CN1414103A - Human serine/threonine protein kitase, its coding sequence, preparation method and use - Google Patents

Abstract

The present invention relates to a human serine/threonine kinase a (hSTKa), the cDNA sequence for the transcription of the said hSTKa, the polypeptide coded by the said nucleotide sequence, and the application and preparing process of the said polynucleotide and polypeptide.

Description

Human serine/threonine protein kinase, its coding sequence, preparation method and use

technical field

The present invention relates to the field of genetic engineering, in particular, the present invention relates to a human gene nucleotide sequence. More specifically, the present invention relates to a cDNA sequence of a human serine/threonine kinase ( human serine / threonine kinase a, hSTKa) and its transcript hSTKc. The present invention also relates to polypeptides encoded by the above nucleotide sequences, applications and production methods of these polynucleotides and polypeptides

Background technique

It is well known that complex signaling pathways exist widely in organisms. Each cell receives signals from the outside world through receptors on the surface, and then transmits the signals from the plasma membrane to the inside of the cell through a series of complex biochemical pathways. Protein phosphorylation and dephosphorylation is one of the most important biochemical pathways ( Bioorg Med Chem. 1997 Sep; 5(9):1751-1773.).

The main function of protein kinases is to catalyze the phosphorylation of proteins, which generally include protein serine/threonine kinases (serine/threonine kinase, referred to as "STK") and protein tyrosine kinases. Protein kinases play an important role in living organisms. In living organisms, they mainly regulate the normal function of proteins at the post-translational level through phosphorylation of proteins or binding to small molecules that play a regulatory role. The expression of serine/threonine protein kinase is affected by various factors such as glucocorticoids, growth factors, cell volume and cell damage.

STK is very conservative in evolution, and there are many conserved regions in its structure, especially in its catalytic function. For example, the N-terminus of the catalytic region is a glycine-rich segment, and there is a lysine near the rich segment. Studies have shown that this region is related to the binding of ATP. The center of the catalytic domain is the conserved aspartic acid, which is closely related to the catalytic activity of the kinase. Serine/threonine protein kinase widely exists in various parts of organisms, including various tissues, organs and serum, and participates in many processes necessary for normal cell function, such as: gene transcription, translation, protein secretion and Cell movement and organelle biosynthesis, etc.

Due to the fundamental role played by serine/threonine protein kinases in the physiological activities of cells, the researches on them, especially the expression of the members of this family in humans, are becoming more and more extensive and in-depth. In 1994, Small D and others cloned human stem cell STK-1 and studied its expression in normal human bone marrow. The results showed that STK-1 may be a growth factor receptor for hematopoietic stem cells (Proc Natl Acad Sci 1994, 91 (2): 459-463). In 1996, Chen, H., et al. studied the mutations of the ninth and tenth exons of STK-1 genome in human skin microvascular endothelial cells. In 1998, Li X. et al. reported that STK-2 was cloned. Brodbeck, D., Nakatani, K., Masure, S., et al. cloned and identified RAC-gamma serine/threonine protein kinase (Akt-3) (J.Biol.Chem.274(14), 9133-9136 (1999); Biochem.Biophys.Res.Commun.257(3), 906-910(1999); Eur.J.Biochem.265(1), 353-360(1999). 2001 Stanchi, F. et al. A serine/threonine protein kinase composed of 603 amino acid residues was also obtained when the human gene similar to the yeast sequence was isolated, and the Genbank accession number is CAA07196, which further proves that the serine/threonine protein kinase is necessary for cell growth Sex (Yeast, 18(1), 69-80).

However, prior to the present invention, no one has disclosed the human serine/threonine kinases hSTKa and hSTKc described in the present invention.

Contents of the invention

One of the objects of the present invention is to provide a polynucleotide sequence encoding a human serine/threonine kinase a ( human serine / threonine kinase a, hSTKa).

The second object of the present invention is to provide a polynucleotide sequence, which encodes a human serine/threonine kinase b ( human serine / threonine kinase c, hSTKc), which is the above-mentioned Transcript splicing of hSTKa.

The third object of the present invention is to provide a protein named human serine/threonine kinase a ( human serine / threonine kinase a, hSTKa).

The fourth object of the present invention is to provide a protein named human serine/threonine kinase c ( human serine / threonine kinase c, hSTKc).

The fifth object of the present invention is to provide a method for producing the hSTKa and hSTKc proteins using recombinant technology.

The sixth object of the present invention is to provide the application of hSTKa and hSTKc nucleic acid sequences and proteins.

The present invention provides an isolated DNA molecule, which includes: a nucleotide sequence encoding a polypeptide having human hSTK protein activity, and the nucleotide sequence has the same nucleotide sequence as that in SEQ ID NO.1 At least 70% homology; or the nucleotide sequence can hybridize with the nucleotide sequence in SEQ ID NO.1 under moderately stringent conditions. Preferably, said sequence encodes a polypeptide having the sequence shown in SEQ ID NO.2 or SEQ ID NO.4. More preferably, the sequence has the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.3.

In another aspect of the present invention, an isolated hSTK protein polypeptide is provided, which includes: a polypeptide having an amino acid sequence of SEQ ID NO.2 or SEQ ID NO.4, or an active fragment thereof, or an active derivative thereof. Preferably, the polypeptide is a polypeptide having the sequence of SEQ ID NO.2 or SEQ ID NO.4.

