WO2001090352A1 - A novel polypeptide, a centrosome nek-2 binding protein 110.12 and the polynucleotide encoding the polypeptide - Google Patents

Abstract

The present invention discloses a novel polypeptide, a centrosome Nek-2 binding protein 110.12, the polynucleotide encoding the polypeptide and the method for producing the polypeptide by DNA recombinant technology. The invention also discloses the uses of the polypeptide in methods for treating various diseases, such as malignant tumour, hemopathy, development disorder, HIV infection, immunological disease, and various inflammations, etc. The invention also discloses the agonists against the polypeptide and the therapeutic action thereof. The invention also discloses the uses of the polynucleotide encoding the novel centrosome Nek-2 binding protein 110.12.

Description

A new polypeptide-centrosome Nek-2 binding protein 110. 12 and a polynucleotide encoding the polypeptide Technical field

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide—the central body Nek-2 binding protein 110.12, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.

# 术 背景

 Centrosome has received attention over 100 years ago, and the regulation of centrosome structure and function is closely related to the cell cycle (Mazia, 1987; Kel logg et al., 1994; Tournier and Bornens,

1994), many centrosome-related components have been obtained. Among them, Nek2 is one of them. It belongs to mammalian cell cycle regulating kinases and is structurally similar to the mitotic regulator NIMA of Aspergillus nidulans (Fry and Nigg, 1995; Lu and Hunter, 1995a; Osmani and Ye,

1996). Centrosome Nek-2 binding protein 1 (centrosoma l Nek2-as sociated protein 1 (C-Napl)) is a human centrosome protein whose carboxy terminus can interact with Nek2, so it was originally called Nek2 interacting protein. It is a core component of mammalian centrosomes.

 C-Napl is a high-molecular-weight (281Kd) coiled-coil protein. Its structure contains a long coiled-coil region with a hinge region in the center of the region. Pro-residue clusters that break the helical structure were found only near the amino and carboxyl terminal and central regions of C-Napl, but did not affect the coiled helix throughout the structure. Some other centrosome proteins were found to contain similar coiled-coil regions (Stearn and Winey, 1997), indicating that protein interactions cannot be separated from this region, and the maintenance of centrosome structure depends on this. Similar to Nek2, C-Napl can also be combined with the centrosome independently without relying on the existence of microtubules, so it also belongs to the core component of the centrosome (Oegema et al.,

1995).

 In human cells, Nek2 can be combined with centrosomes, and it plays a role in the chromatin concentration stage in meiosis (Rhee and Wolgemuth, 1997). However, the overexpression of Nek2 is not conducive to chromatin concentration. Instead, it affects the adhesion between centrioles and can cause obvious centrosome division. C-Napl is a phosphorylated substrate of Nek2, and its carboxyl terminus can be phosphorylated by Nek2 in vitro and in living cells.

 Nek2 expression is tissue-specific and can be expressed in large amounts in the testis, showing a specific expression at the spermatogenesis stage (Rhee and Wolgemuth, 1997; Tanaka et al.,

1997). However, the expression of C-Napl is widespread, and it is found in all tissue cells. When the expression is included in the testis, the expression level is very low, even only a few copies. Gene chip analysis revealed that in the bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibrobla st, growth factor stimulation, 1 024NT, scar-like fc growth factor stimulation, 1013HT, scar Into fc without stimulation with growth factors, 1 013HC, bladder cancer construct cells EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell lines, placenta, spleen, prostate cancer, jejunal adenocarcinoma, and cardia cancer, the present invention The expression profile of the peptide is very similar to that of the centrosome Nek-2 binding protein 1, so the functions of the two may be similar. 12。 The present invention is named centrosome Nek-2 binding protein 110.12.

 As described above, the centrosome Nek-2 binding protein 110.12 protein plays an important role in regulating important functions of the body, such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes. Therefore, the identification of The centrosome Nek-2 binding protein 1 10.12 protein that is involved in many of these processes, especially the amino acid sequence of this protein is identified. The new centrosome Nek-2 binding protein 11 0. 12 The isolation of the protein-coding genes also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for the disease, so it is important to isolate its code for DM. Object of the invention

 It is an object of the present invention to provide an isolated novel polypeptide, the centrosome Nek-2 binding protein 11 0.12, and fragments, analogs and derivatives thereof.

 Another object of the 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 a centrosome Nek-2 binding protein 110.12.

 Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a centrosome Nek-2 binding protein 110.12.

