myeloma patients (MM) was 58.16±9.14 years old, the male to female ratio was 8:3, and all patients obtained informed consent.
2、骨髓瘤诊断标志物筛选2. Screening of myeloma diagnostic markers
利用DIA-MS技术对以上样本进行质谱检测，得到N和MM两组样本的差异蛋白并挑选出潜在的诊断标志物。The above samples were detected by mass spectrometry using DIA-MS technology, and the differential proteins of the N and MM samples were obtained and potential diagnostic markers were selected.
DIA-MS分析步骤如下：The steps of DIA-MS analysis are as follows:
将2μL血清样本加入1.5mL离心管中，用6mol/L的尿素(来自Sigma，美国)的裂解缓冲液稀释，用10mmol/ml的二硫苏糖醇(Dithiothreitol，DTT)在37℃水浴进行二硫化物还原60min；然后在25℃下用500mmol/L的碘乙酰胺(Iodoacetamide，IAA)避光烷基化处理45min；用0.04mg/ml胰蛋白 酶37℃下酶解16h；将酶解的肽用1％的三氟乙酸(Trifluoroacetic acid，TFA，pH＝2-3)进行酸化，然后用C18脱盐柱进行脱盐，将脱盐的肽在真空下干燥后溶于20μL含有0.1％甲酸和2％乙腈的缓冲液中；通过Nanodrop扫描仪在吸光度为A280nm的紫外光下检测肽的浓度，用分析柱(150μm×250mm)分离得到1.5μg肽，注入QE-HF(Q Exactive HF Hybrid Quadrupole OrbitrapTM，Thermo Fisher)质谱仪，DIA采集方案由32个固定窗口组成，采集范围为400-1200质荷比(m/z)，MS1和MS2的分辨率分布为60000和30000。原始文件导入Spectronaut pulsar进行分析，参数为软件默认。Add 2 μL of serum sample into a 1.5 mL centrifuge tube, dilute with 6 mol/L urea (from Sigma, USA) lysis buffer, and use 10 mmol/ml dithiothreitol (DTT) in a water bath at 37 °C for dithiothreitol (DTT). Sulfide reduction for 60 min; then alkylation treatment with 500 mmol/L iodoacetamide (IAA) in the dark for 45 min at 25 °C; enzymatic hydrolysis with 0.04 mg/ml trypsin at 37 °C for 16 h; Use 1% trifluoroacetic acid (Trifluoroacetic acid, TFA, pH = 2-3) to acidify, then desalt with a C18 desalting column, dry the desalted peptide under vacuum and dissolve it in 20 μL containing 0.1% formic acid and 2% acetonitrile The concentration of the peptide was detected by a Nanodrop scanner under ultraviolet light with an absorbance of A280nm, and 1.5 μg of the peptide was separated by an analytical column (150 μm×250 mm), injected into QE-HF (Q Exactive HF Hybrid Quadrupole OrbitrapTM, Thermo Fisher ) mass spectrometer, the DIA acquisition scheme consists of 32 fixed windows, the acquisition range is 400-1200 mass-to-charge ratio (m/z), and the resolution distribution of MS1 and MS2 is 60000 and 30000. The original file is imported into Spectronaut pulsar for analysis, and the parameters are the software defaults.
质谱筛选骨髓瘤诊断标志物的结果如图1所示。The results of mass spectrometry screening of myeloma diagnostic markers are shown in Figure 1.
实施例2：骨髓瘤诊断标志物的抗体芯片分析Example 2: Antibody Chip Analysis of Myeloma Diagnostic Markers
采用特制的抗体芯片对实施例1所得潜在的诊断标志物进行验证。The potential diagnostic markers obtained in Example 1 were verified using a special antibody chip.
1、抗体芯片的制备1. Preparation of antibody chip
(1)实验材料(1) Experimental materials
SERPINF2抗体购自R&D公司，货号：MAB1470-SP。SERPINF2 antibody was purchased from R&D Company, product number: MAB1470-SP.
阴性对照：100μg/mL的BSA和1×PBS。Negative control: 100 μg/mL BSA and 1×PBS.
阳性对照：10μg/mL Alexx-55-羊抗人IgG；100μg/mL的Biotin-IgG。Positive control: 10μg/mL Alexx-55-goat anti-human IgG; 100μg/mL Biotin-IgG.
(2)制备步骤：(2) Preparation steps:
利用英国Arrayjet的Ultra MarathonⅡ点样仪，采用喷点的方式，将各种抗体、阴性对照和阳性对照点置在晶芯高分子三维基片D(博奥生物集团有限公司，北京)上，点制好的芯片置于湿度为60％，温度为25℃的封闭环境中放置2个小时后放在-20℃冰箱储存，备用。Using the Ultra Marathon II sample spotting instrument of Arrayjet in the UK, various antibodies, negative controls and positive controls were placed on the crystal core polymer three-dimensional substrate D (Boao Biological Group Co., Ltd., Beijing) in the way of spraying spots. The prepared chip is placed in a closed environment with a humidity of 60% and a temperature of 25°C for 2 hours, and then stored in a -20°C refrigerator for future use.
