CN1300763A - Polypeptide-hexokinase protien 12 and polynucleotide for coding this polypeptide - Google Patents

Abstract

The invention discloses a new polypeptide-hexokinase protein 12, a polynucleotide encoding the polypeptide and a method for producing the polypeptide through DNA recombination technology. The invention also discloses a method for using the polypeptide to treat various diseases, such as malignant tumors, blood diseases, HIV infection, immune diseases and various inflammations. The invention also discloses an antagonist against the polypeptide and its therapeutic effect. The invention also discloses the application of the polynucleotide encoding the novel hexokinase protein 12.

Description

A New Polypeptide--Hexokinase Protein 12 and the Polynucleotide Encoding the Polypeptide

The invention belongs to the field of biotechnology. Specifically, the invention describes a new polypeptide-hexokinase protein 12, and a polynucleotide sequence encoding the polypeptide. The present invention also relates to the preparation method and application of the polynucleotide and polypeptide. Hexokinase catalyzes the phosphorylation of glucose. This reversible reaction requires the participation of ATP. This reaction is the first step of the glycolysis reaction and also the speed regulation step of the glycolysis reaction. It phosphorylates glucose involved in glycolysis to activate it. There are four main types of hexokinases in vertebrates: Ⅰ, Ⅱ, Ⅲ, Ⅳ. Class IV hexokinase is only expressed in liver cells and spleen β cells, and plays an important role in the secretion of insulin, with a molecular weight of about 50Kd. Class II hexokinase is the predominant form of hexokinase in skeletal muscle. Class I and class III hexokinases have a molecular weight of about 100Kd, and their Km values for glucose are slightly smaller. Structurally they consist of a small N-terminal hydrophobic membrane-binding domain followed by two very similar structures of approximately 450 residues. The first structure has lost its catalytic activity and evolved into a regulatory structure. There are three different hexokinases in yeast: hexokinase PI, PII and glucokinase. The above enzymes all contain a very conserved region, [LIVM]-G-F-[TN]-F-S-[FY]-P-x(5)-[LIVM]-[DNST]-x(3)-[LIVM]-x (2)-W-T-K-x-[LF] In the red blood cells and platelets of patients with non-spherocytic hemolytic anemia, the activity of hexokinase I is only 25% of normal. In rapidly proliferating tumor tissues, the expression level of hexokinase Ⅱ is unusually high, so as to provide the extremely high demand for carbon source supply of tumor cells. Hexokinase Ⅱ may be the pathogenic gene of non-insulin-dependent diabetes mellitus.

Since hexokinase protein 12 proteins play an important role in important functions of the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there is a continuing need in the art to identify more hexokinase protein 12 proteins involved in these processes, In particular, the amino acid sequence of this protein is identified. The isolation of the gene encoding the new hexokinase protein 12 protein also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and/or therapeutic drugs for diseases, so it is very important to isolate its coding DNA.

It is an object of the present invention to provide isolated novel polypeptides - hexokinase protein 12 and fragments, analogs and derivatives thereof.

Another object of the present invention is to provide a polynucleotide encoding the polypeptide.

Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding hexokinase protein 12.

Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding hexokinase protein 12.

Another object of the present invention is to provide a method for producing hexokinase protein 12.

Another object of the present invention is to provide antibodies against the polypeptide of the present invention-hexokinase protein 12. Another object of the present invention is to provide mimetic compounds, antagonists, agonists and inhibitors against the polypeptide of the present invention-hexokinase protein 12.

Another object of the present invention is to provide methods for diagnosis and treatment of diseases associated with hexokinase protein 12 abnormality. The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or its conservative variant, biologically active fragment or derivative. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO:2.

The present invention also relates to an isolated polynucleotide comprising a nucleotide sequence or variant thereof selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No.2;

(b) a polynucleotide complementary to polynucleotide (a);

(c) A polynucleotide having at least 70% identity to the polynucleotide sequence of (a) or (b).

More preferably, the sequence of the polynucleotide is one selected from the following group: (a) having the sequence of 1552-1887 in SEQ ID NO: 1; and (b) having 1-1958 in SEQ ID NO: 1 sequence of bits.

The present invention also relates to a vector containing the polynucleotide of the present invention, especially an expression vector; a host cell genetically engineered with the vector, including transformed, transduced or transfected host cells; a method comprising culturing said Host Cells and Methods of Recovering Expression Products to Produce Polypeptides of the Invention.

The present invention also relates to an antibody capable of specifically binding to the polypeptide of the present invention.

The present invention also relates to a method of screening compounds for simulating, activating, antagonizing or inhibiting the activity of hexokinase protein 12 protein, which comprises using the polypeptide of the present invention. The invention also relates to the compounds obtained by this method.

The present invention also relates to a method for in vitro detection of diseases or disease susceptibility related to abnormal expression of hexokinase protein 12 protein, comprising detection of mutations in the polypeptide or its coding polynucleotide sequence in biological samples, or detection of mutations in biological samples The amount or biological activity of the polypeptide of the present invention.

The present invention also relates to a pharmaceutical composition, which contains the polypeptide of the present invention or its mimetic, activator, antagonist or inhibitor and a pharmaceutically acceptable carrier.

The present invention also relates to the use of the polypeptide and/or polynucleotide of the present invention in the preparation of medicines for treating cancer, developmental diseases or immune diseases or other diseases caused by abnormal expression of hexokinase protein 12.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

Unless otherwise specified, the following terms used in this specification and claims have the following meanings: "Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to genomic or synthetic DNA or RNA, which can be single- or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to oligopeptide, peptide, polypeptide or protein sequences and fragments or portions thereof. When "amino acid sequence" in the present invention refers to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" is not meant to limit the amino acid sequence to the complete natural amino acid associated with said protein molecule .

A protein or polynucleotide "variant" refers to an amino acid sequence or a polynucleotide sequence encoding it having one or more amino acid or nucleotide changes. The alterations may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. A variant may have "conservative" changes in which a substituted amino acid has similar structural or chemical properties to the original amino acid, eg, a leucine is replaced by an isoleucine. Variants may also have non-conservative changes, such as replacing glycine with tryptophan.

"Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or a nucleotide sequence.

"Insertion" or "addition" refers to a change in amino acid sequence or nucleotide sequence that results in the addition of one or more amino acids or nucleotides compared to the naturally occurring molecule. "Substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides.

"Biologically active" refers to a protein that possesses the structural, regulatory, or biochemical functions of a native molecule. Similarly, the term "immunological activity" refers to the ability of a native, recombinant or synthetic protein and fragments thereof to induce a specific immune response and bind to specific antibodies in a suitable animal or cell.

"Agonist" refers to a molecule that, when bound to hexokinase protein 12, causes changes in the protein, thereby modulating the activity of the protein. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules that bind hexokinase protein 12.

"Antagonist" or "inhibitor" refers to a molecule that, when bound to hexokinase protein 12, blocks or modulates the biological or immunological activity of hexokinase protein 12. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecules that bind hexokinase protein 12.