 Another object of the present invention is to provide a method for producing centrosome Nek-2 binding protein 110.12. Another object of the present invention is to provide an antibody against the centrosome Nek-2 binding protein 11 0.12 of the polypeptide of the present invention.

 Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, centrosome Nek-2 binding protein 110.12.

Another object of the present invention is to provide a method for diagnosing and treating diseases associated with centrosome Nek-2 binding protein 110. 12 abnormalities. Summary of invention

 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 a conservative variant, biologically active fragment or derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

 The invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a 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 the polynucleotide (a);

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

 More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 974 to 1252 in SEQ ID NO: 1; and (b) a sequence having 1-1501 in SEQ ID NO: 1 Sequence of bits.

 The present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.

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

 The present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit centrosome Nek-2 binding protein 110. 12 protein activity, which comprises utilizing the polypeptide of the present invention. The invention also relates to compounds obtained by this method.

 The present invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of centrosome Nek-2 binding protein 110. 12 protein, comprising detecting mutations in the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.

 The invention also relates to a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an 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 a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of centrosome Nek-2 binding protein 110. 12 .

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. '' BRIEF DESCRIPTION

The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims. FIG. 1 is a comparison diagram of gene chip expression profiles of centrosome Nek-2 binding protein 110. 12 and centrosome Nek-2 binding protein 1 of the present invention. The top graph is a centroid Nek-2 binding protein 110. 12 expression profile fold graph, and the bottom graph is a centrosome Nek-2 binding protein 1 expression profile histogram. Among them, 1-bladder mucosa, 2- PMA + Ecv304 cell line, 3- LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5- Fibroblas t, growth factor stimulation, 1024NT, 6- scar into fc growth factor Stimulation, 1013HT, 7-scar scar into fc without stimulation with growth factors, 1013HC, 8-bladder cancer cell EJ, 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12- liver cancer cell line, 13-fetus Skin, 14-spleen, 15-prostate cancer, 16-jejunum adenocarcinoma, 17 cardia cancer.

 Figure 2 is the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated centrosome Nek-2 binding protein 110.12. OkDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Summary of the Invention

 The following terms used in this specification and the claims shall have the following meanings unless specifically stated: "Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

 A "variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine. Variants can 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 nucleotide sequence.

 "Insertion" or "addition" means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

 "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.

"Agonist" means that when bound to the centrosome Nek-2 binding protein 110.12, an A molecule that alters the protein to regulate its activity. An agonist may include a protein, a nucleic acid, a carbohydrate or any other molecule that can bind to the centrosome Nek-2 binding protein 110.12.

 "Antagonist" or "inhibitor" refers to a biological or immunological activity that can block or regulate the centrosome Nek-2 binding protein 110.12 when bound to the centrosome Nek-2 binding protein 110.12. Molecule. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind the centrosome Nek-2 binding protein 110.12.

 "Regulation" refers to changes in the function of centrosome Nek-2 binding protein 110.12, including an increase or decrease in protein activity, changes in binding characteristics, and any other biological properties of centrosome Nek-2 binding protein 110.12 , Functional or immune properties.

 "Substantially pure" means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify centrosome Nek-2 binding protein using standard protein purification techniques.

110. 12. The substantially pure centrosome Nek-2 binding protein 110. 12 produces a single main band on a non-reducing polyacrylamide gel. Centrosome Nek-2 binding protein 110. The purity of the polypeptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable 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 may be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.

"Percent identity" refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi ggins, DG and PM Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The 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: Number of residues matching sequence ^

 Number of residues in sequence ^-number of spaced residues in sequence ^-number of spaced residues in sequence s X

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

"Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitution, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids 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 particular DM or RM sequence. "Antisense strand" refers to a nucleic acid strand that is complementary to a "sense strand."

 "Derivative" refers to HFP or a chemical modification of its nucleic acid. This 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 the main biological properties of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') ₂ and? , It can specifically bind to the centrosome Nek-2 binding protein 110. 12 epitope.

 A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is 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 (for example, its natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.

 As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .

As used herein, "isolated centrosome Nek-2 binding protein 110. 12" means that centrosome Nek-2 binding protein 110. 12 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. 12。 Persons skilled in the art can use standard protein purification techniques to purify centrosome Nek-2 binding protein 110.12. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Center The purity of somatic Nek-2 binding protein 110. 12 polypeptide can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, centrosome Nek-2 binding protein 110. 12, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. The polypeptides of the invention may also include or exclude the initial methionine residue.

