Molecular marker combinations and their uses

Abstract

The present invention relates to three different functional heterogeneous megakaryocyte subpopulations that may exist in mouse megakaryocytes (MK), and the specific molecular markers of these subpopulations are identified respectively, including MYLK4, a MK molecular marker that serves as a microenvironment support cell of HSC; LSP1, CD53 or CD52, a MK molecular marker that performs an immunomodulatory function; and ARNTL, a MK molecular marker that has a plate production function. These molecular markers can be used to separate or mark MKs with different functions, thereby promoting MKs to perform different functions, including supporting HSC quiescence, immunomodulation, plate production, etc.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology and medicine, and specifically relates to a megakaryocyte population and a method for identifying a megakaryocyte population. Further, the present invention also relates to the use of a reagent for detecting a marker molecule or a combination thereof in preparing a kit, and the use of an antibody or a combination thereof in preparing a reagent. Background Art

[0002] Megakaryocytes (MK) are the largest and smallest type of hematopoietic cells in the bone marrow. The diameter of MK can reach 50-100μm, and the number of MK only accounts for 0.05-0.1% of bone marrow cells. MK develops and differentiates from hematopoietic stem cells (HSC). Hematopoietic stem cells first differentiate into megakaryocyte progenitor cells. Megakaryocyte progenitor cells undergo polyploidization through nuclear mitosis and gradually mature. Eventually, the number of chromosomes in megakaryocytes continues to double, reaching up to 64N in mice.

[0003] Traditionally, the understanding of megakaryocyte function in this field is limited to the production of platelets with hemostatic and coagulation functions. Therefore, the process of megakaryocyte differentiation, maturation and platelet production has always been the focus of research in this field. However, in recent years, experimental studies in mice in vivo and in vitro have found that megakaryocytes can form the microenvironment of HSCs by releasing a large amount of cytokines IGF-1 (insulin-like growth factor 1), PF4 (platelet factor 4), TGF-β (transforming growth factor-β), and FGF1 (fibroblast growth factor 1) to regulate the quiescence and proliferation of HSCs. In addition, more and more evidence shows that some immune-related receptors or surface markers are expressed on the surface of megakaryocytes, including TLR1-6 (Toll-like receptors 1-6), FcγR, MHC I and CD40L. There is also evidence that during viral infection, megakaryocytes can upregulate the expression of IFITM3 (interferon-induced transmembrane protein 3) to enable them to resist viruses. The above findings all suggest that megakaryocytes may also have the function of participating in immune responses. Therefore, megakaryocytes not only include progenitor cells that produce platelets, but may also have multiple functions such as regulating HSC and immune response.

[0004] The multifunctionality of megakaryocytes has brought about new scientific questions: Are the multiple functions of megakaryocytes performed by the same group of megakaryocytes or by megakaryocytes with different functions? In other words, are there functional heterogeneous subpopulations within megakaryocytes? At present, the research on megakaryocyte heterogeneity is mainly based on three levels: megakaryocyte morphology, chromosome ploidy, and surface marker expression. The morphology of megakaryocytes changes during their development, maturation, and platelet production. According to the differences in morphology, megakaryocytes can be divided into primitive megakaryocytes, immature megakaryocytes, granular megakaryocytes, platelet-producing megakaryocytes, and naked nucleus megakaryocytes. At the same time, studies have shown that there are up to 6 types of megakaryocytes with different numbers of chromosomes in mouse bone marrow. Therefore, the concept that the cell maturity gradually increases with the increase of megakaryocyte chromosome ploidy is also widely accepted. In addition, studies have shown that during the maturation of megakaryocytes, the expression of surface markers such as CD41, CD42, and CD61 also gradually increases. Therefore, the expression of these markers can distinguish megakaryocytes at different developmental stages and maturity. However, the heterogeneity of megakaryocytes distinguished by morphology, chromosome ploidy and surface markers does not correspond to the different functions of megakaryocytes.

[0005] Therefore, the functional heterogeneity of megakaryocytes remains essentially unknown. It is well known that megakaryocytes in the bone marrow are extremely small and fragile, making them difficult to obtain, making it difficult to directly study megakaryocytes in vivo. Megakaryocytes with different functions have different transcriptional programs. Therefore, traditional transcriptomics methods cannot effectively distinguish megakaryocytes with different functions.

[0006] Therefore, it is extremely challenging to explore the functional heterogeneity of megakaryocytes. Summary of the invention

[0007] After in-depth research and creative work, the inventors of the present application discovered that there may be three megakaryocyte subpopulations with different functional heterogeneity in mouse megakaryocytes, and identified the specific molecular markers of these subpopulations, including MYLK4 (Myosin Light Chain Kinase4), a molecular marker for MKs that serve as microenvironment support cells for HSCs; LSP1 (Lymphocyte-specific protein 1), CD53 or CD52, a molecular marker for MKs that exercise immunoregulatory functions; and ARNTL (Aryl hydrocarbon receptor nucleartranslocator-like protein 1, also known as Brain and Muscle ARNT-Like 1, BMAL1), a molecular marker for MKs that have plate production functions. These molecular markers can be used to separate or label MKs with different functions, thereby promoting MKs to exercise different functions, including supporting HSC quiescence, immunoregulation, plate production, etc.

[0008] Thus, in a first aspect, the present application provides the use of an antibody or a combination thereof in preparing a reagent for detecting, identifying or isolating a megakaryocyte cell population in a sample, wherein the antibody or the combination thereof is selected from any one or more of (a) to (c):

[0009] (a) anti-MYLK4 antibody;

[0010] (b) anti-LSP1 antibody, anti-CD52 antibody and / or anti-CD53 antibody;

[0011] (c) anti-ARNTL antibodies;

[0012] In certain embodiments, item (b) of the antibody or combination thereof is an anti-LSPl antibody.

[0013] In certain embodiments, item (b) of the antibody or combination thereof is an anti-CD52 antibody.

[0014] In certain embodiments, item (b) of the antibody or combination thereof is an anti-CD53 antibody.

[0015] In certain embodiments, the antibody or combination thereof is an anti-LSPl antibody.

[0016] In certain embodiments, the antibody or combination thereof is an anti-CD52 antibody.

[0017] In certain embodiments, the antibody or combination thereof is an anti-CD53 antibody.

[0018] In certain embodiments, item (b) of the antibody or combination thereof is an anti-CD52 antibody and an anti-CD53 antibody.

[0019] In certain embodiments, item (b) of the antibody or combination thereof is an anti-LSPl antibody, an anti-CD52 antibody, and an anti-CD53 antibody.

[0020] In certain embodiments, the antibody or combination thereof consists of (a) to (c):

[0021] (a) anti-MYLK4 antibody;

[0022] (b) anti-LSP1 antibody, anti-CD52 antibody and / or anti-CD53 antibody;

[0023] (c) Anti-ARNTL antibody.

[0024] In certain embodiments, the MYLK4, LSP1, CD52, CD53 and / or ARNTL are derived from mammals (eg, humans, mice, sheep, horses, pigs, rabbits, etc.).

[0025] In certain embodiments, the nucleotide sequence encoding the MYLK4 is shown as SEQ ID NO: 2 or 12.

[0026] In certain embodiments, the nucleotide sequence encoding the LSP1 is shown as SEQ ID NO: 4 or 14.

[0027] In certain embodiments, the nucleotide sequence encoding the CD52 is shown as SEQ ID NO: 8 or 18.

[0028] In certain embodiments, the nucleotide sequence encoding the CD53 is shown as SEQ ID NO: 10 or 20.

[0029] In certain embodiments, the nucleotide sequence encoding the ARNTL is shown as SEQ ID NO: 6 or 16.

[0030] In certain embodiments, the sample is selected from mammalian whole blood, peripheral blood, bone marrow, umbilical cord blood, or any combination thereof.

[0031] In certain embodiments, the mammal is selected from a human, a dog, a monkey, a cow, a horse, or any combination thereof.

[0032] In certain embodiments, the antibody or combination thereof is selected from any one or more of (a) to (f):

[0033] (a) anti-MYLK4 antibody;

[0034] (b) anti-LSP1 antibody, anti-CD52 antibody and / or anti-CD53 antibody;

[0035] (c) anti-ARNTL antibodies;

[0036] (d) anti-MYLK4 antibody and anti-CD41 antibody;

[0037] (e) anti-LSP1 antibody, anti-CD52 antibody and / or anti-CD53 antibody, and anti-CD41 antibody;

[0038] (f) Anti-ARNTL antibody and anti-CD41 antibody.

[0039] Herein, the antibody refers to an antibody and an antigen-binding fragment thereof (eg, Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragment, etc.).

[0040] In another aspect, the present application provides the use of a marker molecule or a combination thereof in preparing a reagent for detecting, identifying or isolating a megakaryocyte cell population in a sample, wherein the marker molecule or the combination thereof is selected from any one or more of (a) to (c):

[0041] (a) MYLK4;

[0042] (b) LSP1, CD52 and / or CD53;

[0043] (c) ARNTL;

[0044] In certain embodiments, the marker molecules or combinations thereof of item (b) are CD52 and CD53.

[0045] In certain embodiments, the marker molecules or combinations thereof of item (b) are LSP1, CD52 and CD53.

[0046] In certain embodiments, the labeling molecule or a combination thereof consists of (a) to (c):

[0047] (a) MYLK4;

[0048] (b) LSP1, CD52 and / or CD53;

[0049] (c) ARNTL.

[0050] In certain embodiments, the MYLK4, LSP1, CD52, CD53 and / or ARNTL are derived from mammals (eg, humans, mice, sheep, horses, pigs, rabbits, etc.).

[0051] In certain embodiments, the nucleotide sequence encoding the MYLK4 is shown as SEQ ID NO: 2 or 12.

[0052] In certain embodiments, the nucleotide sequence encoding the LSP1 is shown as SEQ ID NO: 4 or 14.

[0053] In certain embodiments, the nucleotide sequence encoding the CD52 is shown as SEQ ID NO: 8 or 18.

[0054] In certain embodiments, the nucleotide sequence encoding the CD53 is shown as SEQ ID NO: 10 or 20.

[0055] In certain embodiments, the nucleotide sequence encoding the ARNTL is shown as SEQ ID NO: 6 or 16.

[0056] In certain embodiments, the labeling molecule or combination thereof is selected from any one or more of (a) to (f):

[0057] (a) MYLK4;

[0058] (b) LSP1, CD52 and / or CD53;

[0059] (c) ARNTL;

[0060] (d) MYLK4 and CD41;

[0061] (e) LSP1, CD52 and / or CD53, and CD41;

[0062] (f) ARNTL and CD41.

[0063] In another aspect, the present application provides a megakaryocyte cell population, which expresses at least one marker molecule selected from MYLK4, LSP1, CD52, CD53 and ARNTL.

[0064] In certain embodiments, the cell population expresses CD52 and CD53 marker molecules.

[0065] In certain embodiments, the cell population expresses LSP1, CD52 and CD53 marker molecules.

[0066] In certain embodiments, the cell population further expresses the CD41 marker molecule.

[0067] In certain embodiments, the cell population consists of any one or more of the cell populations of (a) to (c):

[0068] (a) Cell population expressing MYLK4 and CD41;

[0069] (b) a cell population expressing LSP1 and CD41, a cell population expressing CD52 and CD41, and / or a cell population expressing CD53 and CD41;

[0070] (c) Cell population expressing ARNTL and CD41.

[0071] In certain embodiments, the MYLK4, LSP1, CD52, CD53 and / or ARNTL are derived from mammals (eg, humans, mice, sheep, horses, pigs, rabbits, etc.).

[0072] In certain embodiments, the nucleotide sequence encoding the MYLK4 is shown as SEQ ID NO: 2 or 12.

[0073] In certain embodiments, the nucleotide sequence encoding the LSP1 is shown as SEQ ID NO: 4 or 14.

[0074] In certain embodiments, the nucleotide sequence encoding the CD52 is shown as SEQ ID NO: 8 or 18.

[0075] In certain embodiments, the nucleotide sequence encoding the CD53 is shown as SEQ ID NO: 10 or 20.

[0076] In certain embodiments, the nucleotide sequence encoding the ARNTL is shown as SEQ ID NO: 6 or 16.

[0077] In certain preferred embodiments, the cell population expressing MYLK4 is capable of regulating HSC quiescence or proliferation.

[0078] In certain preferred embodiments, the cell population expressing LSP1, the cell population expressing CD52, the cell population expressing CD53, the cell population expressing CD52 and CD53, and / or the cell population expressing LSP1, CD52 and CD53 have the ability of immunomodulation (anti-inflammatory), for example, they can respond to pathogen stimulation, have the ability of pathogen phagocytosis, pathogen digestion, antigen presentation and stimulation of T cell proliferation, etc.

[0079] In certain preferred embodiments, the cell population expressing ARNTL and CD41 has platelet-producing ability.

[0080] In certain preferred embodiments, the above-mentioned megakaryocyte populations with different functions are screened and separated by combining the marker molecules as described above, so that the megakaryocyte populations can be used for subsequent treatment (for example, bone marrow transplantation, or cell therapy such as using cytokines to directly or indirectly kill cancer cells).

[0081] On the other hand, the present application provides a method for identifying, isolating or detecting a megakaryocyte cell population as described above, the method comprising: (1) contacting the cell population with at least one reagent (for example, one, two, three or more reagents), and (2) detecting the cell population.

[0082] In certain embodiments, the reagent detects the labeled molecule by a protein detection method.

[0083] In certain embodiments, the protein detection method is selected from immunological methods, chromatography, mass spectrometry, spectroscopy, or any combination thereof.

[0084] In certain embodiments, the protein detection method is an enzyme-linked immunosorbent assay, a chemiluminescent immunoassay, a fluorescent immunoassay, a radioimmunoassay, an electrochemical immunosensor, a peptide mass fingerprinting method, or any combination thereof.

[0085] In certain embodiments, the reagents each independently comprise an antibody capable of specifically binding to the labeled molecule, an antigen-binding fragment thereof, and a detectable label.

