WO2007022668A1

WO2007022668A1 - Important lymphocyte activation regulatory protein pkd2 and uses thereof

Info

Publication number: WO2007022668A1
Application number: PCT/CN2006/000200
Authority: WO
Inventors: Ying Luo; Jun Wu; Qing Li
Original assignee: Shanghai Genomics, Inc.
Priority date: 2005-08-24
Filing date: 2006-02-06
Publication date: 2007-03-01
Also published as: CN1920014A

Abstract

An isolated protein kinase PKD2, nucleic acid sequences encoding the PKD2, vectors comprising the nucleic acid sequences, and host cells comprising the vectors are provided. A preparing method of the PKD2 and uses of the PKD2 are also provided.

Description

Important regulatory protein P D2 in lymphocyte activation and its application

Technical field

The invention relates to the field of biotechnology. More particularly, the present invention relates to a novel phosphokinase PKD2 polypeptide, its coding sequences and antibodies, and the use of such polynucleotides, polypeptides and antibodies thereof, and methods of preparing such polypeptides. Background technique

The "high quality" drug target gene (referred to as drug target) is the source of new drug development. Although the completion of the Human Genome Project has given humans a promising prospect for treating diseases, the genes themselves are not necessarily drug targets except for macromolecular protein drugs. From genes to new drugs, this chain still lacks many essential links. Among them, gene function research, because it can reveal the mysteries of human health and disease at the molecular level, find the most important pathogenic genes, and become a key step in determining whether a gene can become a drug target.

Large foreign pharmaceutical companies have found that genetic sequence data and bioinformatics analysis can only find a large number of potential drug target genes, but such genes can only be classified as "low quality" drug targets. Drug developers face a large number of low-quality drug targets that are at a loss, and a large number of genetic function studies are urgently needed to validate the “high-quality” targets that new drug development can rely on. Therefore, functional genomics research has great application value and business prospects.

The drug research and development of domestic biomedical enterprises is still based on imitation, lacking a complete drug development platform and research and development system, and basically not looking for new drug target genes. Neglecting the development of drug targets has directly led to the fact that most of the new drugs in China do not have independent intellectual property rights protection, and they face vicious competition in China and cannot enter the international market. As in foreign countries, domestic biopharmaceutical companies urgently need fully authenticated and original drug target genes. Only in this way can the cost and risk of new drug development be reduced, and new drugs with independent intellectual property rights can be developed.

Among the 5,000 genes currently available as targets, the sequence of the kinase kinase has great conservatism, and is a recognized drug screening gene target, with phosphatase and protease. And various types of receptors are collectively referred to as a type of target. Phosphoric kinases transfer the phospholipids of the ATP or GTP sites to the amino acid residues of the substrate proteins, catalyzing protein phosphorylation, and phosphorylation and dephosphorylation of proteins are important ways in which proteins regulate their function/activity. For example, MAPK and the transcription factors CREB, Jun, etc., are active in the phosphorylated state, but not in the non-phosphorylated state; whereas the transcription factor I κ B α and the like are opposite, and there is no activity in the phosphorylated state, but in the non- It is active in the phosphorylated state.

Phosphorylation-dephosphorylation of proteins is an important part of the process of cell signal transduction. Cell signal transduction refers to the stimulation of extracellular information molecules by a cell through a receptor located in the cell membrane or intracellular, through a complex intracellular letter. The transformation of the transduction system exerts biological effects, which in turn regulates almost all processes of life activities, including cell proliferation, development and differentiation, neural activity, muscle contraction, metabolism, and tumorigenesis. Many human diseases are closely related to the signal transduction pathway. For example, in the inflammation reaction caused by tumor necrosis factor (TNF), tumor necrosis factor interacts with a series of protein phosphokinases by binding to receptors. Activation of NF- _K B causes an inflammatory response. Biochemists and many companies are exploring key parts of the signaling pathway by studying the interactions between proteins and developing drugs that affect signaling to achieve disease treatment. The smooth progress of the signal transduction process depends on the phosphorylation of various protein substrates, including tyrosine phosphorylation, phosphorylation of threonine residues, and phosphorylation of serine residues, which is catalyzed by phosphokinase. .