 The invention also includes fragments, derivatives and analogs of centrosome Nek-2 binding protein 110.12. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the centrosome Nek-2 binding protein 110. 12 of the present invention with the same biological function or activity. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (Π) such a type in which one or more amino acid residues are substituted with other groups to include a substituent; or (III) such One, in which 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) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences). As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.

 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 1501 bases, and its open reading frame 974-1252 encodes 92 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile to the centrosome Nek-2 binding protein 1, and it can be inferred that the centrosome Nek-2 binding protein 110. 12 has a similarity to the centrosome Nek-2 binding protein 1. Features.

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DM. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding a mature polypeptide may be the same as the coding region sequence 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 coding region that encodes a protein or polypeptide having SEQ ID NO: 2 but is identical to the coding region shown in SEQ ID NO: 1 Sequences with different nucleic acid sequences.

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

 The term "polynucleotide encoding a polypeptide" refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.

 The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide can 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 that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

 The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 ° / »Fi col 1, 42 ° C, etc .; or (3) only in two sequences Crosses occur only when the identity between them is at least 95%, and more preferably 97%. In addition, 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, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 nucleotides. Nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding centrosome Nek-2 binding protein 110.12.

 The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. The specific polynucleotide sequence encoding the centrosome Nek-2 binding protein 110. 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 polynucleosides with common structural characteristics Acid fragments.

The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DM sequence from genomic DM; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide. Of the methods mentioned above, genomic DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the separation of the CDM sequences. The standard method for isolating the CDM of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cMA library. There are many mature techniques for fflRM extraction, and kits are also commercially available (Qiagene). And cMA library construction is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spring Harbor Laboratory. New York, 1989) ₀ Commercially available cDNA libraries, such as different cDNAs from Clontech library. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

These genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DM or DNA-RM hybridization; ( ² ) the presence or loss of marker gene function; (3) determination of the centrosome Nek-2 binding protein 110. 12 transcript Level; (4) detecting protein products of gene expression by immunological techniques or measuring biological activity. The above methods can be used singly or in combination.

 In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and has a length of at least 10 nucleotides, preferably at least 3G 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 probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the (4) method, the protein product expressed by the centrosome Nek-2 binding protein 110. 12 gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).

 A method (Sa iki, et al. Science 1985; 230: 1350-1354) for amplifying DM / RNA by PCR is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-Rapid Amplification of cDNA Ends) can be preferably used. The primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

The polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length CDM 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 a centrosome Nek-2 binding protein 110. 12 coding sequence, and the present invention is produced by recombinant technology Methods of the polypeptide.

 In the present invention, a polynucleotide sequence encoding a centrosome Nek-2 binding protein 110. 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 plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio C em. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.

 Methods known to those skilled in the art can be used to construct expression vectors containing DM sequences encoding centrosome Nek-2 binding protein 110. 12 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombination DM technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Labora tory Manual, Cold Spirit Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Retroviral LTRs and other known promoters that 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 and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.

 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, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes. In the present invention, a polynucleotide encoding a centrosome Nek-2 binding protein 110. 12 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector. Host cell. 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: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf9; animal cells such as CH0, COS or Bowes melanoma cells.

Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DM can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Alternatively, MgCl ₂ is used. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

 By conventional recombinant DM technology, the polynucleotide sequence of the present invention can be used to express or produce a recombinant centrosome Nek-2 binding protein 110. 12 (Science, 1984; 224: 1431). Generally, the following steps are taken:

 (1) using a polynucleotide (or variant) encoding the human centrosome Nek-2 binding protein 110. 12 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing host cells in a suitable medium;

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

 In step (2), depending on the host cell used, the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted into a fine extracellular. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, 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 polypeptides of the present invention and the antagonists, agonists and inhibitors of the polypeptides can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and Immune diseases, etc.

 The regulation of centrosome structure and function in mammalian cells is closely related to the cell cycle. Nek2 protein is one of the centrosome-related components, and it belongs to mammalian cell cycle regulating kinases. Centrosome Nek-2 Binding Protein 1 (C-Napl) is a human centrosome protein. It can interact with Nek2. Similar to Nek2, C-Napl can also independently bind to centrosome without relying on it. The existence of microtubules is a core component of mammalian centrosomes.

 Studies have shown that Nek2 can bind to centrosomes in human cells, and it plays a role in the concentration of chromatin during meiosis. However, the overexpression of Nek2 is not conducive to the concentration of chromatin, but instead affects the centrosome. Adhesion between them can cause obvious centrosome division.