"Modulation" refers to changes in the function of hexokinase protein 12, including an increase or decrease in protein activity, changes in binding properties, and changes in any other biological, functional or immunological properties of hexokinase protein 12.

"Substantially pure" means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify hexokinase protein 12 using standard protein purification techniques. Substantially pure hexokinase protein 12 yields a single major band on non-reducing polyacrylamide gels. The purity of the hexokinase protein 12 polypeptide can be analyzed by amino acid sequence.

"Complementary" or "complementary" refers to the natural association of polynucleotides through base pairing under permissive conditions of salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules can be partial or total. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

"Homology" refers to the degree of complementarity, which may be partial or complete. "Partially homologous"refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of such hybridization can be detected by performing hybridization under conditions of reduced stringency (Southern blot or Northern blot, etc.). Substantially homologous sequences or hybridization probes can compete and inhibit binding of a fully homologous sequence to a target sequence under conditions of reduced stringency. This does not mean that conditions of reduced stringency permit non-specific binding, since conditions of reduced stringency require the binding of two sequences to each other for a specific or selective interaction.

"Percent identity" refers to the percentage of sequences that are identical or similar in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, D.G. and P.M. Sharp (1988) Gene 73:237-244). The Cluster method arranges sets of sequences into clusters by examining the distances between all pairs. Clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:

The number of residues matched between sequence A and sequence B

100 (number of residues in sequence A - number of spacer residues in sequence A - number of spacer residues in sequence B)

The percentage identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in emzumology 183:625-645).

"Similarity" refers to the degree of identity or conservative substitution of amino acid residues at corresponding positions when amino acid sequences are aligned. Amino acids for conservative substitutions. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; Amino acids of similar hydrophilicity may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

"Antisense" refers to a nucleotide sequence that is complementary to a specific DNA or RNA sequence. "Antisense strand" refers to the nucleic acid strand that is complementary to the "sense strand."

"Derivative" refers to a chemical modification of HFP or the nucleic acid encoding it. Such chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain key biological properties of the native molecule.

"Antibody" refers to the whole antibody molecule and its fragments, such as Fa, F(ab') ₂ and Fv, which can specifically bind to the epitope of hexokinase protein 12.

"Humanized antibody" refers to an antibody in which the amino acid sequence of the non-antigen-binding region has been replaced to become more similar to a human antibody, but still retains the original binding activity.

The term "isolated" refers to the removal of a substance from its original environment (eg, its natural environment if naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide is separated from some or all of the substances with which it coexists in a natural system is isolated. Such polynucleotides may be part of a vector, or such polynucleotides or polypeptides may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated hexokinase protein 12" means hexokinase protein 12 is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify hexokinase protein 12 using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the hexokinase protein 12 polypeptide can be analyzed by amino acid sequence.

The present invention provides a new polypeptide-hexokinase protein 12, which is basically composed of the amino acid sequence shown in SEQ ID NO:2. The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

Fragments, derivatives and analogs of hexokinase protein 12 are also encompassed by the present invention. As used in the present invention, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the hexokinase protein 12 of the present invention. Fragments, derivatives or analogs of the polypeptide of the present invention may be: (I) one wherein one or more amino acid residues are substituted by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted The amino acid may or may not be encoded by the genetic code; or (II) a substituent in which a certain group on one or more amino acid residues is replaced by another group; or (III) such One, wherein the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused to the mature polypeptide ( Such fragments, derivatives and analogs are considered to be within the purview of those skilled in the art from the description herein such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence.

The invention provides an isolated nucleic acid (polynucleotide) consisting essentially of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:2. The polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1. The polynucleotides of the present invention were discovered from a cDNA library of human fetal brain tissue. The full-length polynucleotide sequence it contains is 1958 bases, and its open reading frame 1552-1887 encodes 111 amino acids. The polypeptide has the characteristic sequence of the hexokinase characteristic sequence, and it can be deduced that the hexokinase protein 12 has the structure and function represented by the hexokinase characteristic sequence.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide can be the same as the sequence of the coding region shown in SEQ ID NO: 1 or a degenerate variant. As used in the present invention, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein or polypeptide having SEQ ID NO:2, but differs from the sequence of the coding region shown in SEQ ID NO:1.

A polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: the coding sequence of the mature polypeptide only; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non- coding sequence.

The term "polynucleotide encoding a polypeptide" is intended to include polynucleotides encoding the polypeptide and polynucleotides including additional coding and/or non-coding sequences.

The present invention also relates to variants of the polynucleotides described above, which encode polypeptides having the same amino acid sequence as those of the present invention or fragments, analogs and derivatives of polypeptides. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The invention also relates to polynucleotides which hybridize to the sequences described above (with at least 50%, preferably 70% identity between the two sequences). The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with Use a denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 95%, more Preferably hybridization occurs above 97%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, the length of "nucleic acid fragment" contains at least 10 nucleotides, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides glycosides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (eg, PCR) to identify and/or isolate polynucleotides encoding hexokinase protein 12.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The specific polynucleotide sequence encoding hexokinase protein 12 of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleotides with common structural features acid fragments.

The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolation of double-stranded DNA sequence from genomic DNA; 2) chemical synthesis of DNA sequence to obtain double-stranded DNA of the polypeptide.

Of the methods mentioned above, isolating genomic DNA is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The method of choice more often is the isolation of cDNA sequences. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that highly express the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature technologies for the method of extracting mRNA, and the kit can also be obtained from commercial channels (Qiagene). The construction of a cDNA library is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as various cDNA libraries from the company Clontech. When combined with polymerase reaction technology, even minimal expression products can be cloned.

These cDNA libraries can be screened for the gene of the present invention by a conventional method. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) marker gene function appearance or loss; (3) measuring the level of transcripts of hexokinase protein 12; (4) The protein product of gene expression is detected by immunological techniques or by measuring biological activity. The above methods can be used alone or in combination with multiple methods.

In the (1) method, the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probes used here are usually DNA sequences chemically synthesized based on the gene sequence information of the present invention. The genes themselves or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioactive isotopes, luciferin or enzymes (such as alkaline phosphatase) and the like.

In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the hexokinase protein 12 gene.

A method of amplifying DNA/RNA using the PCR technique (Saiki, et al. Science 1985; 230:1350-1354) is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain the full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the polynucleotide sequence information of the present invention disclosed herein. selected and synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The polynucleotide sequences of the genes of the present invention obtained as described above, or various DNA fragments, etc., can be determined by conventional methods such as the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74:5463-5467). Such polynucleotide sequence determination can also use commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones before splicing into a full-length cDNA sequence.

The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the hexokinase protein 12 coding sequence, and a method for producing the polypeptide of the present invention through recombinant techniques .