In view of the important and even decisive role of signal transduction pathways in cell proliferation, differentiation and the development of various diseases, and the extremely important role of phosphokinase in signal transduction pathways, the design and development of new drugs targeting phosphokinase, Treating diseases by regulating and controlling signal transduction pathways is undoubtedly an ideal and highly targeted method with potential prospects for treating various diseases. Current kinase-specific drugs include PKC activity modulators, PKA inhibitors, PTK inhibitors, and receptor-mediated calcium channel modulators, some of which have entered clinical trials.

However, the existing target of kinase drugs is still far from meeting the needs of human beings to overcome diseases and overcome tumors. We still need more specific and effective new kinases as drug targets, thus effectively broadening the way to treat diseases and improving human health. . Summary of the invention

In order to achieve the above object, a first aspect of the present invention provides an isolated phosphokinase P D2 polypeptide or an active fragment thereof, wherein the polypeptide has an amino acid sequence selected from the group consisting of:

(a) Sequence Listing The amino acid sequence shown in SEQ ID NO: 4;

(b) an amino acid sequence having one or more amino acid deletions, substitutions or insertions relative to the amino acid sequence of SEQ ID NO: 4 and retaining the biological activity of the phosphokinase PKD2 of SEQ ID NO: 4.

Another aspect of the present invention provides an isolated nucleic acid sequence characterized by having a nucleic acid sequence encoding the above phosphokinase PKD2 polypeptide or an active fragment thereof or a complement thereof. In a preferred embodiment, the nucleic acid sequence has the nucleic acid sequence set forth in SEQ ID NO:3.

The invention also relates to a vector comprising the nucleic acid sequence described above and a host cell transformed with the vector. In another aspect, the present invention provides a method of producing the above-described phosphokinase PKD2 polypeptide or an active fragment thereof, the method comprising:

(a) cultivating said host cell under conditions suitable for expression of a phosphokinase PKD2 protein polypeptide;

(b) Isolating the above-described phosphokinase PKD2 protein polypeptide of the present invention or an active fragment thereof. Another aspect of the invention provides an antibody that specifically binds to the above-described phosphokinase PKD2 protein polypeptide or an active fragment thereof.

Another aspect of the invention relates to the use of a phosphokinase P D2 polypeptide of the invention, or an active fragment thereof, or a nucleic acid sequence thereof, for the preparation of a preparation for stimulating NF-AT channels.

Still another aspect of the invention relates to the use of a phosphokinase PKD2 polypeptide of the invention, or an active fragment thereof, or a nucleic acid sequence thereof, as a tumor marker.

Another aspect of the invention also relates to a pharmaceutical composition comprising a safe and effective amount of a protein of the invention and a pharmaceutically acceptable carrier.

Still another aspect of the invention relates to a method for detecting the presence or absence of cancer cells in a sample, the sample being derived from esophageal or rectal tissue, the method comprising determining the expression level of a nucleic acid sequence of phosphokinase PKD2 in the sample, and The amount of expression in the standard sample is compared, and if the amount of expression in the sample is increased, it indicates that cancer cells are present in the sample.

In another aspect of the present invention, a method of treating diseases such as T cell activation, autoimmunity, and organ transplant rejection, comprising: administering an effective amount of the acid kinase PKD2 polypeptide or a therapeutic agent to a subject in need thereof An active fragment thereof or a nucleic acid sequence encoding the same. The phosphokinase PKD2 of the present invention contains a phosphokinase domain which stimulates NF-AT activity, suggesting that PKD2 plays a key role in immune and inflammatory responses. In addition, the phosphokinase of the present invention is highly expressed in esophageal and rectal cancer tissues, but is not expressed or expressed in normal tissues, suggesting that it can be used as a marker for detecting esophageal and rectal cancer tissues. Other advantages of the invention will be apparent from the following detailed description. DRAWINGS

Figure 1 shows the structure of a 878 amino acid long silk/threonine protein kinase obtained in Example 1 of the present invention, wherein P represents a proline-rich domain; CYS represents a cysteine-rich zinc finger structure. Domain; S represents a serine-rich domain; AC represents an acidic domain; PH represents a pleckstrin homology domain; Kinase represents a kinase catalytic domain.