 The expression profile of the polypeptide of the present invention is consistent with the expression profile of human centrosome Nek-2 binding protein 1, and the two have similar biological functions. The polypeptide of the present invention has a regulating effect on the cell cycle during mitosis and meiosis, and its effect does not depend on the existence of microtubules. Its abnormal expression is usually closely related to the abnormal proliferation and division of cells, and produces related diseases. .

 It can be seen that the abnormal expression of the central body Nek-2 binding protein 110. 12 of the present invention will produce various diseases, especially various tumors, embryonic development disorders, growth disorders, inflammation, and immune diseases. These diseases including but not limited to:

 Tumors of various tissues: stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma

 Fetal developmental disorders: congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness

 Growth and development disorders: mental retardation, brain development disorders, skin, fat and muscular dysplasia, bone and joint dysplasia, various metabolic deficiencies, stunting, dwarfism, Cushing syndrome, Sexual retardation

 Inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations

 Immune diseases: Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome

The abnormal expression of the centrosome Nek-2 binding protein 110.12 of the present invention will also generate certain heredity, Blood diseases.

 The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially various tumors, embryonic development disorders, growth and development disorders, inflammation, and immunity. Sexual diseases, certain hereditary, blood diseases, etc.

 The invention also provides screening compounds to identify elevating (agonist) or repressing (antagonist) centrosomes

Nek-2 binding protein 110. 12 method of medicament. Agonists enhance centrosome Nek-2 binding protein 110. 12 Stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, a mammalian cell or a membrane preparation expressing the centrosome Nek-2 binding protein 110. 12 and a labeled centrosome Nek-2 binding protein 110. 12 can be cultured together in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of centrosome Nek-2 binding protein 110. 12 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of centrosome Nek-2 binding protein 110.12 can bind to and eliminate its function with centrosome Nek-2 binding protein 110.12, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide such that The polypeptide cannot perform biological functions.

 When screening compounds as antagonists, centrosome Nek-2 binding protein 110. 12 can be added to the bioanalytical assay, and the interaction between the centrosome Nek-2 binding protein 110. 12 and its receptor can be determined by determining the compound Influence to determine if a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Peptide molecules capable of binding to centrosome Nek-2 binding protein 110. 12 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, centrosome Nek-2 binding protein 110. 12 molecules should generally be labeled.

 The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against centrosome Nek-2 binding protein 110. 12 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

Polyclonal antibodies can be produced by centrosome Nek-2 binding protein 110. 12 by direct injection in immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc. Techniques for preparing centrosome Nek-2 binding protein 110. 12 monoclonal antibodies include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cells Hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morr i son et al, PNAS, 1985, 81: 6851). The unique technology for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against the centrosome Nek-2 binding protein 110.12.

 Antibodies against centrosome Nek-2 binding protein 110. 12 can be used in immunohistochemistry techniques to detect centrosome Nek-2 binding protein 11 0.12 in biopsy specimens.

 Monoclonal antibodies that bind to the centrosome Nek-2 binding protein 110. 12 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.

 Antibodies can also be used to design immunotoxins that target a particular part of the body. Such as centrosome Nek-2 binding protein 110. 12 High affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol crosslinker such as SPDP, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill the centrosome Nek-2 binding protein 110. 12 positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases related to centrosome Nek-2 binding protein 110.12. Administration of appropriate doses of antibodies can stimulate or block the production or activity of centrosome Nek-2 binding protein 110.12.

 The invention also relates to a diagnostic test method for quantitatively and locally detecting centrosome Nek-2 binding protein 110. 12 level. These tests are well known in the art and include FISH assays and radioimmunoassays. Centrosome-binding protein detected in the test: Π 0.12 level, which can be used to explain centrosome Neit-2 binding protein 110. 12's importance in various diseases and to diagnose centrosome Nek-2 binding protein 110 12 working diseases.

 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.

The polynucleotide encoding centrosome Nek-2 binding protein 110. 12 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell proliferation, development, or metabolism caused by the absence or abnormal / inactive expression of centrosome Nek-2 binding protein 110.12. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated centrosome Nek-2 binding protein 110. 12 to inhibit endogenous centrosome Nek-2 binding protein 110. 12 activity. For example, a variant centrosome Nek-2 binding protein 110. 12 may be a shortened centrosome Nek-2 binding protein 110. 12 that lacks a signaling domain, although it can bind to downstream substrates, but lacks Signaling activity. Therefore, the recombinant gene therapy vector can be used for the treatment of diseases caused by abnormal expression or activity of centrosome Nek-2 binding protein 110.12. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex disease Viruses, parvoviruses, etc. can be used to transfer a polynucleotide encoding centrosome Nek-2 binding protein 110. 12 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a centrosome Nek-2 binding protein 110.12 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding a centrosome Nek-2 binding protein 110.12 can be packaged into liposomes and transferred into cells.

 Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.

 Oligonucleotides (including antisense RM and DM) and ribozymes that inhibit centrosome Nek-2 binding protein 110. 12 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RM molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RM or DNA synthesis technology. For example, solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides has been widely used. Antisense RM molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.

 The polynucleotide encoding centrosome Nek-2 binding protein 110. 12 can be used for diagnosis of diseases related to centrosome Nek-2 binding protein 110. 12. The polynucleotide encoding centrosome Nek-2 binding protein 110. 12 can be used to detect the expression of centrosome Nek-2 binding protein 110. 12 or the abnormal expression of centrosome Nek-2 binding protein 110. 12 in a disease state. . For example, the DNA sequence encoding centrosome Nek-2 binding protein 110. 12 can be used to hybridize biopsy specimens to determine the expression of centrosome Nek-2 binding protein 110. 12. Hybridization techniques include Southe: rn blotting, Nor thern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available. A part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DM chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. Centrosome Nek-2 binding protein 110. 12 specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect centrosome Nek-2 binding protein 110. 12 transcription products.

Detecting centrosome Nek-2 binding protein 110. 12 mutations can also be used to diagnose centrosome Nek-2 binding protein 110. 12-related diseases. The centrosome Nek-2 binding protein 110. 12 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type centrosome Nek-2 binding protein 110. 12 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so use Northern blotting and Wes tern blotting can indirectly determine whether a gene is mutated.

 The sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome 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 a chromosome.

 In short, PCR primers (preferably 15-35bp) are prepared based on cMA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.

 PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, sublocalization can be achieved by a similar method using a set of fragments from a specific chromosome or a large number of genomic clones. 'Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct a chromosome-specific cDM library.

 Fluorescent in situ hybridization (FISH) of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Pres s, New York (1988).

 Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in V. Mckusick, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.

 Next, the difference in cDM or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution Capacity and each 20kb corresponds to a gene).

The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition contains a safe and effective amount of the polypeptide or antagonist and does not affect Pharmaceutically effective carriers and excipients. These compositions can be used as drugs for the treatment of diseases. The present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders authorize them to be administered to humans by government agencies that manufacture, use, or sell them. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Centrosome Nek-2 binding protein 110. 12 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of centrosome Nek-2 binding protein 110. 12 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples

 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Col Harbor Harbor Laboratory Pres s, 1989), or Follow the conditions recommended by the manufacturer.

 Example 1 Cloning of centrosome Nek-2 binding protein 110.12

Human fetal brain total MA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RM using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA forms CDM by reverse transcription. Using the Smart cDM cloning kit (purchased from Clontech), the 00 fragment was inserted into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5a, and the bacteria formed a CDM library. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the CDM sequence of one of the clones 0541H12 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results show that the 0541H12 clone contains a full-length cDNA of 1501bp (as shown in Seq ID N0: l), and has a 278bp open reading frame (0RF) from 974bp to 1252bp, encoding a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-0541H12, and the encoded protein was named centrosome Nek-2 binding protein 110.12. Example 2 The gene encoding centrosome Nek-2 binding protein 110. 12 was cloned by RT-PCR method. The total RM of fetal brain cells was used as a template, and oligo-dT was used as a primer for reverse transcription reaction to synthesize cDNA. A Qiagene kit was used. After purification, PCR amplification was performed with the following primers:

 Pr imer 1: 5'- GGCAGTTTACAAAGGTAAGCCATT -3 '(SEQ ID NO: 3)

 Pr imer2: 5,-GATAATAGAAATTAGAAATAACAA -3, (SEQ ID NO: 4)

 Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;

 Pr imer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.

