WO2001030826A1

WO2001030826A1 - A novel polypeptide-serine/threonine kinase 29 and polynucleotide encoding said polypeptide

Info

Publication number: WO2001030826A1
Application number: PCT/CN2000/000392
Authority: WO
Inventors: Yumin Mao; Yi Xie
Original assignee: Shanghai Bio Road Gene Development Ltd.
Priority date: 1999-10-27
Filing date: 2000-10-27
Publication date: 2001-05-03
Also published as: AU1126801A; CN1303945A

Abstract

The invention discloses a new kind of polypeptide-serine/threonine kinase 29 and polynucleotide encoding the polypeptide and a process for producing the polypeptide by recombinant methods. It also discloses the method of applying the polypeptide for the treatment of various kinds of diseases, such as cancer, hemopathology, HIV infection, immune diseases and inflammation. The antibody and antagonist of the polypeptide and the therapeutic use of the same is also disclosed. In addition, it refers to the use of polynucleotide encoding said serine/threonine kinase 29.

Description

A new peptide-serine / threonine kinase 29 and a polynucleotide encoding this polypeptide

The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, serine / threonine kinase 29, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Protein phosphorylation is a basic regulatory mechanism in the regulation of eukaryotic cells, and it is also the most common way of protein modification. For example, cell phosphorylation occurs during cell division, cell metabolism, cell adhesion and cell migration, cell-to-cell communication, and signaling. To accomplish these functions, 2% to 4% of human-encoded genes encode approximately 2000 different kinases (Hunter, 1996).

During spermatogenesis, many protein kinases participate in the reaction. Among them, serine / threonine kinases are a large family. A mouse testis-specific serine / threonine kinase (tssk) l (Bielke et al., 1994) has been isolated in 1994. This kinase is a new subfamily of serine / threonine kinases. Now, a second mouse testis-specific serine / threonine kinase (tssk) 2 has been isolated, which is also a new subfamily of the serine / threonine kinase family (Peer Kueng et al., 1999) . Immunohistochemical staining indicates that both kinases are located in the cytoplasm of late spermatids, and they have a significant effect on the last stage of sperm cell maturation. Sequence analysis showed that the ts sk2 gene had an open reading frame of 357 amino acids encoded by 1,071 nucleotides. An adenylate transferase recognition signal of about 25 nucleotides upstream of the poly (A) tail of the non-coding region at the 3 'end. The protein's kinase domain is located at the amino terminus of the peptide chain (12-275 amino acids) and contains all conserved amino acids of the serine / threonine kinase family (Hanks and Puinn, 1991). The tssk2 kinase domain has 89% homology with the human gene DGS-G (Gong et al., 1996), and tsskl has 84% homology. This indicates that ts skl, tssk2 and DGS-G form a new serine / threonine kinase subfamily-tssk family. The functional domains of these three kinases are very similar, except that tssk2, the carboxyl terminus of the DGS-G peptide chain is conserved, and ts skl peptide chains have different amino acid sequences and lengths. The entire nucleotide sequence of the tssk2 gene is very similar to that of the DGS-G gene, and DGS-G is also limited to expression in the human testis. Therefore, the tssk2 gene may be an orthologous gene of the DGS-G gene (Gong et al., 1996). DGS-G gene-specific transcription of 11 critical 250-kb DiGeorge critical regions In one recording unit, the gene is located on the 22nd human chromosome.

The polypeptide of the present invention is derived from a human cDNA library, has a high homology with the serine / threonine kinase subfamily-t s sk family, and has similar structural characteristics. It is named Serine / Threonine Kinase 29.

Object of the invention

It is an object of the present invention to provide isolated novel polypeptides-serine / threonine kinase 29 and fragments, analogs and derivatives thereof.

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

It is another object of the present invention to provide a recombinant vector containing a polynucleotide encoding a serine / threonine kinase 29.

Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a serine / threonine kinase 29.

Another object of the present invention is to provide a method for producing serine / threonine kinase 29.