Figure 2 shows the experimental results of the interaction between PKD2 and LCK. The expression of RX317 and LCK was detected by Westen-blot method using anti-flag and anti-myc monoclonal antibodies, respectively. The results are shown in (2) above and below. It can be seen from the figure that the expression of RX317 and LCK is good and the expression is stable. Then, after immunoprecipitation with anti-myc antibody and Protein G, the coprecipitated product was electrophoretically transferred to a nitrocellulose membrane by SDS-PAGE electrophoresis, and immunoblot hybridization was carried out using an enzyme-labeled anti-flag antibody. The results are shown in Figure (2). PKD2 and LCK are combined under physiological conditions.

Figure 3 shows the effect of transfection of PKD2 gene on NFAT activity in Jurkat T cells, wild-type PKD2 NFAT (activated T cell nuclear factor) can be activated under anti-TCR drug treatment conditions, and its kinase function-deficient mutant PK 2 can inhibit the activation of NFAT (activated T cell nuclear factor). detailed description

The present inventors have successfully established a cDNA library of "new" genes synthesized from EST sequences in Genebank without any functional annotation or incomplete annotation, using bioinformatics methods for phosphokinase, phosphatase, protease, single membrane The body was screened preferentially, predicting a new phosphokinase PKD2 that does not yet have any functional annotation. Subsequently, the inventors cloned its full-length cDNA, and according to its cDNA analysis, the protein encoded by PKD2 contains a domain of a phosphokinase. Using real-time fluorescent quantitative PCR (Q-PCR), the inventors examined the expression of PKD2 in various tumors and their corresponding normal tissues. The results showed that the expression of PKD2 was elevated in esophageal and rectal cancer tissues, but not in normal tissues.

Since the gene is not directly involved in the physiological and pathological processes of the human body, the protein is a direct embodiment of the life activity. To further clarify the function of the new kinase PKD2, the inventors have developed a study on its protein. Yeast double hybridization is currently the most commonly used method for rapid and rapid study of protein-protein interactions. It has the advantages of simplicity and stability, and no other technology has been available to replace this technology. The present inventors used the yeast two-hybrid system to screen the Hela, lymphoid tissue, and fetal brain libraries with the PKD2 gene as a bait, and obtained a positive clone of LCK gene. Subsequently, the relationship between PKD2 and LCK was further verified in mammals by immunoprecipitation experiments. Finally, both yeast two-hybrid screening and co-immunoprecipitation showed that the phosphokinase PKD2 does have a direct interaction with LCK.

LCK (Homo sapiens lymphocyte-specific protein tyrosine kinase) consists of 509 amino acid residues with a molecular weight of 56K. Members of the protein tyrosine kinase family represented by p60v-src/c-src have tyrosine kinase activity as well as other members of this family. It has now been found that p56 LCK is relatively exclusively present in lymphocytes, especially mature resting T lymphocytes. P56 LCK plays an important role in T cell activation signal transduction and differentiation regulation, and plays a key role in the TCR (T cell receptor) pathway.

The present inventors further utilized the reporter gene system to study the function of the PKD2 gene. The results showed that transfection of PKD2 gene in Jurkat T cells had an effect on Anti-TCR-stimulated NF-AT activity. The NF-AT pathway is an important signal transduction pathway in the TCR pathway, regulating the transcription and expression of interleukins and various cytokines in the immune response. PKD2 stimulates NF-AT activity, suggesting that PKD2 plays a key role in immune and inflammatory responses.