Conditions for the amplification reaction: 50 mmol / L KCl, 1 (kimol / L Tri s-HCl pH 8. 5, 1. 5 mmol / L MgCl ₂ , 2 (mol / L dNTP, lOpmol primer, 1U) Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94.C 30sec; 55.C 30sec; 72 ° C 2min. At RT Β-act in was set as positive control and template blank as negative control at the same time. Amplification products were purified with QIAGEN kit, and TA clone kit was used to connect to pCR vector (Invi trogen company). DNA sequence analysis The results showed that the DNA sequence of the PCR product was exactly the same as that of 1-1501bp shown in SEQ ID NO: 1. Example 3 Northern blot analysis of central body Nek-2 binding protein 110. 12 gene expression The total RM was extracted in one step [Anal Biochera 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, 4M guanidinium isothiocyanate-25raM sodium citrate, 0.2M sodium acetate (pH4.0). The tissue was homogenized, and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) were added. After mixing, centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 volume) and centrifuge the mixture to obtain RM precipitate. Wash the resulting RNA precipitate with 70% ethanol, dry and dissolve in water. Use 2 (^ g RNA , Perform electrophoresis on a 1.2% agarose gel containing 20mM 3- (N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2. 2M formaldehyde. Then transfer to nitric acid cellulose membrane with cc -. ³² P dATP labeled probe apparatus ³² P- DM legal by random primer DNA probes used for PCR amplification shown in FIG central body Nek-2 binding protein 110.12. Sequence of coding region (974bp to 1252bp). 32P-labeled probe (approximately 2 x 10 ⁶ cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred in a solution at 42 ° C overnight, the solution containing 50 % Formamide-25mM KH ₂ P0 ₄ (pH7. 4)-5 x SSC-5 x Denhardt, s solution and 20 (^ g / ml salmon sperm DNA. After hybridization, the filter was placed at 1 X SSC-0. 1 Wash in% SDS for 30 min at 55 ° C. Then, Phosphor Imager was used for analysis and quantification. Example 4 In vitro expression, isolation and purification of the recombinant centrosome Nek-2 binding protein 110. 12 According to the sequence of the coding region shown in SEQ ID NO: 1 and FIG. 1, a pair of specific amplification primers were designed, the sequence is as follows: Pr imer3: 5'-CCCCATATGATGTCTGTGTTTCAGATAAATAAT-3 '(Seq ID No: 5) Pr imer4: 5' -C ATGGATCCTTATATT AAAA AGAT AA AGAT A- 3 '(Seq ID No: 6) Ndel and BamHI restriction sites, followed by the coding sequences of the 5 'and 3' ends of the target gene, respectively. The Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) a selective endonuclease site. The pBS-0541H12 plasmid containing the full-length target gene was used as a template for the PCR reaction. The PCR reaction conditions were as follows: a total volume of 50 μ1 containing 10 pg of pBS-0541H12 plasmid, Primer-3 and Primer-4 were 1 Opmol, and Advantage polymerase Mix (Clontech) 1 μ1, respectively. Cycle parameters: 94. C 20s, 60. C 30s, 68. C 2 min, a total of 25 cycles. Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E. coli DH5a using the calcium chloride method. After being cultured on an LB plate containing kanamycin (final concentration 30 μ _§ / ιη1) overnight, positive clones were selected by colony PCR method and sequenced. A positive clone (PET-0541H12) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 3 (^ g / ml)), the host strain BL21 (pET-0541H12) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of lramol / L. Continue to cultivate for 5 hours. Centrifuge to collect the bacterial cells, decompose by ultrasound, collect the supernatant by centrifugation, and use an affinity chromatography column His. Bind Quick Cartr idge (Novagen) Product) was chromatographed to obtain the purified protein protein Nek-2 binding protein 110. 12. After SDS-PAGE electrophoresis, a single band was obtained at 10 OkDa (Figure 2). The band was transferred to Analysis of the N-terminal amino acid sequence on the PVDF membrane by the Edams hydrolysis method showed that the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 5 Anticentrosome Nek- 2 binding protein 110. 12 antibody production

 The following centrosome Nek-2 binding protein 110. 12 specific

NH2-Met-Ser-Val-Phe-Gln-I le-Asn-Asn-Leu-Asn-Asp-Asn-Lys-Tyr-Gly-C00H (SEQ ID NO: 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. Immunoprecipitation proves that purified antibodies can specifically bind to centrosome Nek-2 binding protein 110. 12 combined. Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe

 Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect 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 a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained. The probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention The polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment. In this example, the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.

 First, the selection of the probe

 The selection of oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:

 1. The preferred range of probe size is 18-50 nucleotides;

 2.The GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;

 3. There should be no complementary regions inside the probe;

4. Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used; 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments. After completing the above analysis, select and synthesize the following two probes:

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

 5-TGTCTGTGTTTCAGATAAATAATTTGAATGACAACAAATAT-3 '(SEQ ID NO: 8) Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:

 5'-TGTCTGTGTTTCAGATAAATCATTTGAATGACAACAAATAT-3 '(SEQ ID NO: 9) For other commonly used reagents and their preparation methods not related to the following specific experimental procedures, please refer to the literature: DM PROBES GHKeller; MMManak; Stockton Press, 1989 (USA ) And more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide" (Second Edition 1998) [US] Sambrook et al., Science Press.