[0086] In certain embodiments, the reagents are each independently a combination of multiple (e.g., 2 or more) antibodies that can specifically bind to a labeled molecule. In certain embodiments, the combination comprises a first antibody as a capture reagent and a second antibody as a detection reagent. In certain embodiments, the second antibody has an antigen binding site different from the first antibody. In certain embodiments, the second antibody carries a detectable label.

[0087] In certain embodiments, the detectable labels are each independently selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound), an electrochemiluminescent agent (e.g., terpyridine ruthenium), an electroactive substance, a fluorescent dye (e.g., allophycocyanin (APC), FITC, TRITC, PE, Texas Red, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), a radionuclide (e.g., radioiodine), or biotin.

[0088] In certain preferred embodiments, the detectable labels contained in each reagent are different. For example, when the detectable label is a fluorescent dye, the fluorescent dye contained in each reagent can emit different fluorescence.

[0089] In certain embodiments, the reagent detects the mRNA of the marker molecule or the cDNA of the marker molecule by a nucleic acid detection method.

[0090] In certain embodiments, the nucleic acid detection method is transcription analysis, fluorescent quantitative PCR, nucleic acid hybridization or any combination thereof.

[0091] In certain embodiments, the reagents each independently comprise at least one primer or probe that is complementary to a portion of the nucleotide sequence of the marker molecule mRNA or cDNA.

[0092] In certain embodiments, the method can be used for therapeutic or diagnostic purposes, for example, cell therapy can be performed on a subject based on the megakaryocyte cell population screened and isolated from a sample.

[0093] In certain embodiments, the methods can be used for non-therapeutic or diagnostic purposes, such as identifying different stages of a cell population using marker molecules.

[0094] In another aspect, the present application provides a kit comprising reagents for identifying, detecting or isolating marker molecules, wherein the molecular markers are selected from MYLK4, LSP1, CD52, CD53, ARNTL, or any combination thereof.

[0095] In certain embodiments, the marker molecule further comprises CD41.

[0096] In certain embodiments, the MYLK4, LSP1, CD52, CD53 and / or ARNTL are derived from mammals (eg, humans, mice, sheep, horses, pigs, rabbits, etc.).

[0097] In certain embodiments, the nucleotide sequence encoding the MYLK4 is shown as SEQ ID NO: 2 or 12.

[0098] In certain embodiments, the nucleotide sequence encoding the LSP1 is shown as SEQ ID NO: 4 or 14.

[0099] In certain embodiments, the nucleotide sequence encoding the CD52 is shown as SEQ ID NO: 8 or 18.

[0100] In certain embodiments, the nucleotide sequence encoding the CD53 is shown as SEQ ID NO: 10 or 20.

[0101] In certain embodiments, the nucleotide sequence encoding the ARNTL is shown as SEQ ID NO: 6 or 16.

[0102] In certain embodiments, the reagent detects the labeled molecule by a protein detection method.

[0103] In certain embodiments, the protein detection method is selected from immunological methods, chromatography, mass spectrometry, spectroscopy, or any combination thereof.

[0104] In certain embodiments, the protein detection method is an enzyme-linked immunosorbent assay, a chemiluminescent immunoassay, a fluorescent immunoassay, a radioimmunoassay, an electrochemical immunosensor, a peptide mass fingerprinting method, or any combination thereof.

[0105] In certain embodiments, the reagent comprises an antibody capable of specifically binding to the labeled molecule, an antigen-binding fragment thereof, and a detectable label.

[0106] In certain embodiments, the reagent comprises a combination of multiple (e.g., 2 or more) antibodies that can specifically bind to a labeled molecule. In certain embodiments, the combination comprises a first antibody as a capture reagent and a second antibody as a detection reagent. In certain embodiments, the second antibody has an antigen binding site different from the first antibody. In certain embodiments, the second antibody carries a detectable label.

[0107] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds), an electrochemiluminescent agent (e.g., terpyridine ruthenium), an electroactive substance, a fluorescent dye (e.g., allophycocyanin (APC), FITC, TRITC, PE, Texas Red, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), a radionuclide (e.g., radioiodine), or biotin.

[0108] In certain preferred embodiments, the detectable labels contained in each reagent are different. For example, when the detectable label is a fluorescent dye, the fluorescent dye contained in each reagent can emit different fluorescence.

[0109] In certain embodiments, the reagent detects the mRNA of the marker molecule or the cDNA of the marker molecule by a nucleic acid detection method.

[0110] In certain embodiments, the nucleic acid detection method is transcription analysis, fluorescent quantitative PCR, nucleic acid hybridization or any combination thereof.

[0111] In certain embodiments, the reagents each independently comprise at least one primer or probe that is complementary to a portion of the nucleotide sequence of the marker molecule mRNA or cDNA.

[0112] In certain embodiments, the kits are used to identify, detect or isolate a megakaryocyte population.

[0113] On the other hand, the present application provides use of the cell population as described above or the kit as described above in the preparation of a drug for treating a disease (e.g., blood-related diseases) and / or an immune-related disease (e.g., an autoimmune disease) caused by abnormalities (e.g., decrease or increase) in megakaryocytes, platelets and / or hematopoietic stem cells in a subject.

[0114] In certain embodiments, the disease includes megakaryocytic lineage developmental disorder, thrombocytopenia of various causes, and thrombocytosis.

[0115] In certain embodiments, the blood-related diseases include hematopoietic malignancies and hematopoietic non-malignant diseases.

[0116] The hematopoietic malignancies include acute leukemia, chronic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myelodysplastic syndrome and multiple myeloma.

[0117] In certain embodiments, the non-malignant disease of the hematopoietic system is aplastic anemia, immune thrombocytopenia, familial thrombocytopenia, chronic thrombocytopenic purpura, paroxysmal nocturnal hemoglobinuria, and myelofibrosis.

[0118] In certain embodiments, the autoimmune-related disease includes rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, polymyositis, dermatomyositis, ankylosing spondylitis, psoriatic arthritis, and gout.

[0119] In certain embodiments, the subject is a mammal (eg, mouse, human, dog, monkey, cow, or horse).

[0120] In certain embodiments, the subject is a human.

[0121] In certain preferred embodiments, platelet and / or hematopoietic stem cell abnormality refers to that the platelets and / or hematopoietic stem cells themselves or their content are different from the platelets and / or hematopoietic stem cells of healthy subjects. For example, the detection value of platelets and / or hematopoietic stem cells is lower than the detection value of healthy subjects.

[0122] Definition of terms

[0123] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0124] As used herein, the terms "marker molecule" and "molecular marker" have the same meaning and can be used interchangeably. "Marker molecule" refers to a class of substances that can be used to distinguish different cell types, which can be expressed extracellularly or as intracellular molecules. In this article, marker molecules can include nucleic acids (RNA, cDNA and DNA), proteins, and variants and fragments thereof.

[0125] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site, respectively. The assignment of amino acids to various regions or domains follows the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0126] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and such polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to a polypeptide.

[0127] As used herein, the term "fluorescent dye" refers to a fluorescent molecule that can re-emit light after being excited with absorbed light of a specific wavelength. Typically, the light emitted by the fluorescent dye has a longer wavelength than the absorbed light. The term "fluorescent dye" can be used interchangeably with the terms "fluorophore", "fluorescent label", "fluorescent marker", "fluorescent probe" and "fluorescent reporter molecule".

[0128] As used herein, the term "subject" refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc. In certain embodiments, the subject is a human.

[0129] As used herein, the term "MYLK4 (myosin light chain kinase 4)" refers to an enzyme encoded by the MYLK2 gene in humans. MYLK4 is a member of the myosin light chain kinase family of serine / threonine-specific protein kinases that phosphorylates the regulatory light chain of myosin II. Diseases associated with MYLK4 include deafness, cardiomyopathy, etc. Its related signaling pathways include the Apelin signaling pathway. As a member of the MYLK family, MYLK4 is also expressed in large quantities in the myocardium, and structural analysis shows that it is a Ca-independent 2+ / calmodulin (CaM) kinase, MYLK4 gene may play an important role in muscle development and cardiac myocyte contractile function. However, the specific role and mechanism are rarely reported. When MYLK4 is mentioned in this article, it includes naturally occurring MYLK4 protein, its fusion protein, and artificially introduced mutations or variations (including but not limited to substitution, deletion and / or addition) that do not affect its biological function.

[0130] As used herein, the term "LSP1 (Leukocyte-specific protein 1)" refers to an intracellular F-actin binding protein encoded by the LSP1 gene. The protein is expressed in lymphocytes, neutrophils, macrophages and endothelium, and plays a role in the regulation of neutrophil activation and chemotaxis. Endothelial cell LSP1 can regulate the migration of neutrophils across endothelial cells, thereby exerting an anti-inflammatory effect. In addition, in tumor cells, the expression pattern and effect of LSP1 and the regulation of the immunosuppressive microenvironment still need to be further explained. When referring to LSP1 in this article, it includes naturally occurring LSP1 protein, its fusion protein, and proteins with artificially introduced mutations or variations (including but not limited to substitution, deletion and / or addition) but do not affect its biological function.

[0131] As used herein, the term "ARNTL (Aryl Hydrocarbon Receptor Nuclear Translocator Like)" refers to a helix-loop-helix protein encoded by the Arntl gene, which forms a heterodimer with CLOCK. Defects in this gene are associated with infertility, gluconeogenesis and lipogenesis abnormalities. Diseases associated with Arntl include delayed sleep phase disorder and Rem sleep behavior disorder. Its related pathways include neuroscience and circadian rhythm. Arntl is a core component of the circadian rhythm network, and the circadian clock guides metabolism, cell division, sleep wakefulness, circadian rhythm and seasonal cycles. Arntl is related to sleep, mood, hormone levels and metabolic abnormalities. When Arntl is mentioned in this article, it includes naturally occurring ARNTL proteins, their fusion proteins, and proteins that artificially introduce mutations or variations (including but not limited to substitutions, deletions and / or additions) but do not affect their biological functions.

[0132] As used herein, the term "CD52 (CD52 molecule)" is a protein encoded by the CD52 gene. Diseases associated with CD52 include lymphocytic leukemia and hairy cell leukemia. Its related pathways include protein metabolism and post-translational modification synthesis of GPI-anchored proteins. CD52 is present on the surface of lymphocytes, monocytes, dendritic cells and sperm cells. CD52 is a 12-amino acid peptide anchored to glycosylphosphatidylinositol (GPI). Its function is anti-adhesion and regulating the free movement of cells. When CD52 is mentioned in this article, it includes naturally occurring CD52 protein, its fusion protein, and proteins with artificially introduced mutations or variations (including but not limited to substitutions, deletions and / or additions) but do not affect its biological functions.

[0133] As used herein, the term "CD53" refers to a protein encoded by the CD53 gene, which is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. Most of these members are cell surface proteins characterized by the presence of four hydrophobic domains. The CD53 protein plays a role in the regulation of cell development, activation, growth and movement. The encoded protein is a cell surface glycoprotein that forms a complex with integrins. It helps to transduce signals generated by CD2 in T cells and natural killer cells, playing a role in growth regulation. Familial defects in this gene are associated with immunodeficiency associated with recurrent infectious diseases caused by bacteria, fungi and viruses. Its related pathways are associated with the innate immune system. When CD53 is mentioned in this article, it includes naturally occurring CD53 proteins, their fusion proteins, and proteins that artificially introduce mutations or variations (including but not limited to substitutions, deletions and / or additions) but do not affect their biological functions.

[0134] As used herein, the terms "megakaryocyte cell population" or "cell population" have the same meaning and can be used interchangeably. It is a part or collective composed of many individual megakaryocytes. In this article, the megakaryocyte cell population includes one or more megakaryocyte cell subpopulations.

[0135] Beneficial effects of the present invention:

[0136] This application uses transcriptome analysis to separate megakaryocyte subpopulations with different functions and identifies marker molecules for these subpopulations. By isolating or marking megakaryocytes with different functions, the different functions of megakaryocytes can be regulated (e.g., promoted or inhibited), including supporting HSC quiescence (CD41 + MYLK4 + MK), immune regulation (CD41 + LSP1 + MK、CD41 + CD53 + MK or CD41 + CD52 + MK), production board (CD41 + ARNTL + MK) et al. Further, it was demonstrated that CD53 +The use of megakaryocytes in immunoregulation, and the use of megakaryocytes in the treatment of immune diseases. In summary, the present invention provides a new solution for the clinical transformation of stem cells, the in vitro production of megakaryocytes for clinical transplantation, and the treatment of platelet abnormalities, immune abnormalities and other related diseases. In particular, the marker molecules of the present application have also been verified in the human megakaryocyte system, and the megakaryocyte subpopulation (i.e. Cluster 3) marked by CD53, CD52 and / or LSP1 has a strong anti-inflammatory (immune) ability. In addition, CD52 and CD53 are molecules on the surface of cells, which are more conducive to screening and isolating functional megakaryocyte subpopulations, so as to use megakaryocyte subpopulations for subsequent treatment (for example, cell therapy, bone marrow transplantation, immunoregulation, anti-inflammatory or cell therapy methods such as using cytokines to directly or indirectly kill cancer cells).

[0137] Sequence information

[0138] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0139] Table 1: Description of sequences

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 The results of the presence of transcriptome heterogeneous subpopulations in megakaryocytes are shown. Among them, Figure 1A is the strategy for obtaining single-cell transcriptomes of mouse megakaryocytes with different chromosome ploidy; Figure 1 B is the flow cytometry results of megakaryocyte sorting of different chromosome multiples in mice (SSC is side scattered light).

[0149] Figure 2 The results of the presence of transcriptome heterogeneous subpopulations in megakaryocytes are shown. Among them, Figure 2A is an unsupervised clustering diagram of mouse megakaryocytes with different chromosome ploidy; Figure 2 B is a statistical diagram of megakaryocyte subpopulations contained in mouse megakaryocytes with different chromosome multiples; Figure 2 C is a heat map of differentially expressed genes in different mouse megakaryocyte subsets and their enriched entries; Figure 2 D is the expression of cytokine genes Pf4 and Igf1 in different megakaryocyte subsets; Figure 2 E is the expression of specific markers of different megakaryocyte subsets.