Based on the above findings, one aspect of the present invention provides an isolated phosphokinase PKD2 polypeptide or an active fragment thereof, the polypeptide having an amino acid sequence selected from the group consisting of:

(a) Sequence Listing The amino acid sequence shown in SEQ ID NO: 4;

(b) having one or more amino acid deletions, substitutions or insertions relative to the amino acid sequence of SEQ ID NO:4 The amino acid sequence of the biological activity of the phosphokinase PKD2 of SEQ ID NO: 4 is retained.

In the present invention, the term "isolated" when used in reference to a nucleic acid or protein means that the nucleic acid or protein is substantially free of other cellular components that are related in nature, preferably in a homogeneous state, but may also be dry. Or an aqueous solution. Purity and homogeneity can generally be determined by analytical chemistry such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

The terms "protein", "peptide" or "polypeptide" are used interchangeably. They refer to chains of two or more amino acids joined together by peptide bonds or amide bonds, whether or not post-translationally modified (for example, glycosylation or phosphorylation). Antibodies are specifically included in the scope of this definition. Polypeptides of the invention may also comprise more than one subunit, one for each subunit encoded by a separate DNA sequence.

The term "pineate kinase PKD2 (protein) polypeptide" of the present invention refers to a polypeptide represented by SEQ ID NO: 4 having a phosphokinase PKD2 protein activity. The meaning of the term also includes variant forms of the sequence of SEQ ID NO: 4 having the same biological activity or function as PKD2. These variants include, but are not limited to: a number of sequences relative to SEQ ID NO: 4 (typically 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) deletions, insertions and/or substitutions of amino acids. In addition, the deletion or insertion (increase) may also occur at the C-terminus and/or N-terminus (usually within 20, preferably within 10, more preferably within 5 or less). In the art, when substituted with amino acids of similar or similar properties, the function of the protein is usually not altered. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are mutually conservatively substituted: aliphatic: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I); Family: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur: methionine (Μ), cysteine (C); alkaline: arginine) Lysine (Κ:), histidine (H); Acidity: Aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).

The term also encompasses fragments, derivatives or fusion proteins of the PKD2 protein, preferably the fragment or derivative retains the biological activity of the PKD2 protein. In addition to the nearly full length polypeptide, the present invention also encompasses soluble fragments of the PKD2 polypeptide. Typically, the fragment has at least about 10 contiguous amino acids of the PK 2 polypeptide sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, optimally at least about 100 consecutive amino acids.

As used herein, the term "having a biological activity of (retaining) phosphokinase PKD2" means that the protein or the like can interact with the LCK protein and stimulate NF-AT activity.

The invention also relates to analogs of PKD2 proteins or polypeptides. The difference between these analogs and the native PKD2 polypeptide can be a difference in amino acid sequence and/or a difference in the modified form that does not affect the sequence. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology. Analog Analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, Υ-amino acids) are included. It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

Modifications (usually without altering the primary structure) include: chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. The present invention also encompasses polypeptides which have been modified to enhance their anti-proteolytic properties or to optimize solubility properties.

Another aspect of the present invention provides an isolated nucleic acid sequence characterized by having a nucleic acid sequence encoding the above phosphokinase PKD2 polypeptide or an active fragment thereof or a complement thereof.