2、抗体芯片的检测流程2. Detection process of antibody chip
(1)实验材料(1) Experimental materials
样本稀释液1×PBS(137mMNaCl，2.7mMKCl，10mM Na 2HPO 4，2mM KH 2PO 4)； Sample diluent 1×PBS (137mMNaCl, 2.7mMKCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 );
清洗液0.05％PBST(0.05％Tween，1×PBS)，Tween购自美国Amresco公 司；Cleaning solution 0.05% PBST (0.05% Tween, 1 × PBS), Tween was purchased from American Amresco company;
5％牛奶封闭液(5％牛奶，1×PBST)，脱脂奶粉购自美国BD公司；5% milk blocking solution (5% milk, 1 × PBST), skimmed milk powder was purchased from BD Company in the United States;
生物素标记试剂(NHS-PEG4-Biotin)、荧光染料(Streptavidin,R-Phycoerythrin Conjugate(SAPE))购自美国Thermo Fisher Scientific公司；Biotin labeling reagent (NHS-PEG4-Biotin) and fluorescent dye (Streptavidin, R-Phycoerythrin Conjugate (SAPE)) were purchased from Thermo Fisher Scientific, USA;
BSA(Albumin from bovine serum)购自美国Sigma-Aldrich公司；BSA (Albumin from bovine serum) was purchased from Sigma-Aldrich Company of the United States;
生物芯片扫描仪(4300A)，购自美国Molecular Devices仪器公司；Biochip scanner (4300A), purchased from U.S. Molecular Devices instrument company;
孵育盘(3/5)购自德国PEPperPRINT公司；The incubation plate (3/5) was purchased from PEPperPRINT, Germany;
温度控制混匀器(MixMate)和台式离心机(Centrifuge 5810R)，购自德国Eppendorf股份公司；Temperature control mixer (MixMate) and desktop centrifuge (Centrifuge 5810R), purchased from Germany Eppendorf AG;
微型分离柱Bio-Spin6，购自美国Bio-Rad公司；Micro-separation column Bio-Spin6, purchased from American Bio-Rad company;
PCR-384M2-C微孔板，购自美国Axygen公司。PCR-384M2-C microplates were purchased from Axygen, USA.
(2)实验步骤：(2) Experimental steps:
血清样本的标记：Labeling of serum samples:
采用Xu,M.,Deng,J.,Xu,K.et al.In-depth serum proteomics reveals biomarkers of psoriasis severity and response to traditional Chinese medicine.Theranostics.2019,9,2475-2488.中所述的方法用生物素标记所有血清蛋白质分子。如图2所示，将10μL血清用90μL过滤的1×PBS稀释，然后用1μL的生物素标记试剂标记血清，室温孵育1h后，用Bio-Spin6分离柱(Bio-Rad，美国)在1000×g转速下离心2min除去过量的生物素分子，将收集的生物素化蛋白质溶于500μL含有5％脱脂牛奶的1×PBS中，并在-20℃下储存。Using the method described in Xu, M., Deng, J., Xu, K. et al. In-depth serum proteomics reveals biomarkers of psoriasis severity and response to traditional Chinese medicine. Theranostics. 2019, 9, 2475-2488. All serum protein molecules were labeled with biotin. As shown in Figure 2, 10 μL of serum was diluted with 90 μL of filtered 1×PBS, and then 1 μL of biotin-labeled reagent was used to label the serum. Centrifuge at g speed for 2 min to remove excess biotin molecules, dissolve the collected biotinylated protein in 500 μL of 1×PBS containing 5% skim milk, and store at -20°C.
血清样本的检测：Testing of serum samples:
将抗体芯片从-20℃冰箱拿出，在室温放置30min后固定在孵育盘上(PEPperPRINT，德国)，每一个阵列对应一个围栏；Take the antibody chip out of the -20°C refrigerator, place it at room temperature for 30 minutes, and fix it on an incubation plate (PEPperPRINT, Germany), and each array corresponds to a fence;
(1)封闭：将500μL 5％的脱脂牛奶(溶剂为含0.05％PBST)离心后缓慢加入孵育盘的每个围栏内，室温下孵育1h；(1) Sealing: Centrifuge 500 μL of 5% skimmed milk (solvent containing 0.05% PBST) and slowly add it to each enclosure of the incubation plate, and incubate at room temperature for 1 hour;
(2)加样：真空泵吸去孵育盘内的牛奶，将处理好的血清标记物加入孵育盘的每个围栏内，在摇床上4℃条件下过夜反应；(2) Adding samples: suck the milk in the incubation plate with a vacuum pump, add the processed serum markers into each enclosure of the incubation plate, and react overnight at 4°C on a shaker;
(3)洗涤：真空泵吸去孵育盘内的溶液，0.05％PBST(含0.05％Tween的1×PBS)洗涤三次，每次10min，然后用去离子水洗涤三次，每次5min；(3) Washing: the solution in the incubation tray was sucked by a vacuum pump, washed three times with 0.05% PBST (1×PBS containing 0.05% Tween), each time for 10 min, and then washed three times with deionized water, each time for 5 min;
(4)加荧光染料：将2mg/L的荧光染料(溶剂为5％脱脂牛奶)加入到芯片的阵列中，室温避光孵育1h；(4) Add fluorescent dye: add 2 mg/L fluorescent dye (solvent is 5% skim milk) to the array of the chip, and incubate at room temperature for 1 hour in the dark;
(5)避光洗涤：操作同步骤(3)；(5) Washing in the dark: the operation is the same as step (3);
(6)芯片检测：将芯片晾干，荧光芯片扫描仪进行扫描并进行荧光信号数据提取。(6) Chip detection: dry the chip, scan it with a fluorescent chip scanner and extract the fluorescent signal data.