Another object of the present invention is to provide an antibody against the polypeptide of the present invention, serine / threonine kinase 29.

Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention-serine / threonine kinase 29.

It is another object of the present invention to provide a method for diagnosing and treating diseases related to abnormalities of serine / threonine kinase 29.

Summary of invention

In a first aspect of the present invention, a novel isolated serine / threonine kinase 29 is provided. The polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2; or a conservative variant polypeptide thereof; Or its active fragment, or its active derivative, analog. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

In a second aspect of the present invention, there is provided a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the above-mentioned serine / threonine kinase 29; (b) a polynucleotide complementary to the polynucleotide (a). Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2. More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1069-1650 in SEQ ID NO: 1; and (b) a sequence having 1-2345 in SEQ ID NO: 1 Sequence of bits. In a third aspect of the present invention, there are provided a vector containing the above polynucleotide, and a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above polynucleotide.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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 amino acid sequence homology between serine / threonine kinase 29 and murine protein kinase of the present invention. The upper sequence is serine / threonine kinase 29 and the lower sequence is murine protein kinase. Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".

Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated serine / threonine kinase 29. 21 kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.

Summary of the Invention

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 serine / threonine kinase 29" means that serine / threonine kinase 29 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify serine / threonine kinase 29 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the serine / threonine kinase 29 peptide can be analyzed by amino acid sequence.

The present invention provides a novel polypeptide-serine / threonine kinase 29, 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 invention can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

The invention also includes fragments, derivatives and analogs of serine / threonine kinase 29. As the invention As used, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the serine / threonine kinase 29 of the invention. 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) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (Π Ι) Such a polypeptide sequence in which the mature polypeptide is fused with 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 into the mature polypeptide (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) 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 2345 bases, and its open reading frame (1069-1650) encodes 193 amino acids. According to the amino acid sequence homology comparison, it was found that this peptide is 48% homologous to murine protein kinase, and it can be deduced that the serine / threonine kinase 29 has similar structure and function to murine protein kinase.

The polynucleotide of the present invention may be in the D form or the R form. The D form includes cD, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but having a sequence different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

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 present invention also relates to a variant of the polynucleotide described above, which encodes the same amino group as the present invention. Acid sequences of polypeptides 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 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 present 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) A denaturant was added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42. C, etc .; or (3) hybridization occurs only when the identity between the two sequences 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 herein, 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 More than nucleotides. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding serine / threonine kinase 29.

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 serine / threonine kinase 29 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 D fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.

Of the methods mentioned above, genome D is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction, and kits are also commercially available (Q i agene). The construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua 1, Cold Harbor Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very few Expression products can also be cloned.

The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DM-DNA or DM-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of serine / threonine kinase 29 transcripts; ( 4) Detecting gene-expressed protein products by immunological techniques or by 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 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 probe used here is generally 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, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the serine / threonine kinase 29 gene.

A method (Sa iki, et al. Science 1985; 230: 1350-1354) using PCR technology to amplify DNA / RNA 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 D fragments and the like obtained as described above can be determined by a conventional method such as a 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. To obtain the full-length cD sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

The present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a serine / threonine kinase 29 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.

In the present invention, a polynucleotide sequence encoding a serine / threonine kinase 29 may be inserted into a vector, In order 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 Na thans, J Bio Chera. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in a 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 well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a serine / threonine kinase 29 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, A Laboratory Manua, Cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an 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, and 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 and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, 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 on the late side of the origin of replication, and adenovirus enhancers.

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 serine / threonine kinase 29 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineered gene containing the polynucleotide or the recombinant vector. Programmed host cells. 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 Sf 9; 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 D 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 D may be in the exponential growth phase were harvested, treated with CaC l ₂ method used in steps well known in the art. The alternative is to use MgC l ₂ . 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.