 Sample Preparation:

 1.Extract DNA from fresh or frozen tissue

Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the precipitate (about 10 ml / g) with a cold homogenization buffer (0.25 mol / L sucrose; 25 mmol / L Tris-HCl, pH 7.5; 25 cryptool / L NaCl; 25 mmol / L MgCl ₂ ). 4) at 4. C Use an electric homogenizer to homogenize the tissue suspension at full speed until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (add 1-5 ml per 0.1 g of the original tissue sample), and centrifuge at 1000 _g for 10 minutes. 7) Resuspend the pellet in lysis buffer (1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.

 2, MA phenol extraction method

Steps: 1) Wash cells with 1-10 ml of cold PBS and centrifuge at 1000g for 10 minutes. 2) Resuspend the pelleted cells with cold cell lysate (lx10 ⁸ cells / ml). Use a minimum of 100ul 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 particularly 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 shake gently at 37 ° C overnight. 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 should be clearly separated, otherwise centrifuge again. 7) Transfer the water 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 aqueous phase containing DM to a new tube. Purification of DM and ethanol precipitation were then performed. 3.Purification and ethanol precipitation of DM

Steps: 1) Add 180 vols of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DNA, 1- 5χ10 ⁶ cells per extracted plus about lul.

 The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.

8) Add RMase A to the DNA solution to a final concentration of 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K to the final concentration of 0.5% and 100ug / nil, 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 and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 12) Carefully remove the aqueous phase, add 1/10 volume of 2mol / L sodium acetate and 2.5 volumes of cold ethanol, and mix well--20 ° C for 1 hour. 13) Wash the pellet 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 ₂₆ . And A ₂₈ . To check the purity and yield of DM. 15) Store at -20 ° C after dispensing.

 Preparation of sample film:

 1) Take 4x2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are required for each probe, so that they can be used in the following experimental steps. The film was washed with high-strength conditions and strength conditions, respectively.

 2) Pipette and control 15 microliters each, spot on the sample film, and dry at room temperature.

 3) Place on filter paper infiltrated with 0.1 mol / L NaOH, 1.5raol / L NaCl for 5 minutes (twice), dry and place in 0.5raol / L Tris-HCl (pH7.0), 3mol / L NaCl filter paper for 5 minutes (twice) and allowed to dry.

 4) Clamped in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours.

 Labeling of probes

1) 3μ1 Probe (0.1OD / Ιθμΐ), add 2μ1 Kinase buffer, 8-10 uCi γ- ³² P-dATP + 2U Kinase, to make up to a final volume of 20μ1.

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of Bromophenol Blue Indicator (BPB).

4) Pass through a Sephadex G-50 column. 5) Start collecting the first peak before ³² P-Probe washes out (moni tor can be used for monitoring;).

 6) 5 drops / tube, collect 10-15 tubes.

 7) Monitor the amount of isotope with a liquid scintillator.

8) were collected after the first peak were combined to ³² P- Probe (second peak to prepare the desired free γ- ³² P - dATP) ₀

 Pre-hybridization

 The sample membrane was placed in a plastic bag, and 3-1 Omg pre-hybridization solution (OxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After sealing the bag, shake at 68 ° C for 2 hours.

 Cross

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

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0. Γ / oSDS, 40. C wash for 15 minutes (twice).

 3) Wash in 0.1xSSC, 0.1% SDS at 40 ° C for 15 minutes (twice).

 4) Wash in 0.1xSSC, 0.1% SDS at 55 ° C for 30 minutes (twice), and dry at room temperature. Low-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

 3) 0.1xSSC, 0.1% SDS, 37. C Wash for 15 minutes (twice).

 4) 0.1xSSC, 0.1% SDS, 40. Wash for 15 minutes (twice) and dry at room temperature.

X-ray autoradiography:

 -70 ° C, X-ray autoradiography (compression time depends on the radioactivity of the hybrid spot).