抗体芯片检测结果如图2所示。The results of antibody chip detection are shown in Figure 2.
质谱和抗体芯片检测结果的火山图如图3所示。The volcano plot of the mass spectrometry and antibody chip detection results is shown in Figure 3.
实施例3：抗体芯片的临床检测准确性Embodiment 3: The clinical detection accuracy of antibody chip
图4为抗体芯片检测的代表性结果，其中，A图是健康人抗体芯片的检测结果，B图是骨髓瘤患者的检测结果，发明人发现B图中点的信号普遍较A图的信号强。初步说明骨髓瘤患者较健康人血清中有较多的上调蛋白。Figure 4 is a representative result of antibody chip detection, wherein, picture A is the detection result of the antibody chip of healthy people, and picture B is the test result of myeloma patients. The inventors found that the signal of the point in picture B is generally stronger than that of picture A . It is preliminarily shown that there are more up-regulated proteins in the serum of myeloma patients than in healthy people.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention within.
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。The aforementioned embodiments and methods described in the present invention may vary based on the ability, experience and preference of those skilled in the art.
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。In the present invention, only listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims (10)

  1. 一种检测SERPINF2基因或SERPINF2蛋白的产品在制备用于骨髓瘤的诊断和/或预后的工具中的应用。Application of a product for detecting SERPINF2 gene or SERPINF2 protein in the preparation of tools for diagnosis and/or prognosis of myeloma.
  2. 如权利要求1所述的应用，其特征在于，所述产品包括检测SERPINF2基因或SERPINF2蛋白的表达水平的产品。The application according to claim 1, wherein the product includes a product for detecting the expression level of the SERPINF2 gene or the SERPINF2 protein.
  3. 如权利要求1所述的应用，其特征在于，所述产品包括能够结合SERPINF2基因的核酸或能够结合SERPINF2蛋白的物质。The use according to claim 1, characterized in that the product comprises a nucleic acid capable of binding SERPINF2 gene or a substance capable of binding SERPINF2 protein.
  4. 如权利要求1所述的应用，其特征在于，所述产品选自：试剂、试剂盒、试纸、基因芯片、高通量测序平台、抗体芯片、仪器平台；The application according to claim 1, wherein the product is selected from the group consisting of reagents, kits, test strips, gene chips, high-throughput sequencing platforms, antibody chips, and instrument platforms;
    优选地，所述仪器平台包含测量模块，用于测量待测样本中SERPINF2蛋白的含量。Preferably, the instrument platform includes a measurement module for measuring the content of the SERPINF2 protein in the sample to be tested.
  5. 如权利要求1所述的应用，其特征在于，所述骨髓瘤包括单发性骨髓瘤和多发性骨髓瘤。The use according to claim 1, wherein the myeloma comprises single myeloma and multiple myeloma.
  6. 如权利要求1所述的应用，其特征在于，用于所述SERPINF2基因或SERPINF2蛋白的检测的样本为受试者的血液或其级分，优选为血清。The application according to claim 1, characterized in that the sample used for the detection of the SERPINF2 gene or SERPINF2 protein is the subject's blood or its fraction, preferably serum.
  7. 一种用于骨髓瘤的诊断和/或预后的工具，其包含检测SERPINF2基因或SERPINF2蛋白的产品。A tool for diagnosis and/or prognosis of myeloma, comprising a product for detecting SERPINF2 gene or SERPINF2 protein.
  8. 一种SERPINF2基因或SERPINF2蛋白的抑制剂在制备治疗骨髓瘤的药物中的应用。The application of an inhibitor of SERPINF2 gene or SERPINF2 protein in the preparation of medicine for treating myeloma.
  9. 如权利要求8所述的应用，其特征在于，所述抑制剂能够抑制SERPINF2上游或下游途径的物质的表达或活性。The use according to claim 8, characterized in that the inhibitor can inhibit the expression or activity of substances in the upstream or downstream pathway of SERPINF2.
  10. 一种治疗骨髓瘤的药物，其包含SERPINF2基因或SERPINF2蛋白的抑制剂。A medicine for treating myeloma, comprising an inhibitor of SERPINF2 gene or SERPINF2 protein.
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