[0150] Figure 3 The results of the presence of transcriptome heterogeneous subpopulations in megakaryocytes are shown. Among them, Figure 3A is an unsupervised clustering diagram of human megakaryocytes; Figure 3 B is a heat map of differentially expressed genes in different megakaryocyte subsets in mice and humans and their enriched entries; Figure 3 C is the expression of LSP1, CD52, and CD53 in human megakaryocyte subsets.

[0151] Figure 4 The immunofluorescence results of three markers of megakaryocyte subpopulations are shown. Among them, 4A is the immunofluorescence result of the megakaryocyte subpopulation marked by MYLK4 in the mouse bone marrow; 4B is the immunofluorescence result of the megakaryocyte subpopulation marked by LSP1 in the mouse bone marrow; 4C is the immunofluorescence result of the megakaryocyte subpopulation marked by ARNTL in the mouse bone marrow; 4D is the ratio of the megakaryocyte subpopulations marked by the three markers in the mouse bone marrow; 4E is the cell diameter statistics of the megakaryocyte subpopulations marked by the three markers.

[0152] Figure 5 The immunofluorescence results of ARNTL-labeled megakaryocytes distributed near blood vessels are shown. 5A is ARNTL in mouse bone marrow. + Immunofluorescence images of megakaryocytes and blood vessels; 5B is the distance statistics from megakaryocyte subsets labeled with three markers to blood vessels.

[0153] Figure 6 The results show the detection of megakaryocytes directly contacting HSCs with MYLK4-labeled megakaryocytes. Among them, 6A is the statistical proportion of HSCs at different distances from MK in mouse bone marrow; 6B is the statistical proportion of MYLK4 in mouse bone marrow. + Immunofluorescence images of megakaryocytes and HSCs; 6C is the proportion of megakaryocytes in direct contact with HSCs expressing three markers (MYLK4, LSP1 and ARNTL).

[0154] Figure 7The results of ARNTL-labeled megakaryocytes in the production of precursor platelets are shown. Among them, 7A is an immunofluorescence image of precursor platelets produced by mouse bone marrow explants and megakaryocytes labeled with three markers; Figure 7 B is the statistical proportion of the three markers (MYLK4, LSP1 and ARNTL) expressed in megakaryocytes that produce precursor platelets.

[0155] Figure 8 The results are shown in Figure 8A, which shows the potential of CD53-labeled megakaryocytes in antigen presentation in response to LPS, phagocytosis of pathogens and stimulation of T cell proliferation. 8B shows the ratio of megakaryocytes and CD53-labeled megakaryocytes in mouse bone marrow after LPS stimulation; 8C shows the ratio of CD53-labeled megakaryocytes in mouse bone marrow after LPS stimulation. + Megakaryocytes and CD53 - Immunofluorescence images of megakaryocyte bacterial phagocytosis experiments; Figure 8 D is CD53 + Megakaryocytes and CD53 - Statistical chart of the proportion of megakaryocytes that have the ability to phagocytose bacteria.

[0156] Fig. 9 The results show that CD53-labeled megakaryocytes have antigen presentation potential in response to LPS, phagocytosis of pathogens and stimulation of T cell proliferation. Fig. 9 A is CD53 + Megakaryocytes and CD53 - Flow cytometry analysis of megakaryocyte-stimulated T cell proliferation, where 10 μg / ml CD3 + 2 μg / ml CD28 was the positive control group, and 6 μg / ml CD3 + 2 μg / ml CD28 was the basal group; Fig. 9 B is the statistical proportion of T cell proliferation under different conditions. DETAILED DESCRIPTION

[0157] The present invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0158] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE (Animal Cell Culture, 2nd edition). CULTURE) (RI Freshney, ed. (1987)).

[0159] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0160] Example 1 - Single cell analysis found four megakaryocyte subsets and corresponding markers

[0161] The specific experimental steps and experimental results of this embodiment are as follows:

[0162] 1. Experimental Materials

[0163] (1) Experimental animals

[0164] C57BL / 6 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and maintained at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0165] (2) Donor bone marrow sample

[0166] Human bone marrow samples were obtained from four healthy male volunteers from the Institute of Hematology, Peking University People's Hospital. Acceptance of bone marrow samples from healthy volunteers was approved by the Ethics Committee of Peking University People's Hospital and complied with the regulations of the World Medical Association Declaration of Helsinki.

[0167] (3) Experimental reagents

[0168] Anti-mouse CD41 APC and anti-human CD41 APC antibodies were purchased from eBioscience; Hoechst 33342 was purchased from Invitrogen; fetal bovine serum (FBS) was purchased from Gibco; and reserpine was purchased from TargetMol.

[0169] 2. Experimental Methods

[0170] (1) Sorting of mouse megakaryocytes with different chromosome ploidy

[0171] After the mice were killed by cervical dislocation, the femur, tibia and ilium of the mice were obtained by dissection, and the bone marrow was flushed with PBS + 2% FBS + 2mM EDTA, stained with 0.3mg / ml anti-mouse CD41-APC antibody at 4℃ in the dark for 30min, washed once with PBS and centrifuged to remove the supernatant, and then washed with PBS + 2% FBS + 2mM EDTA + 10μM Hoechst 33342 + 5μM reserpine solution (1×10 6 The cells were resuspended in PBS+2% FBS+2mM EDTA and the megakaryocytes with different chromosome ploidy were sorted by MoFlo XDP flow cytometer.

[0172] (2) Construction and sequencing of mouse megakaryocyte single-cell transcriptome library

[0173] About 700 megakaryocytes of each chromosome multiple (2N, 4N, 8N, 16N, 32N) were sorted, and single cells were picked up with a mouth pipette under a stereoscope and put into a single cell lysis buffer for single cell transcriptome library construction. For the specific steps of single cell transcriptome library construction, see: Li, L., Dong, J., Yan, L., Yong, J., Liu, X., Hu, Y., Fan, X., Wu, X., Guo, H., and Wang, X. (2017). Single-cell RNA-seq analysis maps development of human germline cells and gonadal niche interactions. Cell Stem Cell 20, 858-873.e854. In brief, cells were first lysed, and then mRNA was reverse transcribed into cDNA using primers containing cell barcode and molecular barcode (Unique Molecular Identifier, UMI), and then cDNA was amplified by PCR (polymerase chain reaction) using template-switching oligo (TSO); then cDNA amplified from 96 cells was mixed together and subjected to biotin-labeled PCR reaction, and then cDNA was sheared into DNA fragments of about 300 bp, and biotin-labeled cDNA fragments were enriched by streptavidin magnetic beads, and then DNA library was constructed for the enriched cDNA using KAPA HyperDNA library construction kit. Finally, the constructed libraries were subjected to double-end sequencing (150 bp) to obtain double-end sequencing data containing read 1 and read 2.

[0174] (3) Single-cell transcriptome analysis of mouse megakaryocytes

[0175] According to the cell barcode information in read 2, the single-cell transcriptome sequence is assigned to each cell, the molecular barcode sequence (UMI) in read2 is added to read 1, the template switching primer sequence (TSO) and poly A sequence in read 1 are removed, and then the read 1 sequence is aligned to the mouse genome (mm10) using Tophat2. For each gene, duplicate transcripts with the same UMI are removed, and then the number of transcripts aligned to each gene is counted to obtain the number of transcripts for each gene in each single cell. Then, Seurat software is used to perform unsupervised clustering of single-cell transcriptomes of mouse megakaryocytes with different chromosome multiples, find differentially expressed genes in each subgroup, and perform functional enrichment analysis on differentially expressed genes to determine the potential cell type or function of each subgroup.

[0176] (4) Sorting of human megakaryocytes of different ploidy levels and single-cell transcriptome library construction and analysis

[0177] According to the method for sorting mouse megakaryocytes of different ploidies as described above, human megakaryocytes of different ploidies were sorted from the bone marrow of healthy humans, and then single-cell transcriptome library construction and analysis were performed using the same method as for mice.

[0178] 3. Experimental results

[0179] The megakaryocyte-specific expression gene CD41 and the live cell DNA dye Hoechst 33342 were used to sort out high-purity megakaryocytes with different chromosome multiples in mice. Then, highly active megakaryocytes were randomly picked manually with a mouth pipette. Finally, 795 megakaryocyte single-cell transcriptomes containing different chromosome multiples (2N-32N) were obtained by combining the improved Smart-seq2 single-cell transcriptome sequencing method ( Figure 1 A and 1B).

[0180] Using Seurat unsupervised clustering, four transcriptome heterogeneity subgroups were identified ( Figure 2 A). The results showed that Cluster 1 contained megakaryocytes of all chromosome multiples (2N-32N) and highly expressed genes related to DNA replication and cell cycle ( Figure 2 B and 2C). Therefore, it is believed that Cluster 1 subgroup may be megakaryocytes in the cell cycle state or in the process of polyploidization. Cluster 2 subgroup is mainly composed of high-ploid megakaryocytes (≥8N), which highly express genes related to cell-to-cell signaling and stem cell maintenance; at the same time, this subgroup highly expresses known cytokines secreted by megakaryocytes when regulating HSC, such as Pf4 and Igf1 ( Figure 2 B, 2C, and 2D). This suggests that Cluster 2 may be the microenvironmental cells of HSC, so it is defined as HSC microenvironmental megakaryocytes. Analysis of Cluster 3 revealed that Cluster 3 was mainly composed of low-ploidy megakaryocytes (≤8N) and highly expressed genes related to inflammatory response. Therefore, Cluster 3 may be megakaryocytes related to inflammatory response ( Figure 2 Similar to Cluster 2, Cluster 4 is also mainly composed of high-ploidy megakaryocytes (≥8N), but this subgroup highly expresses genes related to platelet production and function ( Figure 2 B and 2C). This suggests that Cluster 4 may be a megakaryocyte subpopulation that produces platelets.

[0181] Therefore, the above results indicate that there may be three megakaryocyte subpopulations with different functional heterogeneity in mouse megakaryocytes.

[0182] To further investigate these functionally heterogeneous megakaryocyte subsets, we identified specific markers for Cluster 2, 3, and 4, respectively: MYLK4, LSP1, CD52, CD53, and ARNTL ( Figure 2 E). The amino acid sequences of the above five mouse-derived markers are shown in SEQ ID NOs: 1, 3, 5, 7, and 9, respectively, and the nucleotide sequences are shown in SEQ ID NOs: 2, 4, 6, 8, and 10, respectively.

[0183] To determine whether similar heterogeneity exists in human megakaryocytes, we used the same method to obtain human single-cell transcriptomes at different multiples. Unsupervised cluster analysis revealed that human megakaryocytes also have four different subpopulations, and their differentially expressed genes are enriched in items similar to those of mouse megakaryocyte subpopulations ( Figure 3 A and 3B), suggesting that human and mouse megakaryocytes have similar transcriptome heterogeneity. In addition, we also found that the mouse Cluster 3 subgroup-specific expression genes Lsp1, Cd52, and Cd53 were also specifically highly expressed in the human Cluster 3 subgroup ( Figure 3 C), suggesting that these genes are species conserved in human and mouse megakaryocytes. The amino acid sequences of the five human-derived markers, MYLK4, LSP1, CD52, CD53 and ARNTL, are shown in SEQ ID NOs: 11, 13, 15, 17, and 19, respectively, and the nucleotide sequences are shown in SEQ ID NOs: 12, 14, 16, 18, and 20, respectively.

[0184] Example 2 - Molecular markers can be used to mark megakaryocyte subsets

[0185] The specific experimental steps and experimental results of this embodiment are as follows:

[0186] 1. Experimental Materials

[0187] (1) Experimental reagents

[0188] Anti-mouse CD105 PE and anti-mouse CD41 APC were purchased from eBioscience; rabbit polyclonal ARNTL antibody was purchased from Bio-Techne; anti-LSP1 antibody was purchased from Abcam; anti-MYLK4 polyclonal antibody was purchased from Cloud-Clone; MYLK4 rabbit polyclonal antibody was purchased from Proteintech; goat anti-rabbit IgG AF488 and goat anti-rabbit IgG AF555 were purchased from Invitrogen; donkey anti-rabbit IgG DyLight 550 was purchased from Pierce; biotin anti-mouse Lineage Panel, biotin anti-mouse CD48 and BrilliantViolet 421 Streptavidin were purchased from BioLegend; 4% paraformaldehyde (PFA) was purchased from YEASEN; sucrose was purchased from Amresco; EDTA was purchased from GIBCO; OCT cryosection embedding medium was purchased from Sakura; polyvinyl pyrrolidone was purchased from Sigma-Aldrich; gelatin was purchased from Merck millipore.

[0189] 2. Experimental Methods

[0190] (1) Preparation of 10 μm and 100 μm sections of mouse bone marrow

[0191] The femur and tibia of the mouse were fixed in 4% paraformaldehyde (PFA) solution at 4°C overnight, and then transferred to a mixture of 30% sucrose and 10% EDTA for decalcification and dehydration at 4°C for 48 hours. The treated tibia and femur were embedded in OCT embedding medium, and 10 μm and 100 μm mouse bone marrow sections were prepared.

[0192] (2) Preparation of 30 μm sections of mouse bone marrow

[0193] The anesthetized mice were perfused and fixed with 0.9% saline and 4% PFA, respectively. The removed mouse femur was placed in a 4% PFA solution again and fixed at 4°C for 2 hours. The mouse femur was then placed in a 4°C environment for decalcification and dehydration: decalcification was performed using 0.5M EDTA for 48 hours, and then the mouse femur was dehydrated in a mixed solution of 15% sucrose and 2% polyvinyl pyrrolidone for 24 hours. Finally, the dehydrated mouse femur was embedded in a mixed solution of 8% gelatin + 20% sucrose + 2% polyvinyl pyrrolidone to prepare 30μm mouse bone marrow sections.