In the present invention, the term "nucleic acid (sequence)" means a deoxyribonucleic acid or ribonucleic acid and a polymer thereof in a single- or double-stranded form. Unless specifically limited otherwise, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar biological activities or properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. . The term "nucleic acid" is used interchangeably with genes, cDNA and gene-encoded mRNA. In addition, the nucleic acid sequences of the invention can be modified to use codons that are preferred for particular cell classes. The nucleic acid sequence of the present invention further includes a nucleic acid fragment or a nucleic acid subsequence as long as the nucleic acid fragment or nucleic acid subsequence has the same biological activity as the nucleic acid sequence of the present invention. Moreover, the nucleic acid sequences of the invention also include conservatively modified variants and complementary sequences thereof. "Conservative modifications" of a particular nucleic acid sequence refers to those nucleic acids which encode identical or substantially identical amino acid sequences, or which have substantially the same sequence when the nucleic acid does not encode an amino acid sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode a given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA and AGG all encode the amino acid arginine. Thus, at each arginine position determined by the codon, the codon can be changed to any of the corresponding codons described above without altering the encoded polypeptide. This nucleic acid change is a "sudden change", which is one of the "conservative modification changes." One of skill in the art will recognize that each codon in a nucleic acid (other than AUG, which is typically the only codon for methionine) can be modified to produce functionally equivalent molecules using standard techniques. Thus, each sequence is meant to include a "silent change" of a nucleic acid encoding a polypeptide.

Further, a nucleic acid sequence which is identical (homologous) or substantially identical (homologous) to the nucleic acid sequence shown in SEQ ID NO: 3 is also encompassed within the scope of the nucleic acid sequence of the present invention. For example, it comprises a nucleic acid sequence having at least 60%, more preferably 70%, even preferably 80%, 90%, or even more than 95% homology or identity to the nucleic acid sequence of SEQ ID NO:3. In the case of two or more nucleic acid or polypeptide sequences, the terms "same" or "percent identity" refer to the comparison of alignment and alignment to its maximum identity (using one of the following sequence alignment algorithms (or one skilled in the art) When other algorithms are available) or visually observed, two or more sequences or subsequences are identical, or There are a specified percentage of identical amino acid residues or nucleotides. The term "substantially identical" means that two or more sequences or subsequences have at least 60%, when compared and aligned to their maximum identity (determined by one of the following sequence alignment algorithms or by visual observation). Good 80%, optimal 90-95% nucleotide or amino acid residue identity. Such "substantially identical" sequences are generally considered to be homologous. To compare sequences and determine homology, one sequence is typically compared to a test sequence as a reference sequence. When using the sequence comparison algorithm, enter the test and reference sequences into the computer, specify the subsequence coordinates, and if necessary, specify the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. The optimal sequence alignment for sequence comparison can be achieved, for example, by the local homology algorithm in Smith and Waterman, Adv. Appl. Math. 2:482 (1981), Needleman and Wunsch, J. Mol. Biol. 48:443 (1970) Homology ranking algorithm, Pearson and Lipman Proc. Natl. Acad. Sci (USA) 85: 2444 (1988) similarity search methods, computerized operations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, The Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI).

Another indication that two nucleic acid sequences are substantially identical/homologous is that the two nucleic acid sequences hybridize to each other under stringent conditions. The term "specifically hybridizes to" means that when a sequence is present in a complex mixture of DNA or RNA (eg, total cells), the molecule binds to a particular nucleotide sequence under stringent conditions, forming a double strand or Hybrid. In nucleic acid hybridization experiments, "stringent hybridization conditions" and "stringent hybridization wash conditions" depend on the sequence and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. Generally, highly stringent hybridization and washing conditions are selected to be about 5 ° C lower than the thermal melting temperature (Tm) of a particular sequence at a defined ionic strength and pH. Thus, the nucleic acid sequences of the present invention also encompass nucleic acid sequences that hybridize under high stringency conditions to the nucleic acid sequences of SEQ ID NO: 3 under moderately stringent conditions.

The invention also encompasses antisense sequences encoding nucleic acid sequences of phosphokinase PKD2 polypeptides and fragments thereof. This antisense sequence can be used to inhibit the expression of P D2 in cells. The invention also encompasses nucleic acid molecules useful as probes and primers, typically having from about 8 to about 100, preferably from about 15 to about 50, consecutive nucleotides of the nucleic acid sequence set forth in SEQ ID NO: 3. The probe can be used to detect the presence or absence of a nucleic acid molecule encoding PKD2 in a sample.