In the present invention, the polynucleotide sequence encoding hexokinase protein 12 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Carriers applicable in the present invention include, but are not limited to: the expression vector based on the T7 promoter expressed in bacteria (Rosenberg, et al. Gene, 1987, 56:125); the pMSXND expression vector expressed in mammalian cells ( Lee and Nathans, J Bio Chem.263:3521, 1988) and vectors derived from baculovirus expressed in insect cells. In short, any plasmid and vector can be used to construct a recombinant expression vector as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational regulatory elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding hexokinase protein 12 and appropriate transcription/translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retroviruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Inserting an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA expression, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc.

Those of ordinary skill in the art will know how to select appropriate vectors/transcriptional regulatory elements (such as promoters, enhancers, etc.) and selectable marker genes.

In the present invention, the polynucleotide encoding the hexokinase protein 12 or the recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS or Bowes melanoma cells, etc.

Transformation of host cells with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. An alternative is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The polynucleotide sequence of the present invention can be used to express or produce recombinant hexokinase protein 12 by conventional recombinant DNA technology (Science, 1984; 224:1431). Generally speaking, there are the following steps:

(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding human hexokinase protein 12 of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2). Culturing the host cells in a suitable medium;

(3). Isolate and purify proteins from culture medium or cells.

In step (2), the medium used in the culture can be selected from various conventional mediums according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

In step (3), the recombinant polypeptide can be encapsulated in the cell, expressed on the cell membrane, or secreted out of the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. These methods include but are not limited to: conventional refolding treatment, protein precipitant treatment (salting out method), centrifugation, osmotic destruction, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The polypeptide of the present invention and its antagonists, agonists and inhibitors can be directly used in the treatment of diseases, for example, can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infection and immune diseases.

Hexokinase catalyzes the phosphorylation of glucose. This reversible reaction requires the participation of ATP. This reaction is the first step of the glycolysis reaction and also the speed regulation step of the glycolysis reaction. There are currently four classes of hexokinases. Class IV hexokinase is only expressed in liver cells and spleen β cells, and plays an important role in insulin secretion; class II hexokinase is the main form of hexokinase in skeletal muscle. The Km values of class I and class III hexokinases for glucose are slightly smaller. They all have specific conserved sequences that form their active motifs.

Existing studies have shown that in the red blood cells and platelets of patients with non-spherical hemolytic anemia, the activity of hexokinase I is only 25% of normal. In rapidly proliferating tumor tissues, the expression level of hexokinase Ⅱ is unusually high, so as to provide the extremely high demand for carbon source supply of tumor cells. Hexokinase Ⅱ may be the pathogenic gene of non-insulin-dependent diabetes mellitus.

It can be seen that the abnormal expression of the specific hexokinase motif will lead to the abnormal function of the polypeptide containing this motif, which will lead to the abnormal glycolysis process, and further affect the metabolic process of matter and energy, and can also affect the secretion of insulin. Produce information regulation, or play a role in the process of tumor growth and proliferation, and produce related diseases such as material and energy metabolism disorders, non-insulin-dependent diabetes, non-spherical red blood cell hemolytic anemia, tumors, embryonic development disorders, growth developmental disabilities, etc.

It can be seen that the abnormal expression of the hexokinase protein 12 of the present invention will produce various diseases, especially energy metabolism disorders, non-insulin-dependent diabetes mellitus, non-spherical erythrocytic hemolytic anemia, tumors, embryonic development disorders, growth Developmental disabilities, including but not limited to:

Disorders of material and energy metabolism: isovaleric acidemia, propionic acidemia, methylmalonic aciduria, combined carboxylase deficiency, glutaric acidemia type Ⅰ, phenylketonuria, albinism, tryptamine Hyperemia, metabolic deficiency of urea cycle, histidine metabolism deficiency, lysine metabolism deficiency, orotic aciduria, hyperlipoproteinemia, galactosemia, fructose metabolism deficiency, glycogen storage disease

Tumors of various tissues: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioma, Colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymus tumor, nasal cavity and sinus tumor, nasal Pharyngeal cancer, laryngeal cancer, tracheal tumor, fibroma, fibrosarcoma, lipoma, liposarcoma, leiomyoma

Embryodevelopmental disorders: congenital abortion, cleft palate, absence of limbs, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney disease, double ureter, hidden, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias , hermaphroditism, atrial septal defect, ventricular septal defect, pulmonary stenosis, patent ductus arteriosus, neural tube defect, congenital hydrocephalus, iris defect, congenital cataract, congenital glaucoma or cataract, congenital deafness

Growth and development disorders: mental retardation, cerebral palsy, brain developmental disorders, familial cranial nucleus hypoplasia syndrome, skin, fat and muscle dysplasia diseases such as congenital skin laxity, progeria, congenital horn dysplasia, cretinism, dwarfism, sexual retardation

Abnormal expression of the hexokinase protein 12 of the present invention will also cause some hereditary, blood diseases, immune system diseases and the like.

The polypeptide of the present invention and its antagonists, agonists and inhibitors can be directly used in the treatment of diseases, for example, can treat various diseases, especially material and energy metabolism disorders, non-insulin-dependent diabetes mellitus, non-spherical red blood cell Sexual hemolytic anemia, tumors, embryonic development disorders, growth and development disorders, some hereditary, blood diseases and immune system diseases, etc.

The invention also provides methods of screening compounds to identify agents that increase (agonists) or repress (antagonists) hexokinase protein 12. Agonists enhance biological functions of hexokinase protein 12 such as stimulating cell proliferation, while antagonists prevent and treat disorders associated with excessive cell proliferation such as various cancers. For example, mammalian cells or membrane preparations expressing hexokinase protein 12 can be incubated with labeled hexokinase protein 12 in the presence of a drug. The ability of the drug to enhance or repress this interaction is then determined.

Antagonists of hexokinase protein 12 include screened antibodies, compounds, receptor deletions and analogs. The antagonist of hexokinase protein 12 can combine with hexokinase protein 12 and eliminate its function, or inhibit the production of the polypeptide, or combine with the active site of the polypeptide so that the polypeptide cannot perform biological functions.

In screening compounds for antagonists, hexokinase protein 12 can be added to a bioanalytical assay to determine whether a compound is an antagonist by determining the compound's effect on the interaction between hexokinase protein 12 and its receptor. Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds. The polypeptide molecule capable of binding to the hexokinase protein 12 can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to solid phases. During screening, the hexokinase protein 12 molecule should generally be labeled.

The present invention provides methods for producing antibodies using polypeptides, fragments, derivatives, analogs thereof, or cells thereof as antigens. These antibodies can be polyclonal or monoclonal. The present invention also provides antibodies against the epitope of hexokinase protein 12. These antibodies include, but are not limited to: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by a Fab expression library.

The production of polyclonal antibodies can be obtained by direct injection of hexokinase protein 12 into immunized animals (such as rabbits, mice, rats, etc.), and various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc. . Techniques for preparing monoclonal antibodies to hexokinase protein 12 include but are not limited to hybridoma technology (Kohler and Milstein. Nature, 1975,256:495-497), triple tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology etc. Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81:6851). The existing technology for producing single-chain antibodies (U.S. Pat No. 4946778) can also be used to produce single-chain antibodies against hexokinase protein 12.