[0194] (3) Immunofluorescence staining experiment

[0195] For 10μm and 100μm mouse bone marrow sections, after washing with PBS, first place the membrane in 0.5% Triton X-100 (prepared with PBS) at room temperature for 5 minutes, then use 3% BSA to block for 1 hour at room temperature, and block the primary antibody at 4°C overnight: (1:200 diluted rabbit anti-BMAL1; 1:100 diluted rabbit anti-LSP1; 1:100 diluted rabbit anti-MYLK4; 1:100 diluted anti-mouse CD105 PE and 1:500 diluted anti-mouse CD41 APC), wash away the excess primary antibody, incubate with secondary antibody at room temperature for 1 hour (1:500 goat anti-rabbit, Alexa Fluor Plus 488; 1:500 goat anti-rabbit, Alexa Fluor Plus 555), wash away the excess secondary antibody, and stain the nucleus with DAPI at room temperature for 10 minutes.

[0196] HSC used 30μm mouse bone marrow sections, first pretreated with 1g / ml protease at 37℃ for 15 minutes, then blocked with 10% goat serum at room temperature for 1 hour. Blocked with primary antibody CD150 (1:50) at 4℃ overnight, washed off the excess primary antibody, incubated with secondary antibody at room temperature for 1 hour (1:1000 dilution of donkey anti-rabbit IgG (H+L) Cross Adsorbed Secondary Antibody, DyLight 550conjugate); then incubated with primary antibody Lineage and CD48 at room temperature for 2 hours (1:50 dilution of biotin anti-mouse Lineage Panel; 1:100 dilution of biotin anti-mouse CD48) and its secondary antibody at room temperature for 1 hour (1:1000 dilution of Brilliant Violet 421Streptavidin), finally washed off the excess secondary antibody, and DAPI stained the nucleus at room temperature for 10 minutes.

[0197] 3. Experimental results

[0198] (1) Megakaryocytes labeled with three markers are present in mouse bone marrow

[0199] The presence of these transcriptome-heterogeneous subsets was confirmed in mouse bone marrow by immunofluorescence using antibodies against CD41 and markers for each functionally relevant megakaryocyte subset ( Figure 4 For each megakaryocyte subset, at least 668 MKs (CD41 + ), found ARNTL + Megakaryocytes (Cluster 4) accounted for the highest proportion (24.5±1.1%), which is consistent with the understanding that platelet production is the main function of megakaryocytes; MYLK4 + Megakaryocytes (Cluster 2) were the second most abundant subpopulation (17.2±0.38%), and LSP1 +Megakaryocytes (Cluster 3) accounted for the smallest proportion (4.1±0.94%) ( Figure 4 D) By counting the cell diameter, we also found that ARNTL + Megakaryocytes (Cluster 4) and MYLK4 + Megakaryocytes (Cluster 2) were mainly composed of megakaryocytes with larger diameters (average diameters were 31.5 μm and 29.7 μm, respectively), while LSP1 + Megakaryocytes (Cluster 3) have a smaller cell diameter (average diameter 7.4 μm), which is consistent with their chromosome ploidy composition ( Figure 4 E).

[0200] In summary, the results of this example combined with the results of Example 1 indicate that these three markers can distinguish megakaryocyte subpopulations at the mRNA and protein levels and are present in mouse bone marrow.

[0201] (2) ARNTL-labeled megakaryocytes (Cluster 4) are distributed close to blood vessels in the bone marrow

[0202] Megakaryocytes need to come into contact with blood vessels to produce platelets, so it is speculated that platelet-producing megakaryocytes should be distributed close to blood vessels in the bone marrow space.

[0203] To verify this hypothesis, antibodies against ARNTL, CD41, and the vascular marker CD105 were used to mark Cluster 4 megakaryocyte subsets and blood vessels, respectively, and three-dimensional immunofluorescence technology was used for detection.

[0204] The results showed that ARNTL + Megakaryocytes (Cluster 4) directly contact blood vessels (0.34μm) ( Figure 5 A and 5B).

[0205] (3) Megakaryocytes that are in direct contact with HSCs are MYLK4 +

[0206] Megakaryocyte regulation of HSC requires direct contact with HSC, so it is speculated that HSC microenvironment megakaryocytes should be distributed close to HSC in the bone marrow space.

[0207] Using three markers (MYLK4, ARNTL and LSP1), CD41 and HSC marker (Lin - CD48 - CD150 + ) antibodies, and found that 20% of HSCs were in direct contact with megakaryocytes by three-dimensional immunofluorescence technology ( Figure 6A), which is consistent with the reports in the literature. It was further found that almost all megakaryocytes in contact with HSCs were MYLK positive ( Figure 6 B and 6C).

[0208] Combining the above results, it was found that megakaryocytes marked by ARNLT and MYLK4 have a unique spatial distribution in the mouse bone marrow space, which is consistent with their respective functions.

[0209] Example 3-Megakaryocytes labeled with molecular markers have the functions suggested by the transcriptome

[0210] 1. Experimental Materials

[0211] Anti-mouse CD53 FITC was purchased from BioLegend; lipopolysaccharide LPS was purchased from Sigma; rabbit polyclonal ARNTL antibody was purchased from Bio-Techne; anti-LSP1 antibody was purchased from Abcam; anti-MYLK4 polyclonal antibody was purchased from Cloud-Clone; MYLK4 rabbit polyclonal antibody was purchased from Proteintech; goat anti-rabbit IgG AF488 and goat anti-rabbit IgG AF555 were purchased from Invitrogen; donkey anti-rabbit IgG DyLight 550 was purchased from Pierce; cell proliferation detection kit Cell Trace CFSE CellProliferation Kit and bacterial particles pHrodo Red E.coli BioParticles were purchased from Invitrogen.

[0212] 2. Experimental Methods

[0213] (1) LPS stimulation experiment

[0214] C57BL / 6 mice were intraperitoneally injected with 4 mg / kg LPS, and the bone marrow of mice was collected at 0 h, 6 h, 12 h, 24 h, and 30 h after treatment. The bone marrow was stained with CD41 and CD53 antibodies, and CD53 was detected by flow cytometry. + CD41 + Cell ratio.

[0215] (2) Bone marrow explant experiment

[0216] After obtaining complete mouse bone marrow, the experiment was performed according to Eckly, A., Strassel, C., Cazenave, J.-P., Lanza, F., Léon, C., and Gachet, C. (2012). Characterization of megakaryocyte development in the native bone marrow environment. In Platelets and Megakaryocytes (Springer), pp. 175-192. Briefly, the complete bone marrow was gently flushed out of the femur and tibia of the mouse and cut into 0.5 mm thick cross sections. 4-5 sections were transferred to one well of a 48-well plate, and benchtop solution containing 5% bovine serum albumin (Bovine Serum Albumin BSA) was added and cultured at 37°C for 2-4 hours. CD41, MK subset-specific markers (ARNTL / MYLK4 / LSP1) and DAPI were further stained. Confocal microscopy was used to observe and count LSP1 in megakaryocytes in precursor platelet production + CD41 + , MYLK4 + CD41 + and ARNTL + CD41 + The proportion of cells.

[0217] (3) Bacterial phagocytosis experiment

[0218] Draw peripheral blood from mice (heart blood or tail vein blood), let it stand at room temperature for half an hour, and collect serum. Use DPBS or saline to prepare 10% mouse serum conditioning solution, add 100 μl conditioning solution to 5 μl bacterial particle stock solution, mix evenly, place in a 37°C metal bath at 900 rpm for 30 minutes to condition the bacterial particles. 300-1500 CD41 cells obtained by flow cytometry + CD53 + and CD41 + CD53 - Megakaryocytes were suspended in a medium consisting of modified Dulbecco's medium (IMEM) and 10% fetal bovine serum (FBS). The cell suspension was incubated with E. coli particles at a ratio of 1:100 at 37°C for 30 min. The cells were further stained with CD53, CD41 and DAPI, and counted under confocal microscopy.

[0219] (4) T cell proliferation assay

[0220] 10μg / ml (positive control) and 6μg / ml CD3 antibody solution were added to a round-bottom 96-well plate with protein adsorption capacity, 100μl per well, and incubated at 37 degrees for 2 hours. + T cells were stained with 5-hydroxyfluorescein succinimidyl ester (CFSE) at 37°C for 5 min. At the same time, 3000-5000 CD53 T cells were isolated from the bone marrow of mice treated with 4 mg / ml LPS for 12 h. - MK or CD53 + MKs were co-cultured with T cells in a 37°C incubator for 72 hours, and T cell proliferation was detected by flow cytometry.

[0221] 3. Experimental results

[0222] (1) ARNTL-labeled megakaryocytes (Cluster 4) are the main platelet-producing population

[0223] To determine the platelet-producing capacity of megakaryocyte subsets, we used bone marrow explant experiments and found that approximately 93% of precursor platelet-producing megakaryocytes were ARNTL + , only about 10% are MYLK4 + , LSP1 was not observed + The phenomenon of megakaryocytes producing platelet precursors ( Figure 7 A and 7B ), indicating that the ARNTL-labeled megakaryocyte (Cluster 4) subset is the major platelet-producing population.

[0224] (2) CD53-labeled megakaryocytes (Cluster 3) can respond to pathogen stimulation and have the ability to phagocytose bacteria and stimulate T cell proliferation

[0225] In order to better track Cluster 3 subpopulation and verify the immune response ability of Cluster 3 subpopulation megakaryocytes, we first identified the cell surface marker CD53 ( Figure 2 E) Flow cytometry analysis revealed that CD53 + Megakaryocytes account for about 5% of all megakaryocytes. Figure 8 A), this ratio is similar to LSP1 + After LPS stimulation, CD53 + Rapid expansion of megakaryocyte populations ( Figure 8 A and 8B). To further explore the immune function of Cluster 3, we sorted CD53 + and CD53 -Megakaryocytes were tested for bacterial particle phagocytosis and approximately 30% of CD53 + Megakaryocytes undergo phagocytosis, and CD53 - The proportion of megakaryocytes that undergo phagocytosis is only 1% ( Figure 8 C and 8D); in addition, we also sorted CD53 + and CD53 - Megakaryocytes were co-cultured with T cells and found to be associated with CD53 - T cells co-cultured with megakaryocytes underwent a lower level of proliferation (2.81%), whereas T cells co-cultured with CD53 + T cells co-cultured with megakaryocytes underwent significant proliferation, with newly generated T cells accounting for 20% of the entire T cell population ( Fig. 9 A and 9B).

[0226] The above results indicate that CD53-labeled (Cluster 3) megakaryocytes can respond to LPS stimulation and carry out immune response by phagocytizing bacteria and stimulating T cell proliferation.