The polynucleotide sequence of the present invention can be used to express or produce recombinant serine / threonine kinase 29 by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding human serine / threonine kinase 29 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), the medium used in the culture may be selected from various conventional mediums according to the host cells used. 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 outside the cell. If desired, recombinant proteins can be separated and purified by various separation methods using their 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 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 malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, H IV infection and immunity Sexually transmitted diseases.

The serine / threonine kinase 29 of the present invention can also be expressed in the myocardium of early embryonic tissues. This indicates that the polypeptide of the present invention may be involved in cardiac development. At the same time, the serine / threonine kinase 29 of the present invention may also have a cascading effect on tumorigenesis. Deletion of the serine / threonine kinase 29 gene of the present invention will likely lead to Digeorge syndrome and facial deformity syndrome. These syndromes are manifested as developmental disorders such as hypodevelopment of the thymus and parathyroid glands, cardiovascular disorders, and mild craniofacial deformities.

The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) serine / threonine kinase 29. Agonists enhance biological functions such as serine / threonine kinase 29 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing serine / threonine kinase 29 can be cultured with labeled serine / threonine kinase 29 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.

Antagonists of serine / threonine kinase 29 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of serine / threonine kinase 29 can bind to serine / threonine kinase 29 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot exert its biology Features.

When screening compounds as antagonists, serine / threonine kinase 29 can be added to bioanalytical assays to determine whether a compound is Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to serine / threonine kinase 29 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the serine / threonine kinase 29 molecule 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 directed against a serine / threonine kinase 29 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.

Polyclonal antibodies can be produced by direct injection of serine / threonine kinase 29 into 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 'S adjuvant and so on. Techniques for preparing monoclonal antibodies to serine / threonine kinase 29 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497) _t -tumor technology human B-cell hybridoma Technology * EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against serine / threonine kinase 29.

Antibodies against serine / threonine kinase 29 can be used in immunohistochemical techniques to detect serine / threonine kinase 29 in biopsy specimens.

Monoclonal antibodies that bind to serine / threonine kinase 29 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. For example, serine / threonine kinase 29 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 cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill serine / threonine kinase 29 positive cell.

The antibodies of the present invention can be used to treat or prevent diseases related to serine / threonine kinase 29. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of serine / threonine kinase 29.

The invention also relates to a diagnostic test method for quantitative and localized detection of serine / threonine kinase 29 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. Serine / threonine kinase 29 levels measured in the test can be used to explain the importance of serine / threonine kinase 29 in various diseases and to diagnose diseases in which serine / threonine kinase 29 plays a role.

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.

Polynucleotides encoding serine / threonine kinase 29 can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat abnormalities in cell proliferation, development, or metabolism caused by non-expression or abnormal / inactive expression of serine / threonine kinase 29. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant serine / threonine kinase 29 to inhibit endogenous serine / threonine kinase 29 activity. For example, a variant serine / threonine kinase 29 can be shortened and lack signaling The domain of serine / threonine kinase 29, although it can bind to downstream substrates, lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of serine / threonine kinase 29. Expression vectors derived from viruses ^ Retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, parvoviruses, and the like can be used to transfer a polynucleotide encoding a serine / threonine kinase 29 into a cell. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a serine / threonine kinase 29 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding serine / threonine kinase 29 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 RNA and DM) and ribozymes that inhibit serine / threonine kinase 29 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific R. Its mechanism of action is that the ribozyme molecule specifically hybridizes to a complementary target RNA for endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA 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 phosphorothioate or peptide bonds instead of phosphodiester bonds.