Antibodies against hexokinase protein 12 can be used in immunohistochemical techniques to detect hexokinase protein 12 in biopsy specimens.

The monoclonal antibody binding to hexokinase protein 12 can also be labeled with radioactive isotopes, and its location and distribution can be tracked when injected into the body. This radiolabeled antibody can be used as a non-invasive diagnostic method for localization of tumor cells and judgment of metastasis.

Antibodies can also be used to design immunotoxins that target a particular site in the body. For example, high-affinity monoclonal antibodies against hexokinase protein 12 can be covalently combined with bacterial or plant toxins (such as diphtheria toxin, ricin, rhododine, etc.). A common method is to use a sulfhydryl cross-linking agent such as SPDP to attack the amino group of the antibody, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill hexokinase protein 12 positive cells.

The antibody of the present invention can be used to treat or prevent diseases related to hexokinase protein 12. Administration of appropriate doses of antibodies can stimulate or block hexokinase protein 12 production or activity.

The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of the hexokinase protein 12. These assays are well known in the art and include FISH assays and radioimmunoassays. The level of hexokinase protein 12 detected in the test can be used to explain the importance of hexokinase protein 12 in various diseases and to diagnose diseases in which hexokinase protein 12 plays a role.

The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cut physically, chemically or enzymatically, and then subjected to one-dimensional, two-dimensional or three-dimensional gel electrophoresis analysis, and better mass spectrometry analysis.

Polynucleotides encoding hexokinase protein 12 may also be used for various therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell proliferation, development or metabolism due to non-expression or abnormal/inactive expression of hexokinase protein 12. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated hexokinase protein 12 to inhibit endogenous hexokinase protein 12 activity. For example, a mutated hexokinase protein 12 may be a shortened hexokinase protein 12 lacking a signal transduction domain, although it can bind to a downstream substrate, it lacks signal transduction activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of hexokinase protein 12. Expression vectors derived from viruses such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the polynucleotide encoding hexokinase protein 12 into cells. The method for constructing a recombinant viral vector carrying a polynucleotide encoding hexokinase protein 12 can be found in existing literature (Sambrook, et al.). In addition, the recombinant polynucleotide encoding hexokinase protein 12 can be packaged into liposomes and transferred into cells.

Methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.), and then transplanting the cells Wait in the body.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit hexokinase protein 12 mRNA are also within the scope of the invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform an endonucleic cut. Antisense RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoamide chemical synthesis of oligonucleotides, which has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector downstream of the RNA polymerase promoter. In order to increase the stability of nucleic acid molecules, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the connection between ribonucleosides should use phosphothioester bonds or peptide bonds instead of phosphodiester bonds.

The polynucleotide encoding hexokinase protein 12 can be used for the diagnosis of diseases related to hexokinase protein 12. The polynucleotide encoding the hexokinase protein 12 can be used to detect the expression of the hexokinase protein 12 or the abnormal expression of the hexokinase protein 12 in a disease state. For example, the DNA sequence encoding hexokinase protein 12 can be used to hybridize the biopsy specimen to determine the expression status of hexokinase protein 12. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be used as probes to be immobilized on microarrays (Microarray) or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. Hexokinase protein 12 transcripts can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification with hexokinase protein 12-specific primers.

Detection of mutations in the hexokinase protein 12 gene can also be used to diagnose hexokinase protein 12-related diseases. The mutated form of hexokinase protein 12 includes point mutation, translocation, deletion, recombination and any other abnormalities compared with the normal wild-type hexokinase protein 12 DNA sequence. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

The sequences of the invention are also valuable for chromosome identification. The sequence will be specific for a particular location on a human chromosome and can hybridize thereto. Currently, there is a need to identify the specific site of each gene on the chromosome. Currently, only a few chromosomal markers based on actual sequence data (repeat polymorphisms) are available to mark chromosomal positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on chromosomes.

In short, PCR primers (preferably 15-35bp) are prepared according to the cDNA, and the sequence can be positioned on the chromosome. These primers were then used for PCR screening of somatic heterozygous cells containing individual human chromosomes. Only those cells heterozygous for the human gene corresponding to the primer will produce an amplified fragment.

The PCR mapping method of somatic heterozygous cells is a quick method to locate DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, sublocalization can be achieved using a set of fragments from a particular chromosome or a large number of genomic clones by a similar method. Other similar strategies that can be used for chromosome mapping include in situ hybridization, chromosome prescreening by flow sorting with markers, and hybridization preselection to construct chromosome-specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of cDNA clones to metaphase chromosomes allows precise chromosomal mapping in a single step. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to an exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with gene map data. These data can be found, for example, in V. Mckusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between the gene and the disease that has been mapped to the chromosomal region.

Next, the cDNA or genome sequence differences between affected and non-affected individuals need to be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping techniques, the cDNA that is pinpointed to a disease-associated chromosomal region can be one of 50 to 500 potential disease-causing genes (assuming 1 megabase mapping resolution capacity and each 20kb corresponds to a gene).

The polypeptides, polynucleotides and their mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with appropriate pharmaceutical carriers. These carriers can be water, dextrose, ethanol, salts, buffers, glycerol and combinations thereof. The composition contains safe and effective doses of polypeptides or antagonists as well as carriers and excipients that do not affect the drug effect. These compositions can be used as medicine for disease treatment.

The invention also provides kits or kits comprising one or more containers containing one or more ingredients of the pharmaceutical compositions of the invention. Along with these containers, there may be an indicative notice given by the governmental regulatory agency that manufactures, uses or sells the drug or biological product reflecting its approval for human administration by the governmental regulatory agency that manufactures, uses or sells the drug or biological product. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes of administration. Hexokinase protein 12 is administered in an amount effective to treat and/or prevent the particular indication. The amount and dosage range of hexokinase protein 12 administered to a patient will depend on many factors, such as the mode of administration, the health condition of the person to be treated, and the judgment of the diagnosing physician.

The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.

Fig. 1 is a comparison diagram of the amino acid sequence homology of the hexokinase protein 12 of the present invention with a total of 58 amino acids at 10-67 and the characteristic sequence of the structural domain hexokinase. The upper sequence is hexokinase protein 12, and the lower sequence is the characteristic sequence of domain hexokinase. The same amino acid between the two sequences is represented by a single character amino acid:, and the similar amino acid is represented by "+".