[0227] Those skilled in the art should be aware that the scope of the present invention is not limited to the technical solutions formed by a specific combination of the technical features described in the present invention, but should also cover other technical solutions formed by any combination of the technical features described in the present invention or their equivalent features. Sequence Listing <110> Beijing Institute of Genomics, Chinese Academy of Sciences (National Center for Bioinformatics) <120> Molecular marker combinations and their uses <130> IDC200332 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 656 <212> PRT <213> artificial <220> <223> Amino acid sequence of mouse MYLK4 <400> 1 Met Pro Thr Pro Gln Ser Arg Ala Gly Ser Thr Gly Asn Phe Lys Ser 1 5 10 15 Gly Cys Ala Arg Pro Gly Gln Gly Arg Cys Pro Gln Ser Gln Gly Leu 20 25 30 His Ser Leu Ser Gln Arg Met Asp Ser Thr Leu Arg Arg Lys Asp Leu 35 40 45 Trp Ile Val Gly Ser Val Cys Leu Leu Ser Ser Ser Val Phe Trp Arg 50 55 60 Leu Phe Arg Ser Val Ala Ala Trp Gly Gly Pro Lys Gln Glu Pro Ala 65 70 75 80 Leu Glu Asn Ala Gln Ala Glu Glu Asn Val Trp Ser Asn Arg Ala Leu 85 90 95 Asp Glu Arg Ile Ile Val Lys Gly Gly Arg Thr Ser Ala Leu Thr Glu 100 105 110 Lys Thr Asp Gln Asn Asp Gln Phe Ser Ser Glu Lys Glu Lys Ile Thr 115 120 125 Lys Leu Asn Lys Lys Asn Ser Glu Lys Thr Ser Ala Leu Glu Val Gln 130 135 140 Arg Ser Cys Val Ile Cys His Val Glu Arg Gln Asp Lys Ile Glu Phe 145 150 155 160 Val Gln Arg Asn Ser Thr Glu Leu Arg Glu Asn Ala Gly Ala Lys Asp 165 170 175 Ala Pro Cys Ile Glu Ser Leu Glu Lys Gln Glu Ser Asn Asn Leu Arg 180 185 190 Asp Lys Gln Asp Thr Glu Asp Asn Asn Gly Gly Glu Asn Gly Ile Ser 195 200 205 Ser Lys Asp Asp Ser Arg Gly Val Glu Ala Glu Asn Arg Ala Ser Ala 210 215 220 Ala Ala Ser Gly Ala Val Leu Asp Lys His Ser Thr Glu Ser Gln His 225 230 235 240 Thr Glu Asp Met Asn Gly Asp Pro Ser His Glu Arg Cys Ile Ser Asn 245 250 255 Ile Lys Thr Thr Ser Thr Pro Gly Ile Leu Gly Asn Ser Ala Ser Gly 260 265 270 Ser Thr Phe Gln Asn Ala Glu Gln Thr Glu Ser Thr Gly Ser Ser Lys 275 280 285 Arg Arg Val Ala Glu Glu Cys Phe Leu Ser Glu Asp Asn Lys Lys Ser 290 295 300 Arg Ile Asp Val Val Arg Arg Gly Glu Glu Pro Thr Ala Arg Thr Glu 305 310 315 320 Glu Thr Arg Gln Ala Arg Ser Leu Gly Asp Arg Leu Gln Asp Gly Met 325 330 335 Thr Thr Ser Arg Glu Gln Asp Leu Glu Val Pro Leu Asp Asp Ile Pro 340 345 350 Ala Pro Ala Ala Pro Phe Asp His Arg Met Val Met Ala Lys His Ala 355 360 365 Ser Val Asp Asn Leu Tyr Thr Val Ser Lys Ser Glu Ile Leu Gly Gly 370 375 380 Gly Arg Phe Gly Gln Val His Lys Cys Glu Glu Lys Ala Thr Gly Leu 385 390 395 400 Lys Leu Ala Ala Lys Ile Ile Lys Thr Arg Gly Ala Lys Asp Lys Glu 405 410 415 Asp Val Lys Asn Glu Ile Ser Val Met Asn Gln Leu Asp His Val Asn 420 425 430 Leu Ile Gln Leu Tyr Asp Ala Phe Glu Ser Lys His Asp Ile Ile Leu 435 440 445 Val Met Glu Tyr Val Glu Gly Gly Glu Leu Phe Asp Arg Ile Ile Asp 450 455 460 Glu Asn Cys Asn Leu Thr Glu Leu Asp Thr Ile Leu Phe Met Lys Gln 465 470 475 480 Ile Cys Glu Gly Ile Arg Tyr Met His Gln Met Tyr Ile Leu His Leu 485 490 495 Asp Leu Lys Pro Glu Asn Ile Leu Cys Val Asn Arg Asp Ala Lys Gln 500 505 510 Ile Lys Ile Ile Asp Phe Gly Leu Ala Arg Arg Tyr Lys Pro Arg Glu 515 520 525 Lys Leu Lys Val Asn Phe Gly Thr Pro Glu Phe Leu Ala Pro Glu Val 530 535 540 Val Asn Tyr Asp Phe Val Ser Phe Ser Thr Asp Met Trp Ser Val Gly 545 550 555 560 Val Ile Thr Tyr Met Leu Leu Ser Gly Leu Ser Pro Phe Leu Gly Asp 565 570 575 Asn Asp Ala Glu Thr Leu Thr Asn Ile Leu Ala Cys Arg Trp Asp Leu 580 585 590 Glu Asp Glu Glu Phe Gln Asp Ile Ser Glu Glu Ala Lys Glu Phe Ile 595 600 605 Ser Lys Leu Leu Ile Lys Glu Lys Ser Trp Arg Ile Ser Ala Ser Glu 610 615 620 Ala Leu Lys His Pro Trp Leu Ser Asp His Lys Leu His Ser Arg Leu 625 630 635 640 Ser Ala Gln Lys Asn Cys Asn Ser Gly Val Leu Asn Leu Thr Thr Lys 645,650,655 <210> 2 <211> 1971 <212> DNA <213> artificial <220> <223> See more of Mylk4 <400> 2 atgcccactc ctcagagtag agcagggagc acagggact tcaaagtgg ctgtgctaga 60 ccaggggcagg gaaggtgccc aaagccag ggactcaca gcctccca gagaatggac 120 tccacgctc gagaaaaga cctctggatt gtgggtagcg tgtgccttct gtcgtcttcg 180 gtcttctggc gcctgtttcg gtcagtggcg gcatggggg gccccaagca ggaacctgca 240 cttgagaatg cacaggctga ggaaatgta tggtccaacc gtgcactaga tgaaggata 300 attgtcaaag gaggaggac atcagccctc acagagaaaa ctgaccagaa tgaccaattc 360 tcttccgaga aagaaagat taccaactt aaaaaaaaa attcagagaa gaccagtgca 420 ttagaagtgc agaggattg tgtcatatgt cacgtggaaa gatagaca gatagaattt 480 gttcagagga attccacaga gctaagagag aatgcaggtg caaagatgc tccatgcata 540 gaaagtctgg agaaacaaga aagcaacaac ctgagagata aacaagacac tgaagataac 600 aatgggagg aaaatgggat ttcttctaag gacgattcca gaggagtaga agctgagaac 660 720 actgaggaca tgaatggtga cccgtcccat gaaaggtgca tcagtaatat aaagaccact 780 tctacaccag gaatccttgg aaattcagca tcaggatcca ctttccagaa tgctgaacag 840 agagagtcta ctggcagcag taagcgcagg gtggcagagg agtgcttcct cagtgaggac 900 aacaaaaaga cgaagaataga tgtagtgaga agagggaag agcccacagc taggacagag 960 gaaaccaggc aagccagaag cctgggtgat agactccagg atggaatgac caccttaga 1020 gagcaagacc tggaggtccc actggatgac atccctgctc ctgccgctcc atttgatcat 1080 cggatggtaa tggccaaaca tgcttcagtg gacaatctgt acaccgtgag caaatctgag 1140 atcctaggag gggggcgttt tggtcaggta cataagtgtg aagagaaagc aacaggcctg 1200 aagctggctg ctaaaatcat taagactaga ggtgcaaagg acaaggaga tgtgaaac 1260 gagatcagcg tcatgaacca gctgaccac gtgaacctca ttcagctta tgatgccttt 1320 gagtccaagc atgacatcat ccttgtcatg gagtatgtgg agggtgggga gctctttgac 1380 cgcatcattg atgaaactg caactgaca gagctggaca ccatcctgtt catgaagcag 1440 atctgtgagg ggatacggta catgcaccag atgtacatcc tccattgga ttchcagcct 1500 gagaacatcc tgtgtgtgaa tcgggatgct aagcaaataa aattattga ttttggattg 1560 gccagaagat acaagcccag agagaagctg aaggtgaact ttggacccc agaatttctt 1620 gcccctgaag ttgtgaacta tgatttcgtt tccttttcta cagatgtg gagtgtgggc 1680 gtcatcacat acatgctact cagtggcttg tcccctttt tgggagaca tgatgccgag 1740 acactgacca acatcctggc gtgccggtgg gatttagaag atgaggaatt tcaggatatc 1800 tcggaggagg catcaggag ctcctgatta aggagag tggagata 1860 agtgccagcg aagctctca gcaccatgg ttgtcagacc acagctcca ctccaggctg 1920 agtgcacaga agactgcaa ctctggtgtt ctgaacctca cgaccaaata a 1971 <210> 3 <211> 330 <212> PRT <213> artificial <220> <223> Amino acid sequence of mouse LSP1 <400> 3 Met Ala Glu Ala Ala Ile Asp Pro Arg Cys Glu Glu Gln Glu Glu Leu 1 5 10 15 His Ala Glu Asp Ser Glu Gly Leu Thr Thr Gln Trp Arg Glu Glu Asp 20 25 30 Glu Glu Glu Ala Ala Arg Glu Gln Arg Gln Arg Glu Arg Glu Arg Gln 35 40 45 Leu Gln Asp Gln Asp Lys Asp Lys Glu Asp Asp Gly Gly His Ser Leu 50 55 60 Glu Gln Pro Gly Gln Gln Thr Leu Ile Ser Leu Lys Ser Ser Glu Leu 65 70 75 80 Asp Glu Asp Glu Gly Phe Gly Asp Trp Ser Gln Lys Pro Glu Pro Arg 85 90 95 Gln Gln Phe Trp Gly Asn Glu Gly Thr Ala Glu Gly Thr Glu Pro Ser 100 105 110 Gln Ser Glu Arg Pro Glu Glu Lys Gln Thr Glu Glu Ser Ser His Gln 115 120 125 Ala Lys Val His Leu Glu Glu Ser Asn Leu Ser Tyr Arg Glu Pro Asp 130 135 140 Pro Glu Asp Ala Val Gly Gly Ser Gly Glu Ala Glu Glu His Leu Ile 145 150 155 160 Arg His Gln Val Arg Thr Pro Ser Pro Leu Ala Leu Glu Asp Thr Val 165 170 175 Glu Leu Ser Ser Pro Pro Leu Ser Pro Thr Thr Lys Leu Ala Asp Arg 180 185 190 Thr Glu Ser Leu Asn Arg Ser Ile Lys Lys Ser Asn Ser Val Lys Lys 195 200 205 Ser Gln Pro Thr Leu Pro Ile Ser Thr Ile Asp Glu Arg Leu Gln Gln 210 215 220 Tyr Thr Gln Ala Thr Glu Ser Ser Gly Arg Thr Pro Lys Leu Ser Arg 225 230 235 240 Gln Pro Ser Ile Glu Leu Pro Ser Met Ala Val Ala Ser Thr Lys Thr 245 250 255 Leu Trp Glu Thr Gly Glu Val Gln Ser Gln Ser Ala Ser Lys Thr Pro 260 265 270 Ser Cys Gln Asp Ile Val Ala Gly Asp Met Ser Lys Lys Ser Leu Trp 275 280 285 Glu Gln Lys Gly Gly Ser Lys Ile Ser Ser Thr Ile Lys Ser Thr Pro 290 295 300 Ser Gly Lys Arg Tyr Lys Phe Val Ala Thr Gly His Gly Lys Tyr Glu 305 310 315 320 Lys Val Leu Val Asp Glu Gly Ser Ala Pro 325 330 <210> 4 <211> 993 <212> DNA <213> artificial <220> <223> Lsp1 in the <400> 4 atggcggagg ctgccatcga tcccagatgc gaggaacagg aagagctcca tgcagaagac 60 agcgaagggc tcacaactca gtggagagaa gaagacgagg aagaggctgc ccgtgagcag 120 cgtcaacggg agcgagagag gcagctacag gaccaggaca aagataaaga agatgatggt 180 ggccattccc tggaacagcc aggacagcag acactcatca gcctgaagtc ctctgaactg 240 gatgaagatg agggttttgg tgactggtcc caaaagccgg agccacggca gcaattctgg 300 gggaatgagg ggactgcaga agggactgaa ccctctcaaa gcgagagacc agaggagaaa 360 420. caaacagagg agagttctca ccaagccaa gtccacttgg aggagtcaaa cctgagctac agggagcccg atccagag tgctgttggg ggttctgggg aggcggaga gcatctgata 480 cgtcatcagg tcaggacccc cagccctttg gccttagaag acaccgttga gctgagttca 540 cctcccctga gccctaccac caaactggct gataggaccg agtccctga ccgctccatt aaaaagagca acagtgtga gagtctca ccaaccttgc ccatttccac aattgatga cgcctgcagc agtataccca ggccactgag tcttctggcc gaactcctaa gctgtcccgc 720 cagccctcca tagagctgcc cagcatggcc gtagccagta ccaagactct ttgggaaaca ggagaagtgc agtagtcagtc tgcttctaag acaccctcct gccaggatat agtagctgga gacatgagca agaaaagtct gtgggagcag aaaggaggct ccaagatctc atccaccatc aagagcaccc catctggaaa gcggtacaag ttcgtggcca ctggacatgg gaagtacgag aaagtacttg tggatgaggg ctcagcacca tag <210> 5 <211> 613 <212> PRT <213> artificial <220> <223> Amino acid sequence of mouse ARNTL <400> 5 Met Ala Asp Gln Arg Met Asp Ile Ser Ser Thr Ile Ser Asp Phe Met 1 5 10 15 Ser Pro Gly Pro Thr Asp Leu Leu Ser Gly Ser Leu Gly Thr Ser Gly 20 25 30 Val Asp Cys Asn Arg Lys Arg Lys Gly Ser Ala Thr Asp Tyr Gln Leu 35 40 45 Glu Tyr Ala Glu His Gln Gly Arg Ile Lys Asn Ala Arg Glu Ala His 50 55 60 Ser Gln Ile Glu Lys Arg Arg Arg Asp Lys Met Asn Ser Phe Ile Asp 65 70 75 80 Glu Leu Ala Ser Leu Val Pro Thr Cys Asn Ala Met Ser Arg Lys Leu 85 90 95 Asp Lys Leu Thr Val Leu Arg Met Ala Val Gln His Met Lys Thr Leu 100 105 110 Arg Gly Ala Thr Asn Pro Tyr Thr Glu Ala Asn Tyr Lys Pro Thr Phe 115 120 125 Leu Ser Asp Asp Glu Leu Lys His Leu Ile Leu Arg Ala Ala Asp Gly 130 135 140 Phe Leu Phe Val Val Gly Cys Asp Arg Gly Lys Ile Leu Phe Val Ser 145 150 155 160 Glu Ser Val Phe Lys Ile Leu Asn Tyr Ser Gln Asn Asp Leu Ile Gly 165 170 175 Gln Ser Leu Phe Asp Tyr Leu His Pro Lys Asp Ile Ala Lys Val Lys 180 185 190 Glu Gln Leu Ser Ser Ser Asp Thr Ala Pro Arg Glu Arg Leu Ile Asp 195 200 205 Ala Lys Thr Gly Leu Pro Val Lys Thr Asp Ile Thr Pro Gly Pro Ser 210 215 220 Arg Leu Cys Ser Gly Ala Arg Arg Ser Phe Phe Cys Arg Met Lys Cys 225 230 235 240 Asn Arg Pro Ser Val Lys Val Glu Asp Lys Asp Phe Ala Ser Thr Cys 245 250 255 Ser Lys