Accordingly, the present invention also encompasses a method of detecting a PKD2 nucleotide sequence in a sample which comprises hybridizing to a sample using the probe described above and then detecting whether the probe has bound. In a preferred method, the detection can be carried out using a real-time quantitative PCR method. Alternatively, the sample is preferably a product after PCR amplification, wherein the PCR amplification primer corresponds to a sequence encoding a PKD2 polypeptide and may be located on either or both sides of the coding sequence. Primers are typically 20-50 nucleotides in length.

The present invention also provides a method for detecting the presence or absence of cancer cells in a sample in vitro, the sample being derived from esophageal or rectal tissue, the method comprising determining the expression level of the PKD2 nucleic acid sequence in the sample, and using the standard sample In comparison with the amount of expression in the sample, if the amount of expression in the sample is increased, it indicates that cancer cells are present in the sample. In a preferred method, the detection can be performed using a real-time quantitative PCR method.

Accordingly, still another aspect of the present invention relates to the use of the phosphokinase PKD2 polypeptide of the present invention or an active fragment thereof or a nucleic acid sequence thereof as a tumor marker for detecting a tumor, such as esophageal cancer or rectal cancer.

The full length sequence of the P D2 nucleotide of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art. The library is used as a template to amplify the relevant sequences. When the sequence is long, amplification can usually be performed by overlapping, for example, two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order. Once the relevant sequences have been obtained, the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.

Another aspect of the invention relates to a vector or construct comprising the above-described nucleic acid sequence of the invention or a fragment thereof. In a preferred embodiment, the vector is an expression vector comprising the nucleic acid sequence of the invention described above and an expression control sequence operably linked to the nucleic acid sequence. The term "expression control sequence" as used herein generally refers to sequences involved in controlling the expression of a nucleic acid sequence. Expression control sequences include promoter and termination signals operably linked to a target nucleic acid sequence. They also typically include sequences required for proper translation of the nucleic acid sequence. "Operatively linked" means that portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if a promoter or enhancer increases the transcription of a coding sequence, it is operably linked to the coding sequence.

In a preferred embodiment, the expression control sequence comprises a constitutively high expression promoter. Expression control sequences suitable for high or overexpression in the target tissue, such as promoters (e.g., 35S promoter), are selected. In addition, the presence of a booster element (enhancer), combined with the above-described promoter elements, also generally increases the level of expression.

Another aspect of the invention also relates to a host cell transformed with the above vector. In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cell, such as a CHO cell, a COS cell or the like. The term "transformation" as used herein, refers to the direct introduction of an expression vector containing a nucleic acid of interest into a host cell by methods well known to those skilled in the art. Transformation methods vary by host cell type and typically include: electroporation; transfection with calcium chloride, DEAE-dextran or other substances; microprojectile bombardment; lipofection; infection and other methods (see Sambrook et al. The Guide to Molecular Cloning, 2nd Edition, 1989). Another aspect of the present invention provides a method for producing the above phosphokinase P D2 polypeptide or an active fragment thereof, which comprises: (a) cultivating the above host cell under conditions suitable for expression of a phosphokinase PKD2 protein polypeptide; (b) The phosphokinase PKD2 protein polypeptide or an active fragment thereof is isolated.

The phosphokinase PKD2 polypeptide of the present invention or an active fragment thereof can be produced by direct synthesis of a peptide by solid phase techniques, in addition to being produced by recombinant methods (Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Meri-ifield J. (1963) J. Am Chera. Soc 85: 2149-2154). The synthesis of proteins in vitro can be carried out manually or automatically. For example, peptides can be synthesized automatically using Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA). The fragments of the inventive protein can be separately chemically synthesized and then chemically linked to produce a full length molecule.