Fig. 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of the separated hexokinase protein 12. 12KDa is the molecular weight of the protein. Arrows indicate isolated protein bands.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions. Example 1: Cloning of Hexokinase Protein 12

Total RNA was extracted from human fetal brain by one-step method of guanidine isothiocyanate/phenol/chloroform. Poly(A) mRNA was isolated from total RNA with Quik mRNA Isolation Kit (product of Qiegene). 2ug poly(A) mRNA was reverse transcribed to form cDNA. The cDNA fragment was directional inserted into the multiple cloning site of the pBSK (+) vector (product of Clontech) using the Smart cDNA cloning kit (purchased from Clontech), transformed into DH5α, and the bacteria formed a cDNA library. The sequences of the 5' and 3' ends of all clones were determined with Dyeterminate cycle reaction sequencing kit (product of Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one of the clones, 0664a04, was a new DNA. The insert cDNA fragment contained in this clone was determined bidirectionally by synthesizing a series of primers. The results show that the full-length cDNA contained in the 0664a04 clone is 1958bp (as shown in Seq ID NO: 1), and there is a 336bp open reading frame (ORF) from the 1552bp to 1887bp, encoding a new protein (as shown in Seq ID NO :2). We named this clone pBS-0664a04, and named the encoded protein as hexokinase protein 12.

Example 2: Domain analysis of cDNA clones

The sequence of the hexokinase protein 12 of the present invention and the protein sequence encoded thereof are used in the profilescan program (Basiclocal Alignment search tool) in GCG [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10 ], domain analysis in databases such as prosite. The hexokinase protein 12 of the present invention has homology with the characteristic sequence of domain hexokinase at 10-67, the homology result is shown in Figure 1, the homology rate is 30%, the score is 17.16; the threshold is 14.79. Embodiment 3: Cloning the gene encoding hexokinase protein 12 by RT-PCR method

The total RNA of fetal brain cells was used as a template, and oligo-dT was used as a primer to carry out reverse transcription reaction to synthesize cDNA. After purification with a Qiagene kit, PCR amplification was performed with the following primers:

Primer1: 5'-GGAGTTGAACCCTGACTGTATGAC-3' (SEQ ID NO: 3)

Primer2: 5'-AAAAATACAATGTCATCTCTATTC-3' (SEQ ID NO: 4)

Primer1 is the forward sequence starting from the 1st bp at the 5' end of SEQ ID NO:1;

Primer2 is the 3' reverse sequence in SEQ ID NO:1.

Amplification reaction conditions: 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/L MgCl ₂ , 200μmol/L dNTP, 10pmol primers, 1U Taq DNA in a 50μl reaction volume Polymerase (product of Clontech Company). On a PE9600 DNA thermal cycler (Perkin-Elmer), the reaction was performed for 25 cycles under the following conditions: 94°C for 30 sec; 55°C for 30 sec; 72°C for 2 min. During RT-PCR, β-actin was set as positive control and template blank was set as negative control. The amplified product was purified with a kit from QIAGEN, and connected to a pCR vector (product of Invitrogen) with a TA cloning kit. The DNA sequence analysis results showed that the DNA sequence of the PCR product was completely identical to 1-1958bp shown in SEQ ID NO:1. Embodiment 4: Northern blot analysis of the expression of the hexokinase protein 12 gene:

Total RNA was extracted by one-step method [Anal. Biochem 1987, 162, 156-159]. The method includes acid guanidine thiocyanate phenol-chloroform extraction. That is, use 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0) to homogenate the tissue, add 1 times the volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49:1 ), mixed and centrifuged. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. 20 µg of RNA was electrophoresed on a 1.2% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transfer to nitrocellulose membrane. ³² P-labeled DNA probes were prepared by the random primer method using α- ³² P dATP. The DNA probe used is the PCR-amplified hexokinase protein 12 coding region sequence (1552bp to 1887bp) shown in FIG. 1 . The 32P-labeled probe (approximately 2×10 ⁶ cpm/ml) was hybridized with the RNA-transferred nitrocellulose membrane at 42°C overnight in a solution containing 50% formamide-25mM KHxPO ₄ (pH7. 4) - 5 x SSC - 5 x Denhardt's solution and 200 μg/ml salmon sperm DNA. After hybridization, the filters were washed in 1×SSC-0.1% SDS at 55° C. for 30 min. Then, analysis and quantification were performed with Phosphor Imager. Example 5: In vitro expression, isolation and purification of recombinant hexokinase protein 12

According to SEQ ID NO:1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:

Primer3:5'-CATGCTAGCATGCAGCATGGTGTTTATGAAGAC-3'(Seq ID No:5)

Primer4:5'-CCCGAATTCCTATTTAGAAATCACAAATGGACA-3'(Seq ID No:6)

The 5' ends of these two primers contain NdeI and EcoRI restriction sites respectively, followed by the coding sequences of the 5' and 3' ends of the target gene respectively, and the NdeI and EcoRI restriction sites correspond to the expression vector plasmid pET-28b( +) Selective endonuclease site on (Novagen product, Cat.No.69865.3). The pBS-0664a04 plasmid containing the full-length target gene was used as a template for PCR reaction. The PCR reaction conditions were as follows: 10 pg of pBS-0664a04 plasmid contained in a total volume of 50 μl, 10 pmol of primers Primer-3 and Primer-4 respectively, and 1 μl of Advantage polymerase Mix (product of Clontech Company). Cycle parameters: 94°C for 20s, 60°C for 30s, 68°C for 2min, a total of 25 cycles. The amplified product and plasmid pET-28(+) were digested with NdeI and EcoRI respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into Escherichia coli DH5α by the calcium chloride method. After culturing overnight on an LB plate containing kanamycin (final concentration 30 μg/ml), positive clones were screened by colony PCR and sequenced. A positive clone (pET-0664a04) with the correct sequence was selected to transform the recombinant plasmid into Escherichia coli BL21(DE3) plySs (product of Novagen) by calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 μg/ml), the host strain BL21 (pET-0664a04) was cultured at 37°C to logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol/L, and the culture was continued for 5 Hour. Bacteria were collected by centrifugation, broken by ultrasonic waves, supernatant was collected by centrifugation, and chromatographically performed with an affinity chromatography column His.Bind Quick Cartridge (product of Novagen Company) capable of binding to 6 histidines (6His-Tag), to obtain The purified target protein hexokinase protein 12 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 12KDa (Figure 2). This band was transferred to PVDF membrane and carried out N-terminal amino acid sequence analysis with Edams hydrolysis method, the result N-terminal 15 amino acid residues and the N-terminal 15 amino acid residues shown in SEQ ID NO:2 are completely identical. Example 6 Production of anti-hexokinase protein 12 antibody

Use a peptide synthesizer (product of PE company) to synthesize the following hexokinase protein 12-specific polypeptide: NH2-Met-Gln-His-Gly-Val-Tyr-Glu-Asp-Leu-Cys-Gly-Leu-Cys- Leu-Asp-COOH

(SEQ ID NO: 7). The polypeptide is respectively coupled with hemocyanin and bovine serum albumin to form a complex. For the method, see: Avrameas, et al. Immunochemistry, 1969; 6:43. Rabbits were immunized with 4 mg of the above-mentioned hemocyanin polypeptide complex plus complete Freund's adjuvant, and then boosted with the hemocyanin polypeptide complex plus incomplete Freund's adjuvant once 15 days later. The titer of antibody in rabbit serum was determined by ELISA using a titer plate coated with 15 μg/ml bovine serum albumin-polypeptide complex. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a Sepharose4B column activated by cyanogen bromide, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. Immunoprecipitation proved that the purified antibody could specifically bind to hexokinase protein 12. Embodiment 7: the application of polynucleotide fragment of the present invention as hybridization probe

Select suitable oligonucleotide fragments from the polynucleotides of the present invention to be used as hybridization probes in many ways, such as using the probes to hybridize with genome or cDNA libraries of normal tissues or pathological tissues from different sources to Identify whether it contains the polynucleotide sequence of the present invention and detect the homologous polynucleotide sequence, and further use the probe to detect the presence of the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology Whether the expression in tissue cells is abnormal.