Lys Lys Ala Asp Arg Lys Ser Phe Cys Thr Ile His Ser Thr 260 265 270 Gly Tyr Leu Lys Ser Trp Pro Pro Thr Lys Met Gly Leu Asp Glu Asp 275 280 285 Asn Glu Pro Asp Asn Glu Gly Cys Asn Leu Ser Cys Leu Val Ala Ile 290 295 300 Gly Arg Leu His Ser His Met Val Pro Gln Pro Ala Asn Gly Glu Ile 305 310 315 320 Arg Val Lys Ser Met Glu Tyr Val Ser Arg His Ala Ile Asp Gly Lys 325 330 335 Phe Val Phe Val Asp Gln Arg Ala Thr Ala Ile Leu Ala Tyr Leu Pro 340 345 350 Gln Glu Leu Leu Gly Thr Ser Cys Tyr Glu Tyr Phe His Gln Asp Asp 355 360 365 Ile Gly His Leu Ala Glu Cys His Arg Gln Val Leu Gln Thr Arg Glu 370 375 380 Lys Ile Thr Thr Asn Cys Tyr Lys Phe Lys Ile Lys Asp Gly Ser Phe 385 390 395 400 Ile Thr Leu Arg Ser Arg Trp Phe Ser Phe Met Asn Pro Trp Thr Lys 405 410 415 Glu Val Glu Tyr Ile Val Ser Thr Asn Thr Val Val Leu Ala Asn Val 420 425 430 Leu Glu Gly Gly Asp Pro Thr Phe Pro Gln Leu Thr Ala Pro Pro His 435 440 445 Ser Met Asp Ser Met Leu Pro Ser Gly Glu Gly Gly Pro Lys Arg Thr 450 455 460 His Pro Thr Val Pro Gly Ile Pro Gly Gly Thr Arg Ala Gly Ala Gly 465 470 475 480 Lys Ile Gly Arg Met Ile Ala Glu Glu Ile Met Glu Ile His Arg Ile 485 490 495 Arg Gly Ser Ser Pro Ser Ser Cys Gly Ser Ser Pro Leu Asn Ile Thr 500 505 510 Ser Thr Pro Pro Pro Asp Ala Ser Ser Pro Gly Gly Lys Lys Ile Leu 515 520 525 Asn Gly Gly Thr Pro Asp Ile Pro Ser Thr Gly Leu Leu Pro Gly Gln 530 535 540 Ala Gln Glu Thr Pro Gly Tyr Pro Tyr Ser Asp Ser Ser Ser Ile Leu 545 550 555 560 Gly Glu Asn Pro His Ile Gly Ile Asp Met Ile Asp Asn Asp Gln Gly 565 570 575 Ser Ser Ser Pro Ser Asn Asp Glu Ala Ala Met Ala Val Ile Met Ser 580 585 590 Leu Leu Glu Ala Asp Ala Gly Leu Gly Gly Pro Val Asp Phe Ser Asp 595 600 605 Leu Pro Trp Pro Leu 610 <210> 6 <211> 1842 <212> DNA <213> artificial <220> <223> Nucleotide sequence of mouse Arntl <400> 6 atggcggacc agagaatgga catttcctca accatcagcg acttcatgtc tccgggcccc 60 accgacctac tctccggttc cctgggcacc agtggtgtgg actgcaatcg caagaggaaa 120 ggcagtgcca ctgactacca gttagaatat gcagaacacc aaggaaggat caagaatgca 180 agggaggccc acagtcagat tgaaaagagg cgtcgggaca aaatgaacag tttcattgat 240 gaattggctt ctttggtacc aacatgcaat gcaatgtcca ggaagttaga taaactcacc 300 gtgctaagga tggctgttca gcacatgaaa actttgagag gtgccaccaa cccatacaca 360 gaagcaaact acaagccaac atttctatca gatgacgaac tgaaacacct aattctcagg 420 gcagcagatg gatttttgtt tgtcgtagga tgtgaccgag ggaagatcct ctttgtctcc 480 gagtctgtct tcaagatcct caattatagc cagaatgacc ttattggcca gagcttgttt 540 gactacctgc atccaaaaga tattgccaaa gttaaggaac agctatcttc ctcggacact 600 gcgccccgggg agcgactcat tgatgccaag actggacttc cggttaaaac ggatataacc 660 cctgggccct cccggctatg ctctggagcc cgccgctt tctctgtag aatgaagtgc 720 aacaggcctt cagtaaggt ggataag gactcgcct ctacctgttc aaagaaaaaa 780 gcagatcgaa aaagctctg cacaatccac agcacaggct atttgaaag ctggccaccc 840 acgaagatgg ggctggacga agacatgag ccagacaacg agggctgcaa cctcagctgc 900 ctcgttgcaa tcggcgcct gcactcgcac atggttccac aaccagcgaa cggggaata 960 cgggtgaaat ctatggagta cgtttctcga cacgcaatag atgggaatt tgtttttgta 1020 gatcagagggg cgacagctat ttggcgtat ctaccacagg aacttctagg tacatcatgt 1080 tatgagtatt ttcatcaaga cgacatagga cacctcgcag atgtcacag gcaagtttta 1140 cagacagag aaagatcac aactattgc tataagtttta agatchaaga tggttctttt 1200 atcacgctac gaagtcgatg gttcagtttc atgaacccgt ggaccaagga agttgaatac 1260 attgtctcaa cccacactgt tgtttagcc attgtcctgg aaggcgggga cccaaccttc 1320 ccgcagctaa cagcaccccc ccacagcatg gacagcatgc tgccctctgg agaaggtggc 1380 ccaaagagga ctcatcccac tgtcccaggc attccagggg gaaccagagc cggagcagga 1440 aaaataggtc gaatgatcgc ggaggaaatc atggaaatcc acaggataag agggtcatcg 1500 ccttccagct gtggctccag cccgctgaac atcacaagta cgcctccccc tgatgcctct 1560 tctccaggag gcaagaagat tctaaatgga gggactccag acattccttc cactggacta 1620 ttaccagggc aggctcagga gaccccaggg tatccctatt ctgatagttc ttctattctt 1680 ggtgagaacc cccacatagg catcgatatg atagataacg accaaggatc aagtagtccc 1740 agtaacgatg aggcagcaat ggctgtcatc atgagcctct tggaagcaga tgcggggctg 1800 ggtggccccg ttgactttag tgacttgcca tggccgctgt ag 1842 <210> 7 <211> 74 <212> PRT <213> artificial <220> <223> Amino acid sequence of mouse CD52 <400> 7 Met Lys Ser Phe Leu Leu Phe Leu Thr Ile Ile Leu Leu Val Val Ile 1 5 10 15 Gln Ile Gln Thr Gly Ser Leu Gly Gln Ala Thr Thr Ala Ala Ser Gly 20 25 30 Thr Asn Lys Asn Ser Thr Ser Thr Lys Lys Thr Pro Leu Lys Ser Gly 35 40 45 Ala Ser Ser Ile Ile Asp Ala Gly Ala Cys Ser Phe Leu Phe Phe Ala 50 55 60 Asn Thr Leu Met Cys Leu Phe Tyr Leu Ser 65 70 <210> 8 <211> 225 <212> DNA <213> artificial <220> <223> Nucleotide sequence of mouse Cd52 <400> 8 atgaagagct tcctcctctt cctcactatc attcttctgg ttgtgattca gatacaaaca 60 ggatccttgg gacaagccac tacggccgct tctggtacta acaaaaacag cacctccacc 120 aaaaaaaccc ccttaaagag tggggcctca tccatcatcg atgcgggtgc ctgcagtttc 180 ctcttctttg ccaatacctt aatgtgcctc ttctacctca gctga 225 <210> 9 <211> 219 <212> PRT <213> artificial <220> <223> Amino acid sequence of mouse CD53 <400> 9 Met Gly Met Ser Ser Leu Lys Leu Leu Lys Tyr Val Leu Phe Ile Phe 1 5 10 15 Asn Leu Leu Phe Trp Val Cys Gly Cys Cys Ile Leu Gly Phe Gly Ile 20 25 30 Tyr Phe Leu Val Gln Asn Thr Tyr Gly Val Leu Phe Arg Asn Leu Pro 35 40 45 Phe Leu Thr Leu Gly Asn Ile Leu Val Ile Val Gly Ser Ile Ile Met 50 55 60 Val Val Ala Phe Leu Gly Cys Met Gly Ser Ile Lys Glu Asn Lys Cys 65 70 75 80 Leu Leu Met Ser Phe Phe Val Leu Leu Leu Ile Ile Leu Leu Ala Glu 85 90 95 Val Thr Ile Ala Ile Leu Leu Phe Val Tyr Glu Gln Lys Leu Asn Thr 100 105 110 Leu Val Ala Glu Gly Leu Asn Asp Ser Ile Gln His Tyr His Ser Asp 115 120 125 Asn Ser Thr Met Lys Ala Trp Asp Phe Ile Gln Thr Gln Leu Gln Cys 130 135 140 Cys Gly Val Asn Gly Ser Ser Asp Trp Thr Ser Gly Pro Pro Ser Ser 145 150 155 160 Cys Pro Ser Gly Ala Asp Val Gln Gly Cys Tyr Asn Lys Ala Lys Ser 165 170 175 Trp Phe His Ser Asn Phe Leu Tyr Ile Gly Ile Ile Thr Ile Cys Val 180 185 190 Cys Val Ile Gln Val Leu Gly Met Ser Phe Ala Leu Thr Leu Asn Cys 195 200 205 Gln Ile Asp Lys Thr Ser Gln Ala Leu Gly Leu 210 215 <210> 10 <211> 660 <212> DNA <213> artificial <220> <223> Nucleotide sequence of mouse Cd53 <400> 10 atgggcatga gcagcctgaa attgctgaaa tatgttctgt ttatctttaa cttgcttttt 60 tgggtctgtg gctgttgcat tttgggcttt ggcatctatt tcctggtcca aaatacctat 120 ggagtactct tccgtaacct tcccttcctg acacttggca acattctggt cattgtggga 180 tccattatca tggtagttgc cttcttgggt tgcatgggct caatcaagga aaataagtgc 240 ctgcttatgt cgttctttgt tctgctgctg attattctcc ttgctgaggt gaccatagcc 300 atcctgctct ttgtgtatga acaaaaactc aacactttag tggctgaggg tctgaatgac 360 agcatccaac attatcactc tgacaacagc actatgaagg catgggactt catccagaca 420 caactgcagt gttgtggtgt aaatggctca agtgattgga ccagtggtcc accatcttcc 480 tgcccatcag gtgcagatgt tcagggttgc tataataagg caaaatcgtg gtttcactcc 540 aatttcttgt atattggaat cattaccatc tgtgtatgtg tgatacaggt gctgggaatg 600 tcctttgcac tgacactcaa ctgccagatt gacaaaacaa gccaggcttt agggctgtga 660 <210> 11 <211> 388 <212> PRT <213> artificial <220> <223> Amino acid sequence of human MYLK4 <400> 11 Met Leu Lys Val Lys Arg Leu Glu Glu Phe Asn Thr Cys Tyr Asn Ser 1 5 10 15 Asn Gln Leu Glu Lys Met Ala Phe Phe Gln Cys Arg Glu Glu Val Glu 20 25 30 Lys Val Lys Cys Phe Leu Glu Lys Asn Ser Gly Asp Gln Asp Ser Arg 35 40 45 Ser Gly His Asn Glu Ala Lys Glu Val Trp Ser Asn Ala Asp Leu Thr 50 55 60 Glu Arg Met Pro Val Lys Ser Lys Arg Thr Ser Ala Leu Ala Val Asp 65 70 75 80 Ile Pro Ala Pro Pro Ala Pro Phe Asp His Arg Ile Val Thr Ala Lys 85 90 95 Gln Gly Ala Val Asn Ser Phe Tyr Thr Val Ser Lys Thr Glu Ile Leu 100 105 110 Gly Gly Gly Arg Phe Gly Gln Val His Lys Cys Glu Glu Thr Ala Thr 115 120 125 Gly Leu Lys Leu Ala Ala Lys Ile Ile Lys Thr Arg Gly Met Lys Asp 130 135 140 Lys Glu Glu Val Lys Asn Glu Ile Ser Val Met Asn Gln Leu Asp His 145 150 155 160 Ala Asn Leu Ile Gln Leu Tyr Asp Ala Phe Glu Ser Lys Asn Asp Ile 165 170 175 Val Leu Val Met Glu Tyr Val Asp Gly Gly Glu Leu Phe Asp Arg Ile 180 185 190 Ile Asp Glu Ser Tyr Asn Leu Thr Glu Leu Asp Thr Ile Leu Phe Met 195 200 205 Lys Gln Ile Cys Glu Gly Ile Arg His Met His Gln Met Tyr Ile Leu 210 215 220 His Leu Asp Leu Lys Pro Glu Asn Ile Leu Cys Val Asn Arg Asp Ala 225 230 235 240 Lys Gln Ile Lys Ile Ile Asp Phe Gly Leu Ala Arg Arg Tyr Lys Pro 245 250 255 Arg Glu Lys Leu Lys Val Asn Phe Gly Thr Pro Glu Phe Leu Ala Pro 260 265 270 Glu Val Val Asn Tyr Asp Phe Val Ser Phe Pro Thr Asp Met Trp Ser 275 280 285 Val Gly Val Ile Ala Tyr Met Leu Leu Ser Gly Leu Ser Pro Phe Leu 290 295 300 Gly Asp Asn Asp Ala Glu Thr Leu Asn Asn Ile Leu Ala Cys Arg Trp 305 310 315 320 Asp Leu Glu Asp Glu Glu Phe Gln Asp Ile Ser Glu Glu Ala Lys Glu 325 330 335 Phe Ile Ser Lys Leu Leu Ile Lys Glu Lys Ser Trp Arg Ile Ser Ala 340 345 350 Ser Glu Ala Leu Lys His Pro Trp Leu Ser Asp His Lys Leu His Ser 355 360 365 Arg Leu Ashn White Light Light Light Asn Arg Gly Ser Asp White White Asp 370 375 380 Phe Val Thr Lys 385 <210> 12 <211> 1167 <212> DNA <213> artificial <220> <223> Take the MYLK4 snowflake switch <400> 12 atgttaaaag tgaagaggct ggaagaattc aacacgtgtt father ccagctggag aaaatggcct tttttcagtg cagggagag gtggagaag tgaagtgttt tctggaaaaa aattctgggg accaggattc aagatctgga catatgagg cgaaggaggt gtggtcaaac gccgacctga cggaaggat gcccgtcaaa agcaaaagga catcagccct cgcagttgac 240 atcccggctc ctccggcccc atttgatcat cgtattgtga cagccaagca aggagcggtc aacagcttct atactgtgag caagacagaa atcctaggag gagggcgttt cggccaggtt cacaagtgtg aggagacggc cacaggtctg aagctggcag ccaaaatcat caagaccaga ggcatgaagg acaaggagga ggtgaagaac gagatcagcg tcatgaacca gctggaccac gcgaacctca tccagctgta cgatgccttc gagtctaaga acgacattgt cctggtcatg 540 gagtatgtgg atggtgggga gctgtttgac cgcatcatcg atgagagcta caatttgacg 600 gagcttgata ccatcctgtt catgaagcag atatgtgagg ggataggca catgcatcag 660 atgtacattc tccacttgga cctgaagcct gagaatatcc tgtgtgtgaa tcgggatgct 720 aagCAAA aaattattga ttttgttg gccagaagat aaacccag aggaagctg 780 aaggtgaact ttggacccc agaatttctc gcccctgaag tgtgaacta tgatttgtt 840 tcattccca ctgacatgtg gagtgtgggg gtcatcgcct atatgctact tagcggttg 900 tcgcctttcc tgggtgacaa tgatgctgag acgctgaaca acatcctggc ctgcaggtgg 960 gacttagagg tgagaatt tcggacatc tcggaggg spacecraft catctctaag 1020 cttctgatta aggagag ttggcgaata agtgcaagcg aagctctcaa gcacccctgg 1080 ttgtcagacc acagctcca ctccagactc aatgcccaga agagaagaa tcgtggctct 1140 gatgcccagg actttgtgac caatag 1167 <210> 13 <211> 277 <212> PRT <213> artificial <220> <223> Amino acid sequence of human LSP1 <400> 13 Met Leu Leu Ser Leu Lys Pro Ser Glu Ala Pro Glu Leu Asp Glu Asp 1 5 10 15 Glu Gly Phe Gly Asp Trp