Another aspect of the invention also encompasses polyclonal and monoclonal antibodies specific for a phosphokinase PKD2 protein polypeptide or an active fragment thereof, preferably a monoclonal antibody. Here, "specificity" refers to an antibody that binds to a phosphokinase PKD2 polypeptide or fragment, preferably an antibody that binds to a phosphokinase PKD2 polypeptide or a fragment thereof but does not recognize and bind to other unrelated antigen molecules. The antibodies of the present invention include those capable of binding to and inhibiting the phosphokinase PKD2 polypeptide, as well as those which do not affect the function of the phosphokinase PKD2 protein. The invention also includes those antibodies that bind to the modified or unmodified form of the phosphokinase PKD2. The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single chain Fv molecules; Or chimeric antibodies. Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, purified phosphokinase PKD2 or its antigenic fragment can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing the phosphokinase PKD2 or an antigenic fragment thereof can be used to immunize an animal to produce antibodies. The antibody of the invention may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol. 6: 511, 1976; ohler et al, Eur. J. Immunol 6:292, 1976; Hammerling et al, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981). The antibodies of the invention include antibodies that block the biological activity of phosphokinase PKD2. The various antibodies of the invention can be obtained by conventional immunological techniques using fragments or functional regions of the PKD2 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. An antibody that binds to an unmodified form of the phosphokinase PKD2 can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (eg, E. coli); an antibody that binds to a post-translationally modified form (eg, a glycosylated or phosphorylated protein) Or a polypeptide), which can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (eg, yeast or insect cell:).

Substances that interact with the phosphokinase PKD2, such as receptors, inhibitors or antagonists, can also be screened by various conventional screening methods. The term "interaction" means a form that includes direct interaction, such as Binding, cutting and indirect interactions. A preferred screening method is a yeast two-hybrid screening method in which a first expression vector encoding a fusion of a first protein and a DNA binding domain and a fusion encoding a second protein and a DNA activation domain are introduced into a yeast cell. The second expression vector can be used to determine protein-protein interactions. As described in the Examples of the present specification, yeast two-hybrid screening revealed that the phosphokinase PKD2 of the present invention has a direct interaction with LCK.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are only intended to illustrate the invention and not to limit the scope of the invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are known to those skilled in the art. These techniques are fully described in the following literature: for example, Sambrook, Guide to Molecular Cloning, 2nd Edition (1989); DNA Cloning, Volumes I and II (DN Glover, 1985); Oligonucleotide Synthesis (Editing by M Gait, 1984); Nucleic Acid Hybridization (edited by BD Hames and SJ Higgins. 1984); Protein Purification: Principles and Practice, 2nd Edition (Springer-Verlag, NY), and the Manual of Experimental Immunology I -IV volume (edited by DC Weir and CC Blackwell 1986). Alternatively, follow the instructions provided by the reagent manufacturer. Example 1. Screening of PKD2 gene:

The present inventors applied a modern bioinformatics PFAM/Profile model to phosphokinase, phosphatase, protease from a cDNA library of an established "new" gene synthesized by EST sequences in Genebank without any functional annotation or incomplete annotation. Single-pass membrane receptors were screened preferentially to find a newly discovered 878 amino acid long silk/threonine protein kinase that can be activated by DAG, the structure of which is shown in Figure 1. Example 2. Acquisition of the PKD2 gene:

In order to obtain the full length of the PKD2 gene, the inventors designed the following primers.

Upstream bow I: 5* acgctGGCCAATCCGGCCATGGCCACCGCCCCCTCTTATCC3' (SEQ ID NO: 1)

Downstream primer: 5' acgctGGCCTCTAAGGCCTCAGAGAACACTGATGCGCTCCGC 3' (SEQ ID NO: 2)

The pair of primers contained a Sfil shuttle cloning site, a start codon and a stop codon, and the full-length PKD2 gene was obtained by a conventional PCR method using human tissue mixed RNA as a template. It is then sequenced. This gene sequence is shown in SEQ ID NO: 3, and its amino acid sequence is shown in SEQ ID NO: 4. Example 3: Screening for proteins that interact with PKD2