A polynucleotide encoding a serine / threonine kinase 29 can be used for the diagnosis of diseases related to serine / threonine kinase 29. A polynucleotide encoding a serine / threonine kinase 29 can be used to detect the expression of serine / threonine kinase 29 or the abnormal expression of serine / threonine kinase 29 in a disease state. For example, a DNA sequence encoding serine / threonine kinase 29 can be used to hybridize biopsy specimens to determine the expression of serine / threonine kinase 29. Hybridization techniques include Southern blotting, Nor thern imprinting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available. Part or all of the polynucleotides of the present invention can be immobilized on a microarray as probes

(Microarray) or DM chip (also known as "gene chip") for differential expression analysis and gene diagnosis of genes in tissues. Serine / threonine kinase 29-specific primers can also be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect serine / threonine kinase 29 transcription products. Detection of mutations in the serine / threonine kinase 29 gene can also be used to diagnose serine / threonine kinase 29-related diseases. The forms of serine / threonine kinase 29 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type serine / threonine kinase 29 DNA sequences. 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. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

The sequences of the invention are also valuable for chromosome identification. The sequence specifically targets 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 cD, 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 locate D to a specific chromosome. Using the oligonucleotide primers of the present invention, in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. 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 chromosome-specific cDNA libraries.

Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: A Manua l of Basic Techniques, Pergamon Press, 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. This data can be found in, for example, V. Mckusick, Mendelian Inherance 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 have been mapped to chromosomal regions.

Next, the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals, and the mutation is not observed 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 the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA 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 comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

The invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell. 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. Serine / threonine kinase 29 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of serine / threonine kinase 29 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 in the following examples are not marked with specific conditions, usually according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Harbor Labora tory Pres s, 1989), or Follow the conditions recommended by the manufacturer.

Example 1 Cloning of serine / threonine kinase 29

Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Quik mRNA Isolat ion Ki t

(Qiegene) Isolate poly (A) mRNA from total RNA. 2ug poly (A) mRNA is reverse transcribed to form cDNA. A Smart cDNA cloning kit (purchased from Clontech) was used to orient the cDNA fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5c. The bacteria formed a cDNA library. With Dye The terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) determined the sequences at the 5 'and 3' ends of all clones. The determined CDM sequence was compared with an existing public DM sequence database (Genebank), and it was found that the CDM sequence of one of the clones 0097dl 0 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 0097dl 0 clone contains a full-length cDNA of 2345bp (as shown in Seq ID N0: l), and has a 193bp open reading frame (0RF) from 1069bp to 1650bp, encoding a new protein (such as Seq ID NO: 2). We named this clone pBS-0097dlO and the encoded protein was named serine / threonine kinase 29. Example 2 Homologous search of cDNA clones

The sequence of the serine / threonine kinase 29 of the present invention and the protein sequence encoded by the serine / threonine kinase 29 of the present invention were analyzed using the Blas t program (Basic local alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403- 10]. Perform homology search in databases such as Genbank and Swissport. The gene most homologous to the serine / threonine kinase 29 of the present invention is a known murine protein kinase, and the accession number of the encoded protein is Gen019 in Genbank. The two are highly homologous, with an identity of 48% and a similarity of 69%. Example 3 Cloning of a gene encoding serine / threonine kinase 29 by RT-PCR

CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:

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

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

Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;

Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.

Conditions for the amplification reaction: 50 μl / L KC1, 10 μl / L Tri s-

Cl, (pH 8. 5), 1.5 mmol / L MgCl ₂ , 200 μmol / L dNTP, 1 Opmol primer, 1U of Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min. During RT-PCR, β-act in was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a PCR vector using a TA cloning kit (Invitrogen). DNA sequence analysis results indicate PCR The DM sequence of the product is exactly the same as 1-23 ⁴ 5bp shown in SEQ ID NO: 1. Example 4 Northern blot analysis of serine / threonine kinase 29 gene expression

Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] „This method involves acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidinium isothiocyanate-25 mM sodium citrate, 0.2 \ 1 Sodium acetate (^ .0) was used to homogenize the tissue, 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) were added, and the mixture was centrifuged. The aqueous layer was aspirated, and isopropyl alcohol ( 0.8 volume) 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 μ ₈ RNA was used in 20 mM 3- (N-morpholino) propanesulfonic acid (PH7 .0) was electrophoresed on a 1.2% agarose gel -5m -ImM EDTA-2.2M sodium acetate, formaldehyde and then transferred to nitrocellulose. Preparation of a- ³² P dATP using ³² P- labeled by random priming SYSTEM DNA probe. The DNA probe used was the PCR-amplified serine / threonine kinase 29 coding region sequence (1069bp to 1650bp) shown in Figure 1. A 32P-labeled probe (about 2 x 10 ⁶ cpm / ml) hybridized with RNA-transferred nitrocellulose membrane at 42 ° C overnight in a solution containing 50% formamide-25mM KH ₂ P0 ₄ (pH7.4) -5 33 (-5 ^ 0 61111 & 1 ^, 3 solution and _20,048 / 1111 salmon sperm 0 hybridization Thereafter, the filters in 1 x SSC- 0.1% SDS are washed at 55 ° C 30min. Then, analyzed and quantified by Phosphor Imager. Example 5 Recombinant Expression, isolation and purification of serine / threonine kinase 29 in vitro

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

Priraer3: 5,-CCCCATATGATGCTAGAGTCTGCCGACGGGAAA-3 '(Seq ID No: 5) Primer4: 5'-CCCGAATTCTCAAGTGCTTGCTAGCCATGGATG-3' (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and EcoRI restriction sites, respectively. The coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively. The Ndel and EcoRI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3) Digestion site. The pBS-0097dl0 plasmid containing the full-length target gene was used as a template for the PCR reaction. The PCR reaction conditions were as follows: 10 pg of pBS-0097dl0 plasmid, primer Primer-3, and Primer_4 were included in a total volume of 50 μl; j was lpmol, Advantage polymerase Mix (Clontech) 1 μ1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Ndel and EcoRI 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 by the calcium chloride method of coliform bacteria DH5 alpha, containing kanamycin After the LB plate (final concentration 30 g / ml) was cultured overnight, positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0097dlO) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) by the calcium chloride method. In containing kanamycin (final concentration 30 g / ml) of LB liquid medium, host strain BL21 _(P ET-0097dlO) were cultured to logarithmic growth phase at 37 ° C, added with IPTG to a final concentration of 1 ol / L. Continue incubation for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation. An affinity column His. Bind Quick Cartridge capable of binding to 6 histidines (6His-Tag) was used.

(Product of Novagen) Chromatography was performed to obtain the purified protein serine / threonine kinase 29. By SDS-PAGE electrophoresis, a single band was obtained at 21 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, 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 6 Production of anti-serine / threonine kinase 29 antibodies

The following peptides specific to serine / threonine kinase 29 were synthesized using a peptide synthesizer (product of PE): NH ₂ -Me t -Leu-G lu-Ser-Ala-As ρ-G 1 y-Ly s-11 e-Cys-Leu-Va 1 -Me t -G 1 u-Leu-COOH (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 hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. 15Using a 15 μg / ml bovine serum albumin peptide complex-coated titer plate for ELISA to determine the antibody titer 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 demonstrated that the purified antibodies specifically bind to serine / threonine kinase 29.

Claims

Profit requirements

1. An isolated polypeptide-serine / threonine kinase 29, 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 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 the 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 represented by SEQ ID NO: 2.

6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises a sequence of positions 1069-1650 in SEQ ID NO: 1 or a sequence of positions 1-2345 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 a polynucleotide according to any one of claims 4-6.

9. A method for preparing a polypeptide having serine / threonine kinase 29 activity, characterized in that the method comprises:

(a) culturing the engineered host cell according to claim 8 under the condition of expressing serine / threonine kinase 29; (b) Isolating a polypeptide having serine / threonine kinase 29 activity 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 serine / threonine kinase 29.

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 serine / threonine kinase 29.

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. Use of a compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of serine / threonine kinase 29 in vivo and in vitro.

14. A method for detecting a disease or susceptibility to a disease associated with a 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 detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

15. Use of a polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of serine / threonine kinase 29; or 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 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 the 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 an abnormality of serine / threonine kinase 29.

18. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Disease, HIV infection and immune diseases and drugs of various inflammations.