Ser Gln Arg Pro Glu Gln Arg Gln Gln His 20 25 30 Glu Gly Ala Gln Gly Ala Leu Asp Ser Gly Glu Pro Pro Gln Cys Arg 35 40 45 Ser Pro Glu Gly Glu Gln Glu Asp Arg Pro Gly Leu His Ala Tyr Glu 50 55 60 Lys Glu Asp Ser Asp Glu Val His Leu Glu Glu Leu Ser Leu Ser Lys 65 70 75 80 Glu Gly Pro Gly Pro Glu Asp Thr Val Gln Asp Asn Leu Gly Ala Ala 85 90 95 Gly Ala Glu Glu Glu Gln Glu Glu His Gln Lys Cys Gln Gln Pro Arg 100 105 110 Thr Pro Ser Pro Leu Val Leu Glu Gly Thr Ile Glu Gln Ser Ser Pro 115 120 125 Pro Leu Ser Pro Thr Thr Lys Leu Ile Asp Arg Thr Glu Ser Leu Asn 130 135 140 Arg Ser Ile Glu Lys Ser Asn Ser Val Lys Lys Ser Gln Pro Asp Leu 145 150 155 160 Pro Ile Ser Lys Ile Asp Gln Trp Leu Glu Gln Tyr Thr Gln Ala Ile 165 170 175 Glu Thr Ala Gly Arg Thr Pro Lys Leu Ala Arg Gln Ala Ser Ile Glu 180 185 190 Leu Pro Ser Met Ala Val Ala Ser Thr Lys Ser Arg Trp Glu Thr Gly 195 200 205 Glu Val Gln Ala Gln Ser Ala Ala Lys Thr Pro Ser Cys Lys Asp Ile 210 215 220 Val Ala Gly Asp Met Ser Lys Lys Ser Leu Trp Glu Gln Lys Gly Gly 225 230 235 240 Ser Lys Thr Ser Ser Thr Ile Lys Ser Thr Pro Ser Gly Lys Arg Tyr 245 250 255 Lys Phe Val Ala Thr Gly His Gly Lys Tyr Glu Lys Val Leu Val Glu 260 265 270 Gly Gly Pro Ala Pro 275 <210> 14 <211> 834 <212> DNA <213> artificial <220> <223> Nucleotide sequence of human LSP1 <400> 14 atgctcctca gcctgaagcc ctcggaggcc cctgaactgg atgaggacga gggctttggc 60 gactggtccc agaggccaga gcagcggcag cagcacgagg gggcgcagg cgccttggac 120 agcggagagc ccccccagtg caggagtcct gagggggagc aagagcag gcccggcctg 180 catgcctacg aaaaggaga cagtgatgaa gtccacctgg aggagttgag tctgagcaag 240 gaggggccag gcccagga cactgtccag gacaacctgg gggccgcagg ggctgaggag 300 gaacgagg agcaccagaa atgtcagcag cccaggacac ccagcccctt ggtcttggag 360 gggaccatcg aacagagctc gcctcccctg agccctacca ccaaactcat cgacaggacc 420 gagtccctaa accgctccat agaagaagt aacagtgtga agaaatccca gccagacttg 480 cccatctcca agattgatca gtggctgggaa caatacaccc aggccatcga gaccgctggc 540 cggaccccca agctagcccg ccaggcctcc atagagctgc ccagcatggc tgtggccagt 600 accaagagtc ggtgggagac gggtgaggta caggctcagt ctgcggccaa gactccgtcc 660 tgcaaggata ttgtggctgg agacatgagc aagaaaagcc tctgggagca gaagggaggc 720 tccaagacct catcaacaat taagagcacc ccatctggga agaggtataa gttttgtggcc 780 accgggcatg ggaagtatga gaaggtgctt gtggaagggg gcccggctcc ctag 834 <210> 15 <211> 625 <212> PRT <213> artificial <220> <223> See page ARNTL <400> 15 Met Ala Asp Gln Arg Met Asp Ile Ser Ser Thr Ile Ser Asp Phe Met 1 5 10 15 Ser Pro Gly Pro Thr Asp Leu Leu Ser Ser Ser Leu Gly Thr Ser Gly 20 25 30 Val Asp Cys Asn Arg Lys Arg Lys Gly Ser Ser Thr Asp Tyr Gln Glu 35 40 45 Ser Met Asp Thr Asp Lys Asp Asp Pro His Gly Arg Leu Glu Tyr Thr 50 55 60 Glu His Gln Gly Arg Ile Lys Asn Ala Arg Glu Ala His Ser Gln Ile 65 70 75 80 Glu Lys Arg Arg Arg Asp Lys Met Asn Ser Phe Ile Asp Glu Leu Ala 85 90 95 Ser Leu Val Pro Thr Cys Asn Ala Met Ser Arg Lys Leu Asp Lys Leu 100 105 110 Thr Val Leu Arg Met Ala Val Gln His Met Lys Thr Leu Arg Gly Ala 115 120 125 Thr Asn Pro Tyr Thr Glu Ala Asn Tyr Lys Pro Thr Phe Leu Ser Asp 130 135 140 Asp Glu Leu Lys His Leu Ile Leu Arg Ala Ala Asp Gly Phe Leu Phe 145 150 155 160 Val Val Gly Cys Asp Arg Gly Lys Ile Leu Phe Val Ser Glu Ser Val 165 170 175 Phe Lys Ile Leu Asn Tyr Ser Gln Asn Asp Leu Ile Gly Gln Ser Leu 180 185 190 Phe Asp Tyr Leu His Pro Lys Asp Ile Ala Lys Val Lys Glu Gln Leu 195 200 205 Ser Ser Ser Asp Thr Ala Pro Arg Glu Arg Leu Ile Asp Ala Lys Thr 210 215 220 Gly Leu Pro Val Lys Thr Asp Ile Thr Pro Gly Pro Ser Arg Leu Cys 225 230 235 240 Ser Gly Ala Arg Arg Ser Phe Phe Cys Arg Met Lys Cys Asn Arg Pro 245 250 255 Ser Val Lys Val Glu Asp Lys Asp Phe Pro Ser Thr Cys Ser Lys Lys 260 265 270 Lys Asp Arg Lys Ser Phe Cys Thr Ile His Ser Thr Gly Tyr Leu Lys 275 280 285 Ser Trp Pro Pro Thr Lys Met Gly Leu Asp Glu Asp Asn Glu Pro Asp 290 295 300 Asn Glu Gly Cys Asn Leu Ser Cys Leu Val Ala Ile Gly Arg Leu His 305 310 315 320 Ser His Val Val Pro Gln Pro Val Asn Gly Glu Ile Arg Val Lys Ser 325 330 335 Met Glu Tyr Val Ser Arg His Ala Ile Asp Gly Lys Phe Val Phe Val 340 345 350 Asp Gln Arg Ala Thr Ala Ile Leu Ala Tyr Leu Pro Gln Glu Leu Leu 355 360 365 Gly Thr Ser Cys Tyr Glu Tyr Phe His Gln Asp Asp Ile Gly His Leu 370 375 380 Ala Glu Cys His Arg Gln Val Leu Gln Thr Arg Glu Lys Ile Thr Thr 385 390 395 400 Asn Cys Tyr Lys Phe Lys Ile Lys Asp Gly Ser Phe Ile Thr Leu Arg 405 410 415 Ser Arg Trp Phe Ser Phe Met Asn Pro Trp Thr Lys Glu Val Glu Tyr 420 425 430 Ile Val Ser Thr Asn Thr Val Val Leu Ala Asn Val Leu Glu Gly Gly 435 440 445 Asp Pro Thr Phe Pro Gln Leu Thr Ala Ser Pro His Ser Met Asp Ser 450 455 460 Met Leu Pro Ser Gly Glu Gly Gly Pro Lys Arg Thr His Pro Thr Val 465 470 475 480 Pro Gly Ile Pro Gly Gly Thr Arg Ala Gly Ala Gly Lys Ile Gly Arg 485 490 495 Met Ile Ala Glu Glu Ile Met Glu Ile His Arg Ile Arg Gly Ser Ser 500 505 510 Pro Ser Ser Cys Gly Ser Ser Pro Leu Asn Ile Thr Ser Thr Pro Pro 515 520 525 Pro Asp Ala Ser Ser Pro Gly Gly Lys Lys Ile Leu Asn Gly Gly Thr 530 535 540 Pro Asp Ile Pro Ser Ser Gly Leu Leu Ser Gly Gln Ala Gln Glu Asn 545 550 555 560 Pro Gly Tyr Pro Tyr Ser Asp Ser Ser Ser Ile Leu Gly Glu Asn Pro 565,570,575 His Ile Gly Ile Asp Met Ile Asp Asn Asp Gln Gly Ser Ser Ser Pro 580,585,590 Sister Donkey Asp Glu Ala Ala Put Ala Val Ile Put Sister Leu Leu Glu Ala 595,600,605 Asp Ala Gly Leu Gly Gly Pro Val Asp Phe Ser Asp Leu Pro Trp Pro 610 615 620 Leu 625 <210> 16 <211> 1878 <212> DNA <213> artificial <220> <223> Pages ARNTL Videos <400> 16 atggcagacc agagaatgga catttcttca accatcagtg atttcatgtc cccgggcccc 60 accgacctgc tttccagctc tcttggtacc agtggtgtgg attgcaaccg caaacggaaa 120 ggcagctcca ctgactacca agaaagcatg gacacagaca aagatgaccc tcatggaagg 180 ttagaatata cagaacacca aggaaggata aaaaatgcaa gggaagctca cagtcagatt 240 gaaaagcggc gtcgggataa aatgaacagt tttatagatg aattggcttc tttggtacca 300 acatgcaacg caatgtccag gaaattagat aaacttactg tgctaaggat ggctgttcag 360 cacatgaaaa cattaagagg tgccaccaat ccatacacag aagcaaacta caaaccaact 420 tttctatcag acgatgaatt gaaacacctc attctcaggg cagcagatgg atttttgttt 480 gtcgtaggat gtgaccgagg gaagatactc tttgtctcag agtctgtctt caagatcctc 540 aactacagcc agaatgatct gattggtcag agtttgtttg actacctgca tcctaaagat 600 attgccaaag tcaaggagca gctctcctcc tctgacaccg caccccggga gcggctcata 660 gatgcaaaaa ctggacttcc agttaaaaca gatataaccc ctgggccatc tcgattatgt 720 tctggagcac gacgttcttt cttctgtagg atgaagtgta acaggccttc agtaaaggtt 780 gaagacaagg acttcccctc tacctgctca aagaaaaaag atcgaaaaag cttctgcaca 840 atccacagca caggctattt gaaaagctgg ccacccacaa agatggggct ggatgaagac 900 aacgaaccag acaatgaggg gtgtaacctc agctgcctcg tcgcaattgg acgactgcat 960 tctcatgtag ttccacaacc agtgaacggg gaaatcaggg tgaaatctat ggaatatgtt 1020 tctcggcacg cgatagatgg aaagtttgtt tttgtagacc agagggcaac agctattttg 1080 gcatatttac cacaagaact tctaggcaca tcgtgttatg aatattttca ccaagatgac 1140 ataggacatc ttgcagaatg tcataggcaa gttttacaga cgagagaaaa aattacaact 1200 aattgctata aattaaaat caaagatggt tcttttatca cactacggag tcgatggttc 1260 agtttcatga acccttggac caaggaagta gaatatattg tctcaactaa cactgttgtt 1320 ttagccaacg tcctggaagg cggggaccca accttcccac agctcacagc atccccccac 1380 agcatggaca gcatgctgcc ctctggagaa ggtggcccaa agaggaccca ccccactgtt 1440 ccagggattc cagggggaac ccgggctggg gcaggaaaaa taggccgaat gattgctgag 1500 gaaatcatgg aaatccacag gataagaggg tcatcgcctt ctagctgtgg ctccagccca 1560 ttgaacatca cgagtacgcc tccccctgat gcctcttctc caggaggcaa gaagatttta 1620 aatggaggga ctccagacat tccttccagt ggcctactat caggccaggc tcaggagaac 1680 ccaggttatc catattctga tagttcttct attcttggtg agaaccccca cataggtata 1740 gacatgattg acaacgacca aggatcaagt agtcccagta atgatgaggc agcaatggct 1800 gtcatcatga gcctcttgga agcagatgct ggactgggtg gccctgttga ctttagtgac 1860 ttgccatggc cgctgtaa 1878 <210> 17 <211> 61 <212> PRT <213> artificial <220> <223> Amino acid sequence of human CD52 <400> 17 Met Lys Arg Phe Leu Phe Leu Leu Leu Thr Ile Ser Leu Leu Val Met 1 5 10 15 Val Gln Ile Gln Thr Gly Leu Ser Gly Gln Asn Asp Thr Ser Gln Thr 20 25 30 Ser Ser Pro Ser Ala Ser Ser Asn Ile Ser Gly Gly Ile Phe Leu Phe 35 40 45 Phe Val Ala Asn Ala Ile Ile His Leu Phe Cys Phe Ser 50 55 60 <210> 18 <211> 186 <212> DNA <213> artificial <220> <223> Nucleotide sequence of human CD52 <400> 18 atgaagcgct tcctcttcct cctactcacc atcagcctcc tggttatggt acagatacaa 60 actggactct caggacaaa cgacaccagc caaccagca gcccctcagc atccacac 120 ataagcggag gcattttcct tttctcgtg gccaatgcca taatccacct cttctgcttc 180 agttga 186 <210> 19 <211> 219 <212> PRT <213> artificial <220> <223> About CD53 <400> 19 Met Gly Met Ser Ser Leu Lys Leu Lys Tyr Val Leu Phe Phe Phe 1 5 10 15 Asn Leu Leu Phe Trp Ile Cys Gly Cys Cys Ile Leu Gly Phe Gly Ile 20 25 30 Tyr Leu Leu Ile His Asn Asn Phe Gly Val Leu Phe His Asn Leu Pro 35 40 45 Ser Leu Thr Leu Gly Asn Val Phe Val Ile Val Gly Ser Ile Ile Met 50 55 60 Val Val Ala Phe Leu Gly Cys Met Gly Ser Ile Lys Glu Asn Lys Cys 65 70 75 80 Your Met Ser Phe Phe Ile You Your You Ile Ile You You Ala Glu 85 90 95 Val Thr Leu Ala Ile Leu Phe Val Tyr Glu Gln Lys Leu Asn Glu 100 105 110 Tyr Val Ala Lys Gly Leu Thr Asp Ser Ile His Arg Tyr His Ser Asp 115 120 125 Asn Ser Thr Lys Ala Ala Trp Asp Ser Ile Gln Ser Phe Leu Gln Cys 130 135 140 Cys Gly Ile Asn Gly Thr Ser Asp Trp Thr Ser Gly Pro Pro Ala Ser 145 150 155 160 Cys Pro Ser Asp Arg Lys Val Glu Gly Cys Tyr Ala Lys Ala Arg Leu 165 170 175 Trp Phe His Ser Asn Phe Leu Tyr Ile Gly Ile Ile Thr Ile Cys Val 180 185 190 Cys Val Ile Glu Val Leu Gly Met Ser Phe Ala Leu Thr Leu Asn Cys 195 200 205 Gln Ile Asp Lys Thr Ser Gln Thr Ile Gly Leu 210 215 <210> 20 <211> 660 <212> DNA <213> artificial <220> <223> Nucleotide sequence of human CD53 <400> 20 atgggcatga gtagcttgaa actgctgaag tatgtcctgt ttttcttcaa cttgctcttt 60 tggatctgtg gctgctgcat tttgggcttt gggatctacc tgctgatcca caacaacttc 120 ggagtgctct tccataacct cccctccctc acgctgggca atgtgtttgt catcgtgggc 180 tctattatca tggtagttgc cttcctgggc tgcatgggct ctatcaagga aaacaagtgt 240 ctgcttatgt cgttcttcat cctgctgctg attatcctcc ttgctgaggt gaccttggcc 300 atcctgctct ttgtatatga acagaagctg aatgagtatg tggctaaggg tctgaccgac 360 agcatccacc gttaccactc agacaatagc accaaggcag cgtgggactc catccagtca 420 tttctgcagt gttgtggtat aaatggcacg agtgattgga ccagtggccc accagcatct 480 tgcccctcag atcgaaaagt ggagggttgc tatgcgaaag caagactgtg gtttcattcc 540 aatttcctgt atatcggaat catcaccatc tgtgtatgtg tgattgaggt gttggggatg 600 tcctttgcac tgaccctgaa ctgccagatt gacaaaacca gccagaccat agggctatga 660