 Experimental results:

The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probes. However, in the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger. To the radioactive intensity of the hybridization spot of another probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues. Example 7 DNA Microarray

 Gene chip or gene microarray (DM Microarray) is a new technology currently being developed by many national laboratories and large pharmaceutical companies. The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature. For example, see DeRis i, JL, Lyer, V. & Brown, PO (1997) Science 278, 680-686. And He lie, RA, Schema, M. , Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.

 (A) spotting

 A total of 4,000 polynucleotide sequences of various full-length cDNAs are used as the target DM, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500 ng / ul after purification, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 μηι. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:

 1. Hydration in a humid environment for 4 hours;

 2. 0.2% SDS was washed for 1 minute;

 3. dd 0 wash twice, 1 minute each time;

4. NaBH _{4 is} blocked for 5 minutes;

 5. 95 ° C water for 2 minutes;

 6. Wash with 0.2% SDS for 1 minute;

7. Rinse twice with ddH ₂ 0;

 8. Dry and store at 25 ° C in the dark for future use.

 (Two) probe marking

Total mRM was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified using Ol igotex mRNA Midi Kit (purchased from QiaGen), and separated by reverse transcription! ] The fluorescent test was also Cy3dUTP (5- Amino-propargyl-2 '-deoxyuridine 5'-triphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) to label mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5 -Amino-propargyl- 2'- deoxyuridine 5'-triphate coupled to Cy5 f luorescent dye, purchased from Amersham Phamacia Biotech The company) labeled the body's specific tissues (or stimulated cell lines) with mRM, and purified them to prepare probes. For specific steps and methods, see:

 Schena, M., Shalon, D., Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614-10619. Schena, M., Shalon, Dari., Davis, RW (1995 ) Science.270 (20): 467-480.

 (Three) cross

 The probes from the above two tissues were hybridized with the chip in a UniHyb ™ Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (lx SSC, 0.2 ° / oSDS) at room temperature and then scanned with ScanArray 3000. The scanner (purchased from General Scanning Company, USA) was used for scanning. The scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.

 The above specific tissues (or stimulated cell lines) are bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scar-fc growth factor stimulation 1013HT, scar into fc without stimulation with growth factors, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, cardia cancer. Based on these 17 Cy3 / Cy5 ratios, a histogram is drawn (Figure 1). It can be seen from the figure that the centrosome Nek-2 binding protein 110.12 and the centrosome Nek-2 binding protein 1 according to the present invention have very similar expression profiles.

Claims

Rights request

1. An isolated polypeptide-centrosome Nek-2 binding protein 110. 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 Thing.

 2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the 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, further comprising 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 that is at least 70% identical to (a) or (b).

 5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an 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 a sequence of positions 974-1252 in SEQ ID NO: 1 or a sequence of positions 1-1501 in SEQ ID NO: 1. .

 7. A recombination vector containing an exogenous polynucleotide, characterized in that it is a recombination constructed by the polynucleotide according to any one of claims 4-6 and a plasmid, virus or a carrier expression vector Carrier.

 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:

 (a) a host cell transformed or transduced with the recombinant vector of claim 7; or

 (b) A host cell transformed or transduced with any of the polynucleotides of claim 4-6.

 9. A method for preparing a polypeptide having centrosome Nek-2 binding protein 110.12 activity, characterized in that the method comprises:

 (a) culturing the engineered host cell of claim 8 under the condition of expressing centrosome Nek-2 binding protein 110.12;

(b) A polypeptide having centrosome Nek-2 binding protein 110. 12 activity was isolated from the culture.

10. An antibody capable of binding to a polypeptide, characterized in that said antibody is an antibody capable of specifically binding to a centrosome Nek-2 binding protein 110.12.

 11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of centrosome Nek-2 binding protein 110.12.

 12. The compound according to claim 11, characterized in that it is an antisense sequence of the polynucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof.

 13. A method of using the compound according to claim 11, characterized in that the compound is used for regulating the centrosome Nek-2 binding protein 110. 12 in vivo and in vitro activity method.

 14. A method for detecting a disease or disease susceptibility related to 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 polypeptide Polypeptide activity, or a nucleotide variation in a 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 a centrosome Nek-2 binding protein 110. 12 mimic, agonist, antagonist or inhibitor ; Or for identification of peptide fingerprints.

 16. The use of a nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or used for manufacturing a gene chip. Or microarray.

 17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist is used Or the inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with centrosome-Nek-2 binding protein 110.12 abnormality.

 18. The use of a polypeptide, polynucleotide or compound as claimed in any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or compound is used for preparing a treatment such as a malignant tumor, Hematological diseases, HIV infection and immune diseases and drugs of various inflammations.