The purpose of this embodiment is to select suitable oligonucleotide fragments as hybridization probes from the polynucleotide SEQ ID NO:1 of the present invention, and to identify whether some tissues contain polynucleosides of the present invention by filter hybridization method acid sequence or its homologous polynucleotide sequence. Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods, all of which use basically the same steps to hybridize after immobilizing the polynucleotide sample to be tested on the filter membrane. These same steps are: the sample-immobilized filter is first prehybridized with probe-free hybridization buffer, so that the non-specific binding sites of the sample on the filter are saturated with the support and synthetic polymer. The prehybridization solution is then replaced with hybridization buffer containing labeled probes and incubated to allow the probes to hybridize to the target nucleic acid. After the hybridization step, unhybridized probes are removed by a series of washing steps. In this embodiment, higher strength membrane washing conditions (such as lower salt concentration and higher temperature) are used to reduce the background of hybridization and retain only highly specific signals. The probes selected in this embodiment include two classes: the first class probes are completely identical or complementary oligonucleotide fragments to the polynucleotide SEQ ID NO:1 of the present invention; the second class probes are partially identical to the present invention The polynucleotide SEQ ID NO: 1 identical or complementary oligonucleotide fragments. In this embodiment, the dot blot method is used to immobilize the sample on the filter membrane. Under the condition of relatively high intensity washing membrane, the hybridization specificity of the first type of probe to the sample is the strongest and thus retained. 1. Selection of probes

From the polynucleotide SEQ ID NO:1 of the present invention, select oligonucleotide fragments as hybridization probes, the following principles and several aspects to be considered should be followed: 1, the preferred range of probe size is 18-50 nuclei nucleotide; 2, GC content is 30%-70%, if it exceeds, non-specific hybridization will increase; 3, there should be no complementary region inside the probe; 4, those that meet the above conditions can be used as primary probes, and then further computer sequence analysis , including the initial

The selected probes are compared with their source sequence region (ie SEQ ID NO:1) and other known genomic sequences and their complementary regions. If the homology with the non-target molecular region is greater than 85% or more than 15% If two consecutive bases are exactly the same, the primary probe should generally not be used; 5. Whether the primary probe is finally selected as a probe with practical application value should be further determined by experiments.

After completing the analysis of the above aspects, select and synthesize the following two probes:

Probe 1 (probe1), belonging to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO:1 (41Nt)

5'-TGCAGCATGGTGTTTATGAAGACCTTTGTGGACTCTGCTTG-3' (SEQ ID NO: 8)

Probe 2 (probe2), belonging to the second type of probe, is equivalent to the substitution mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:

5'-TGCAGCATGGTGTTTATGAAGATCTTTGTGGACTCTGCTTG-3' (SEQ ID NO: 9)

For other unlisted common reagents and their preparation methods related to the following specific experimental steps, please refer to the literature: DNA PROBES G.H.Keller; Experiment Guide" (Second Edition in 1998) [US] Sam Brook et al., Science Press.

Sample preparation: 1. Extract DNA from fresh or frozen tissue

Steps: 1) Put fresh or freshly thawed normal liver tissue into a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. The tissue should be kept moist during operation. 2) Mince the tissue by centrifugation at 1000 g for 10 minutes. 3) Suspend the precipitate (about 10ml/g) with cold homogenization buffer (0.25mol/L sucrose; 25mmol/L Tris-HCl, pH7.5; 25mmol/L NaCl; 25mmol/L MgCl ₂ ). 4) Homogenize the tissue suspension at 4°C with an electric homogenizer at full speed until the tissue is completely broken. 5) Centrifuge at 1000 g for 10 minutes. 6) Resuspend the cell pellet (add 1-5 ml per 0.1 g of the initial tissue sample), and then centrifuge at 1000 g for 10 minutes. 7) Resuspend the pellet with lysis buffer (add 1 ml per 0.1 g of the initial tissue sample), and then follow the following phenol extraction method. 2. Phenol extraction of DNA

Steps: 1) Wash the cells with 1-10ml of cold PBS, and centrifuge at 1000g for 10 minutes. 2) Resuspend the pelleted cells (1×10 ⁸ cells/ml) with cold cell lysate and apply at least 100ul of lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is directly added to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break and reduce the overall yield. This is especially serious when extracting >10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug/ml. 5) Incubate at 50°C for 1 hour or gently shake overnight at 37°C. 6) Extract with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), and centrifuge in a small centrifuge tube for 10 minutes. The two phases were clearly separated, otherwise the centrifugation was repeated. 7) Transfer the aqueous phase to a new tube. 8) Extract with an equal volume of chloroform:isoamyl alcohol (24:1), and centrifuge for 10 minutes. 9) Transfer the DNA-containing aqueous phase to a new tube. DNA purification and ethanol precipitation were then performed. 3. DNA Purification and Ethanol Precipitation

Steps: 1) Add 1/10 volume of 2mol/L sodium acetate and 2 times volume of cold 100% ethanol to the DNA solution, and mix well. Place at -20°C for 1 hour or up to overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour off the ethanol. 4) Wash the precipitate with 500 ul of 70% cold ethanol, and centrifuge for 5 minutes. 5) Carefully aspirate or pour off the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully suck out or pour off the ethanol, then invert it on absorbent paper to drain the remaining ethanol. Air dry for 10-15 minutes to allow surface ethanol to evaporate. Be careful not to dry the pellet completely as it will be difficult to redissolve. 7) Resuspend the DNA pellet with a small volume of TE or water. Vortex at a low speed or blow with a dropper, while gradually increasing TE, mix until the DNA is fully dissolved, add about 1ul for every 1-5×10 ⁶ cells extracted.