Claims

1. Use of an antibody or a combination thereof in the preparation of a reagent for detecting, identifying or separating a megakaryocyte cell population in a sample, wherein: The antibody or combination thereof is selected from any one or more of (a) to (c): (a) anti-MYLK4 antibody; (b) anti-LSP1 antibody, anti-CD52 antibody and / or anti-CD53 antibody; (c) anti-ARNTL antibodies; Among them, the nucleotide sequence encoding the MYLK4 is shown as SEQ ID NO: 2 or 12; the nucleotide sequence encoding the LSP1 is shown as SEQ ID NO: 4 or 14; the nucleotide sequence encoding the CD52 is shown as SEQ ID NO: 8 or 18; the nucleotide sequence encoding the CD53 is shown as SEQ ID NO: 10 or 20; the nucleotide sequence encoding the ARNTL is shown as SEQ ID NO: 6 or 16.

2. The use according to claim 1, wherein the MYLK4, LSP1, CD52, CD53 and / or ARNTL are derived from mammals.

3. The use according to claim 2, wherein the mammal is selected from the group consisting of humans, mice, sheep, horses, pigs and rabbits.

4. The use according to claim 1, wherein the sample is selected from mammalian whole blood, peripheral blood, bone marrow, umbilical cord blood, or any combination thereof.

5. The use according to claim 4, wherein the mammal is selected from humans, dogs, monkeys, cows, horses, or any combination thereof.

6. The use according to claim 1, wherein the antibody or combination thereof is selected from any one or more of (a) to (f): (a) anti-MYLK4 antibody; (b) anti-LSP1 antibody, anti-CD52 antibody and / or anti-CD53 antibody; (c) anti-ARNTL antibodies; (d) anti-MYLK4 antibody and anti-CD41 antibody; (e) anti-LSP1 antibody, anti-CD52 antibody and / or anti-CD53 antibody, and anti-CD41 antibody; (f) Anti-ARNTL antibody and anti-CD41 antibody.

7. Use of a marker molecule or a combination thereof in the preparation of a reagent for detecting, identifying or isolating a megakaryocyte cell population in a sample, wherein: The labeling molecule or combination thereof is selected from any one or more of (a) to (c): (a) MYLK4; (b) LSP1, CD52 and / or CD53; (c) ARNTL; Among them, the nucleotide sequence encoding the MYLK4 is shown as SEQ ID NO: 2 or 12; the nucleotide sequence encoding the LSP1 is shown as SEQ ID NO: 4 or 14; the nucleotide sequence encoding the CD52 is shown as SEQ ID NO: 8 or 18; the nucleotide sequence encoding the CD53 is shown as SEQ ID NO: 10 or 20; the nucleotide sequence encoding the ARNTL is shown as SEQ ID NO: 6 or 16.

8. The use according to claim 7, wherein the MYLK4, LSP1, CD52, CD53 and / or ARNTL are derived from mammals.

9. The use according to claim 8, wherein the mammal is selected from the group consisting of humans, mice, sheep, horses, pigs and rabbits.

10. The use according to claim 7, wherein the labeling molecule or a combination thereof is selected from any one or more of (a) to (f): (a) MYLK4; (b) LSP1, CD52 and / or CD53; (c) ARNTL; (d) MYLK4 and CD41; (e) LSP1, CD52 and / or CD53, and CD41; (f) ARNTL and CD41.

11. A megakaryocyte cell population, wherein the cell population expresses at least one marker molecule selected from MYLK4, LSP1, CD52, CD53 and ARNTL; in, The nucleotide sequence encoding the MYLK4 is shown as SEQ ID NO: 2 or 12; the nucleotide sequence encoding the LSP1 is shown as SEQ ID NO: 4 or 14; the nucleotide sequence encoding the CD52 is shown as SEQ ID NO: 8 or 18; the nucleotide sequence encoding the CD53 is shown as SEQ ID NO: 10 or 20; the nucleotide sequence encoding the ARNTL is shown as SEQ ID NO: 6 or 16.

12. The megakaryocyte cell population according to claim 11, further comprising expressing the CD41 marker molecule.

13. The megakaryocyte cell population according to claim 11, wherein the cell population is composed of any one or more of the cell populations of (a) to (c): (a) Cell population expressing MYLK4 and CD41; (b) a cell population expressing LSP1 and CD41, a cell population expressing CD52 and CD41, and / or a cell population expressing CD53 and CD41; (c) Cell population expressing ARNTL and CD41. The megakaryocyte cell population according to claim 11 , wherein the MYLK4, LSP1, CD52, CD53 and / or ARNTL are derived from mammals.

15. The megakaryocyte cell population according to claim 14, wherein the mammal is selected from the group consisting of humans, mice, sheep, horses, pigs and rabbits.

16. A method for identifying, isolating or detecting the megakaryocyte population according to any one of claims 11 to 15, the method comprising: (1) contacting the cell population with at least one reagent, and (2) performing a test on the cell population.

17. The method of claim 16, wherein: The reagent detects the labeled molecule by a protein detection method.

18. The method of claim 17, wherein the protein detection method is selected from immunological methods, chromatography, mass spectrometry, spectroscopy, or any combination thereof.

19. The method of claim 17, wherein the protein detection method is enzyme-linked immunosorbent assay, chemiluminescent immunoassay, fluorescent immunoassay, radioimmunoassay, electrochemical immunosensor, peptide mass fingerprinting, or any combination thereof.

20. The method of claim 16, wherein the reagents each independently comprise an antibody capable of specifically binding to the labeled molecule, an antigen-binding fragment thereof, and a detectable label.

21. The method of claim 16, wherein each of the reagents independently comprises a combination of multiple antibodies capable of specifically binding to labeled molecules.

22. The method of claim 21, wherein the combination comprises a first antibody as a capture reagent and a second antibody as a detection reagent.

23. The method of claim 22, wherein the second antibody has a different antigen binding site than the first antibody.

24. The method of claim 22, wherein the second antibody is detectably labeled.

25. The method of claim 20, wherein the detectable labels are each independently selected from an enzyme, a chemiluminescent agent, an electrochemiluminescent agent, an electroactive substance, a fluorescent dye, a radionuclide, or biotin.

26. The method of claim 16, wherein: The reagent detects the mRNA of the marker molecule or the cDNA of the marker molecule by a nucleic acid detection method.

27. The method according to claim 26, wherein the nucleic acid detection method is transcription analysis, fluorescent quantitative PCR, nucleic acid hybridization or any combination thereof.

28. The method of claim 27, wherein the reagents each independently comprise at least one primer or probe complementary to a portion of the nucleotide sequence of the marker molecule mRNA or cDNA.

29. A kit comprising reagents for identifying, detecting or isolating a marker molecule, wherein the marker molecule is selected from MYLK4, LSP1, CD52, CD53, ARNTL, or any combination thereof; in, The nucleotide sequence encoding the MYLK4 is shown as SEQ ID NO: 2 or 12; the nucleotide sequence encoding the LSP1 is shown as SEQ ID NO: 4 or 14; the nucleotide sequence encoding the CD52 is shown as SEQ ID NO: 8 or 18; the nucleotide sequence encoding the CD53 is shown as SEQ ID NO: 10 or 20; the nucleotide sequence encoding the ARNTL is shown as SEQ ID NO: 6 or 16.

30. The kit of claim 29, wherein the marker molecule further comprises CD41.

31. The kit of claim 29, wherein the MYLK4, LSP1, CD52, CD53 and / or ARNTL are derived from mammals.

32. The kit of claim 31, wherein the mammal is selected from the group consisting of humans, mice, sheep, horses, pigs and rabbits.

33. The kit of claim 29, wherein the reagent detects the labeled molecule by a protein detection method.

34. The kit of claim 33, wherein the protein detection method is selected from immunological method, chromatography, mass spectrometry, spectroscopy, or any combination thereof.

35. The kit of claim 33, wherein the protein detection method is enzyme-linked immunosorbent assay, chemiluminescent immunoassay, fluorescent immunoassay, radioimmunoassay, electrochemical immunosensor, peptide mass fingerprinting, or any combination thereof.

36. The kit of claim 33, wherein the reagent comprises an antibody capable of specifically binding to the labeled molecule, an antigen-binding fragment thereof and a detectable label. The kit of claim 33 , wherein the reagent comprises a combination of multiple antibodies capable of specifically binding to labeled molecules.

38. The kit of claim 37, wherein the combination comprises a first antibody as a capture reagent and a second antibody as a detection reagent.

39. The kit of claim 38, wherein the second antibody has a different antigen binding site than the first antibody.

40. The kit of claim 38, wherein the second antibody is detectably labeled.

41. The kit of claim 40, wherein the detectable label is selected from an enzyme, a chemiluminescent agent, an electrochemiluminescent agent, an electroactive substance, a fluorescent dye, a radionuclide, or biotin.

42. The kit of claim 29, which is used for identifying, detecting or isolating a megakaryocyte population.

43. The kit of claim 29, wherein the reagent detects the mRNA of the marker molecule or the cDNA of the marker molecule by a nucleic acid detection method.

44. The kit of claim 29, wherein each of the reagents independently comprises at least one primer or probe complementary to a portion of the nucleotide sequence of the marker molecule mRNA or cDNA.

45. The kit of claim 29, which is used for identifying, detecting or isolating a megakaryocyte population.

46. ​​Use of the megakaryocyte population according to any one of claims 11 to 14 or the kit according to any one of claims 29 to 45 in the preparation of a medicament for treating a disease caused by abnormalities in megakaryocytes, platelets and / or hematopoietic stem cells and / or an immune-related disease in a subject.

47. The use according to claim 46, wherein the medicament is used for treating a subject's blood-related diseases and / or autoimmune diseases caused by a decrease or increase in megakaryocytes, platelets and / or hematopoietic stem cells.

48. The use according to claim 46, wherein the disease comprises megakaryoblastic lineage developmental disorder, thrombocytopenia and thrombocytosis caused by various reasons.

49. The use according to claim 47, wherein the blood-related diseases include malignant diseases of the hematopoietic system and non-malignant diseases of the hematopoietic system.

50. The use of claim 49, wherein the hematopoietic malignancies include acute leukemia, chronic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myelodysplastic syndrome and multiple myeloma.

51. The use of claim 49, wherein the non-malignant diseases of the hematopoietic system include aplastic anemia, chronic thrombocytopenic purpura, paroxysmal nocturnal hemoglobinuria, and myelofibrosis.

52. The use of claim 46, wherein the immune-related diseases include rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, polymyositis, dermatomyositis, ankylosing spondylitis, psoriatic arthritis and gout.

53. The use of claim 46, wherein the subject is a mammal.

54. The use according to claim 46, wherein the subject is a mouse, a human, a dog, a monkey, a cow or a horse.

Images