The present inventors constructed a full-length cDNA of PKD2 and used it as a bait gene, using the Gal4 yeast two-hybrid system. The cDNA library of Hela cells was screened, and two positive clones were obtained, which were identified as LCK genes (targets of lymphocytes) by sequencing. Example 4, verifying the interaction between PKD2 and LCK

In order to eliminate the false positives that may be caused by the yeast two-hybrid technique, the present inventors cloned the PKD2 and LCK genes into eukaryotic expression vectors containing Flag, Myc, etc., and transfected into human embryonic kidney 293T cell line, 24-48 hours later. The cells were disrupted and antibodies such as Flag, Myc, etc. were used to determine if they co-precipitated. The experimental results indicate that PKD2 interacts with LCK (Fig. 2). Example 5, Functional Identification of PKD2 Gene

Wild-type or ATP-binding site mutants overexpressing the PKD2 gene in Jurkat T cells were studied using a reporter gene system. Luciferase activity can be obtained 24 hours after transfection. Since the luciferase method is a very sensitive method, it is easy to introduce experimental errors, and it is planned to repeat each experiment data three times or more to obtain an average value. Results Transfection of PKD2 gene in Jurkat T cells had an effect on Anti-TCR (T cell receptor)-stimulated NF-AT activity (see Figure 3 for results). Example 6. Tissue distribution of PKD2

Real-time fluorescent quantitative PCR (Q-PCR) is currently the most sensitive and accurate technique for quantitative analysis of genes. The inventors have constructed a library of frozen tissue with more than 300 pairs of tumor tissues and their corresponding normal tissues, including 18 tumor types. In order to further determine the expression of the PKD2 gene in various tumor tissues, the inventors used the frozen tissue bank to conduct qualitative and quantitative studies by Q-PCR technology.

As shown in the results in Table 1, the expression of the PKD2 gene in human esophageal and rectal cancer tissues was higher than that in normal tissues.

Table 1

Note: The expression elevation is defined as MT/N>5, the expression down-regulation is defined as MT/N<0.2, and the expression is flat at 0.2<MT/N<5. Although the present invention describes specific examples, one thing is to the art. It will be apparent to those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the invention. Therefore, the appended claims are intended to cover all such modifications within the scope of the invention. All publications, patents, and patent applications cited herein are hereby incorporated by reference.

Claims

Rights request

An isolated phosphokinase PKD2 polypeptide or an active fragment thereof, wherein the polypeptide has an amino acid sequence selected from the group consisting of:

(a) Sequence Listing The amino acid sequence shown in SEQ ID NO: 4;

An isolated nucleic acid sequence characterized by comprising a nucleic acid sequence encoding the phosphokinase PKD2 polypeptide of claim 1 or an active fragment thereof, or a complement thereof.

The isolated nucleic acid sequence according to claim 2, wherein the nucleic acid sequence has the nucleic acid sequence shown in SEQ ID NO: 3.

A vector comprising the nucleic acid sequence of claim 2.

A host cell characterized in that it is transformed with the vector of claim 4.

A method of producing the phosphokinase PKD2 polypeptide of claim 1, or an active fragment thereof, wherein the method comprises:

(a) cultivating the host cell of claim 5 under conditions suitable for expression of the acid ester kinase PKD2 protein polypeptide;

(b) isolating the phosphokinase of claim 1 : a PKD2 protein polypeptide or an active fragment thereof.

An antibody which specifically binds to the phosphokinase PKD2 protein polypeptide of claim 1 or an active fragment thereof.

The use of the phosphokinase PKD2 polypeptide of claim 1, or an active fragment thereof, or the nucleic acid sequence thereof according to claim 2, for use in the preparation of a disease for treating T cell activation, autoimmunity, and organ transplant rejection. Drug.

9. Use of the phosphokinase PKD2 polypeptide of claim 1 or an active fragment thereof or the nucleic acid sequence of claim 2 as a tumor marker.

A pharmaceutical composition comprising a safe and effective amount of the protein of claim 1 and a pharmaceutically acceptable carrier.