The following steps 8-13 are only used when the pollution must be removed, otherwise step 14 can be performed directly. 8) Add RNase A to the DNA solution with a final concentration of 100ug/ml, and incubate at 37°C for 30 minutes. 9) Add SDS and proteinase K, the final concentrations are 0.5% and 100ug/ml respectively. Incubate at 37°C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase, re-extract with an equal volume of chloroform:isoamyl alcohol (24:1), and centrifuge for 10 minutes. 12) Carefully remove the water phase, add 1/10 volume of 2mol/L sodium acetate and 2.5 volumes of cold ethanol, mix well and place at -20°C for 1 hour. 13) Wash the precipitate with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid, the process is the same as steps 3-6. 14) Measure A ₂₆₀ and A ₂₈₀ to detect the purity and yield of DNA. 15) After aliquoting, store at -20°C. Preparation of sample membranes: 1) Take 4×2 nitrocellulose membranes (NC membranes) of appropriate size, and lightly mark the spotting position and sample number on them with a pencil. Each probe needs two NC membranes, In order to wash the membrane with high-intensity conditions and intensity conditions in subsequent experimental steps. 2) Pipette 15 microliters each of control and control, spot on sample membrane, and dry at room temperature. 3) Place on filter paper soaked with 0.1mol/LNaOH, 1.5mol/LNaCl for 5 minutes (twice), dry and place on filter paper soaked with 0.5mol/L Tris-HCl (pH7.0), 3mol/LNaCl 5 minutes (twice), let dry. 4) Put it in clean filter paper, wrap it with aluminum foil, and dry it under vacuum at 60-80°C for 2 hours.

Probe labeling 1) 3 μl Probe (0.10D/10 μl), add 2 μl Kinase buffer, 8-10 uCi γ- ³² P-dATP+2UKinase, to make up to a final volume of 20 μl. 2) Incubate at 37°C for 2 hours. 3) Add 1/5 volume of bromophenol blue indicator (BPB). 4) Pass through Sephadex G-50 column. 5) Start to collect the first peak before ³² P-Probe elutes (can be monitored by Monitor). 6) 5 drops/tube, collect 10-15 tubes. 7) Monitor the amount of isotopes with a liquid scintillation instrument. 8) The ³² P-Probe (the second peak is free γ- ³² P-dATP) is prepared after combining the collected liquid of the first peak.

prehybridization

Put the sample membrane in a plastic bag, add 3-10mg pre-hybridization solution (10×Denhardt’s; 6×SSC, 0.1mg/ml CT DNA (calf thymus DNA).), seal the bag, shake in a water bath at 68°C for 2 Hour.

hybridize

Cut off a corner of the plastic bag, add the prepared probe, seal the bag, and shake in a water bath at 42°C overnight.

Membrane Washing: High Strength Membrane Washing:

1) Take out the hybridized sample membrane.

2) Wash in 2×SSC, 0.1% SDS, 40°C for 15 minutes (2 times).

3) Wash in 0.1×SSC, 0.1% SDS, 40°C for 15 minutes (2 times).

4) Wash in 0.1×SSC, 0.1% SDS, 55°C for 30 minutes (twice), and dry at room temperature. Low intensity wash:

1) Take out the hybridized sample membrane.

2) Wash in 2×SSC, 0.1% SDS, 37°C for 15 minutes (2 times).

3) Wash in 0.1×SSC, 0.1% SDS, 37°C for 15 minutes (2 times).

4) Wash in 0.1×SSC, 0.1% SDS, 40°C for 15 minutes (twice), and dry at room temperature.

X-ray autoradiography:

-70°C, X-ray autoradiography (the pressing time depends on the radioactivity of the hybridization spots).

Experimental results:

In the hybridization experiment conducted under low-intensity washing conditions, there was no significant difference in the radioactivity of the hybridization spots of the above two probes; while in the hybridization experiment performed under high-intensity washing conditions, the radioactivity of the hybridization spots of probe 1 was significantly stronger than that of The radioactive intensity of another probe hybridizing spot. Probe 1 can thus be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues. Sequence listing (1) General information: (ii) Invention name: Hexokinase protein 12 and its coding sequence (iii) Number of sequences: 9 (2) Information on SEQ ID NO: 1: (i) Sequence features:

(A) Length: 1958bp

(B) type: nucleic acid

(C) chain: double chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(ⅹⅰ)序列描述：SEQ ID NO:1:1 GGAGTTGAACCCTGACTGTATGACTCCAGGGACCTTGTTCTCATTCACACGTCTGCATCT61 CCATGTTATGTTGAGACATTGCATTCCTAGTAGTTTTACATGATCACATGTAGATCATAC121 CATATGTAAATAGAAAAGTATGCTTGTACATTGTCTTTAACAGCAATTATCAGTATTAGAl81 CAAAGCATAGTTTTGTTTCCCTCCCCCACCCCCCGACCCTGTTCCTGTCTCATAGTTTGA241 AGTTTCCCTTAAGTATTCACAGTTATAGTCCCCAAAGACTTTAGATGCCACGTGGTATGC301 GTAAAGCCAGCATATTCTATTGAAATGGCCGGTATAGTTGCCGAATCAGATGTTTTTAGC361 TTACAGAAATGACAATTTCATATGACATTTCTTTTCATATGTTCAAAGAAATTACTTGAG421 CAAAATGGGAGATTTCTGGAGTAGGCCATAGGATCAAAGGAGTTGCTGGAGGGGACAAGA481 GAGCTTTAGTATCCTCAGAGTTTGGAACCTAGGACACATTCTTTTTATTTCCATCTCATA541 CTTTCTTCTCCCTGGATTTTGGCACATACTGCCTGCAGTGGAATCATTGGTGGTTTCCAG601 TATCTTCTTTGACCAGTCCTGCAGTCTGAAGGAAATAACTTCTGTATTCCAGGATTTGTT661 TGAAAAGATCATAGAGAAGATTAGAATTAGCCTGGCTTGGATCACAGCCCTACACTTTTC721 CAGTCACTGGGCAAGAGGCAGTGATTGGCCTGGTACAGGCCATGTGGACAGGGAGGGAGA781 GGTTATTGCTCTGTGAAGGGAGTAAGAGCTGGGAGGGGCAGAGGGGCAATGCAGACACAG841 CTGTTAGAGCGACTGCTGGGATCCAGCAGCTGCTGAAGCCTGTGTCTGAGATTATCAAGT901 CAAGATATCTAAATGGCAGTTAAATCATGAACATAAAAATAATTTTCAAAGTATGCTTAA 961 TTGTTCAGTTTTTTAATTCAGGAGAATTTTTCTCCTCTGCTAATGACAAGGCAGTCTATA1021 TTAGAGACTGTCAAAATTATTTCTTAAGAAGCATTTAAACAAATAATTTTTGTAGACTGC1081 TATATGCATTTCATTAGACAAATGTATATTCTGCTACAAATTCACATAATCATTTTTCCC1141 TCACTCTACTCAGGACTACTTAGATTGTTTGAAAGAAGGCTTTGGCTAATGGGTATTGGA1201 AGGAATGGCTGATTGTTTTAAAATATCCTGTTGCATACAGGATGTATTTTTTGAAGGGTT1261 AAGAATGAAAATAGGAAGCAAGCATGTTCCCTCTGACTGAAATTACTTTCCTTGAAGCCA1321 TTAGTGTGTAAGTCAGATGCTGAACTTACGATATATAGATATTATATAATATGTTACATA1381 TATACTTGAAAATAAAATTCCAAACAGAATAATGACTGTTGCATGGATATATAATGGAGT1441 AATTTACTATGATGAAGTCTGACTTGATAGCACATCAACACAGTAAGCCTCATTTTGAAC1501 AGTTCTGCTGTTTCCTTTGTGAAATGGTAGTTGGAAGTATTGTCCGCGGTTATGCAGCAT1561 GGTGTTTATGAAGACCTTTGTGGACTCTGCTTGGATCCATTTCCAGGAAAAAAAATTTCT1621 CTGCCAGTGAACTTTTATGTTTCTTTTGCCAGAAAATGGAGAAAGATGAATGTTTCAATT1681 TTTTTTTTAAGTGCCAGGATACTTAGAAGCAGCCTGGTTCAGTGCCAAGATGTAGTAATC1741 AGCTTCCTCAGATGTAGTCATTCATTGACGCTGTTTTCTGATTCACCACCAGTGTCTTGC1801 AAGAGCTTTCACTTTATCTGCCTGCAGCCCTGTTTTAACAGTAGTGTTGACACAACTCAC1861 CTGTGTCCATTTGTGATTTCTAAATAGTTTTTAAATCATAAATATTTGAACCTCAAGGTA1921 ACCCTGTAAAGTATGAATAGAGATGACATTGTATTTTT(3) Information on SEQ ID NO:2:

(i) Sequence features:

(A) Length: 111 amino acids

(B) Type: amino acid

(D) Topology: Linear

(ii) Molecular type: polypeptide

(ⅹⅰ) Sequence description: SEQ ID NO:2:

1 Met  Gln  His  Gly  Val  Tyr  Glu  Asp  Leu  Cys  Gly  Leu  Cys  LeuAsp16 Pro  Phe  Pro  Gly  Lys  Lys  Ile  Ser  Leu  Pro  Val  Asn  Phe  Tyr  Val31 Ser  Phe  Ala  Arg  Lys  Trp  Arg  Lys  Met  Asn  Val  Ser  Ile  Phe  Phe46 Leu  Ser  Ala  Arg  Ile Leu arg seer leuuu gln Cys Gln ASP61 Val Val Ile Serite Leu ARG CYS Serou Thr Leu PHE 76 Ser Pro Pro Val Sering His PHE ILE CYS PHLN PRORN SERR Information on Asp Thr Thr His Leu Cys106 Pro Phe Val Ile Ser Lys(4)SEQ ID NO:3

(i) Sequence features

(A) Length: 24 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(ⅹⅰ) Sequence description: SEQ ID NO:3:GGAGTTGAACCCTGACTGTATGAC 24(5) Information on SEQ ID NO:4

(i) Sequence features

(A) Length: 24 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(ⅹⅰ) Sequence description: SEQ ID NO:4:AAAAATACAATGTCATCTCTATTC 24(6) Information on SEQ ID NO:5

(i) Sequence features

(A) Length: 33 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide (ⅹⅰ) Sequence description: SEQ ID NO:5:CATGCTAGCATGCAGCATGGTGTTTATGAAGAC 33

(7) Information on SEQ ID NO:6

(i) Sequence features

(A) Length: 33 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(ⅹⅰ) Sequence description: SEQ ID NO: 6: CCCGAATTCCTATTTAGAAATCACAAATGGACA 33

(8) Information on SEQ ID NO:7:

(i) Sequence features:

(A) Length: 15 amino acids

(B) Type: amino acid

(D) Topology: Linear

(ii) Molecular type: polypeptide

(ⅹⅰ) Sequence description: SEQ ID NO:7:

Met-Gln-His-Gly-Val-Tyr-Glu-Asp-Leu-Cys-Gly-Leu-Cys-Leu-Asp 15

(9) Information on SEQ ID NO:8

(i) Sequence features

(A) Length: 41 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(ⅹⅰ) Sequence description: SEQ ID NO:8:TGCAGCATGGTGTTTATGAAGACCTTTGTGGACTCTGCTTG 41

(10) Information on SEQ ID NO:9

(i) Sequence features

(A) Length: 41 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(ⅹⅰ) Sequence description: SEQ ID NO:9:TGCAGCATGGTGTTTATGAAGATCTTTGTGGACTCTGCTTG 41

Claims (18)

1. An isolated polypeptide-hexokinase protein 12, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment, analog or derivative thereof. 2. The polypeptide according to claim 1, characterized in that the amino acid sequence of said polypeptide, analog or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO:2. 3. The polypeptide according to claim 2, characterized in that it comprises a polypeptide having the amino acid sequence shown in SEQ ID NO:2. 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof; (b) a polynucleotide complementary to polynucleotide (a); or (c) A polynucleotide having at least 70% identity to (a) or (b). 5. The polynucleotide according to claim 4, characterized in that said polynucleotide comprises a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:2. 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 1552-1887 in SEQ ID NO: 1 or the sequence of positions 1-1958 in SEQ ID NO: 1 . 7. A recombinant vector containing an exogenous polynucleotide, characterized in that it is a recombinant vector constructed from the polynucleotide of any one of claims 4-6 and a plasmid, virus or carrier expression vector carrier. 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from the following host cells: (a) a host cell transformed or transduced with the recombinant vector according to claim 7; or (b) A host cell transformed or transduced with the polynucleotide according to any one of claims 4-6. 9. A method for preparing a polypeptide having hexokinase protein 12 activity, characterized in that the method comprises: (a) cultivating the engineered host cell according to claim 8 under the condition of expressing hexokinase protein 12; (b) Isolating a polypeptide having hexokinase protein 12 activity from the culture. 10. An antibody capable of binding to a polypeptide, characterized in that the antibody is capable of specifically binding to hexokinase protein-12. 11. A class of compounds that mimic or modulate the activity or expression of polypeptides, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of hexokinase protein-12. 12. The compound according to claim 11, characterized in that it is the antisense sequence of the polynucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof. 13. The application of the compound according to claim 11, characterized in that the compound is used in the method of regulating the activity of hexokinase protein 12 in vivo and in vitro. 14. A method for detecting a disease or disease susceptibility associated with the polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide , or detect the nucleotide variation in the polynucleotide that causes abnormal expression or activity of the polypeptide. 15. The use of the polypeptide according to any one of claims 1-3, characterized in that it is used to screen for mimics, agonists, antagonists or inhibitors of hexokinase protein 12; or for peptide fingerprinting Spectrum identification. 16. The application of the nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for nucleic acid amplification reactions, or as a probe for hybridization reactions, or for making gene chips or microarray. 17. The application of the polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or its mimetic, agonist, antagonist Or the safe and effective dose of the inhibitor and a pharmaceutically acceptable carrier can be used as a pharmaceutical composition for diagnosing or treating diseases related to hexokinase protein 12 abnormality. 18. The application of the polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or compound is used to prepare for the treatment of malignant tumors, blood Diseases, HIV infection and immune diseases and various types of inflammation drugs.

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