In another aspect of the present invention, there is provided a vector comprising the above isolated DNA.

In another aspect of the present invention, a host cell transformed with the vector is provided.

In another aspect of the present invention, a method for producing a polypeptide having hSTK protein activity is provided, the method comprising:

(a) The nucleotide sequence encoding the polypeptide having hSTK protein activity is operably connected to the expression control sequence to form an hSTK protein expression vector, and the nucleotide sequence is the same as SEQ ID NO.1 or SEQ ID NO.3 There is at least 70% homology of nucleotide sequences among them;

(b) transferring the expression vector in step (a) into a host cell to form a recombinant cell of the hSTK protein;

(c) cultivating the recombinant cells in step (b) under conditions suitable for expressing the hSTK protein polypeptide;

(d) isolating a polypeptide having hSTK protein activity.

In the present invention, "isolated", "purified" or "substantially pure" DNA means that the DNA or fragment has been separated from the sequences flanking it in its natural state, and also means that the DNA or fragment has been Separated from the components that naturally accompany nucleic acids and that have been separated from the proteins that accompany them in cells.

In the present invention, the term "hSTK protein (or polypeptide) coding sequence" refers to a nucleotide sequence encoding a polypeptide having hSTK protein activity, such as the 3-2009 nucleotide sequence and its degenerate sequence in SEQ ID NO.1 . The degenerate sequence refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the 3-2009 nucleotides of the coding frame of the sequence of SEQ ID NO.1. Due to the degeneracy of codons, a degenerate sequence with a homology as low as about 70% of the 3-2009 nucleotide sequence in SEQ ID NO.1 can also encode the sequence described in SEQ ID NO.2. The term also includes a nucleotide sequence capable of hybridizing to the nucleotide sequence from nucleotide 3-2009 in SEQ ID NO.1 under moderately stringent conditions, preferably under highly stringent conditions. In addition, the term also includes at least 70%, preferably at least 80%, more preferably at least 90% of the nucleosides of the nucleotide sequence from nucleotide 3-2009 in SEQ ID NO.1 acid sequence.

The term also includes variants of the sequence of SEQ ID NO. 1 that encode a protein having the same function as human hSTK. These variations include (but are not limited to): the deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , insertion and/or substitution, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) at the 5' and/or 3' end ) nucleotides.

In the present invention, "substantially pure" protein or polypeptide means that it accounts for at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight) of the total substance of the sample. or wet weight). Purity can be measured by any suitable method, such as measuring the purity of the polypeptide by column chromatography, PAGE or HPLC. A substantially pure polypeptide is substantially free of components that accompany it in its native state.

In the present invention, the term "hSTK protein polypeptide" refers to a polypeptide of SEQ ID NO.2 or SEQ ID NO.4 sequence having hSTK protein activity. The term also includes variant forms of the sequence of SEQ ID NO. 2 or SEQ ID NO. 4 that have the same function as human hSTK. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of hSTK proteins.

Variants of the polypeptide include: homologous sequences, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the hSTK DNA of the present invention under high or low stringency conditions, and using Polypeptide or protein obtained by antiserum against hSTK polypeptide. The invention also provides other polypeptides, such as fusion proteins comprising hSTK polypeptides or fragments thereof. In addition to substantially full-length polypeptides, the present invention also includes soluble fragments of hSTK polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the hSTK polypeptide sequence. consecutive amino acids.

The difference between the analog of hSTK protein or polypeptide referred to in the present invention and the natural hSTK polypeptide may be the difference in amino acid sequence, or the difference in modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

The invention also includes antisense sequences of hSTK polypeptide coding sequences. Such antisense sequences can be used to inhibit the expression of hSTK in cells.

The present invention also includes a probe molecule, which generally has 8-100, preferably 15-50 contiguous nucleotides of the hSTK polypeptide coding sequence. The probe can be used to detect whether there is a nucleic acid molecule encoding hSTK in a sample.

The present invention also includes a method for detecting hSTK nucleotide sequence, which comprises using the above-mentioned probe to hybridize with a sample, and then detecting whether the probe is combined. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the coding sequence of the hSTK polypeptide and can be located on both sides or in the middle of the coding sequence. Primers are generally 20-50 nucleotides in length.

In the present invention, various vectors known in the art, such as commercially available vectors, can be used.

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 cells are eukaryotic cells, such as CHO cells, COS cells and the like.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific for hSTK DNA or polypeptides encoded by its fragments. Here, "specificity" means that the antibody can bind to hSTK gene product or fragment. Preferably, it refers to those antibodies that can bind to hSTK gene products or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules capable of binding and inhibiting hSTK protein, as well as those antibodies that do not affect the function of hSTK protein. The invention also includes antibodies that bind to modified or unmodified forms of the hSTK gene product.

The present invention includes not only complete 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 ( Ladner et al., US Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of a murine antibody but retain portions of the antibody from humans.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified hSTK gene products, or antigenic fragments thereof, can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing hSTK or antigenic fragments thereof can be used to immunize animals to produce antibodies. Antibodies of the invention may also be monoclonal antibodies. 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; Kohler 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 present invention include antibodies capable of blocking the function of hSTK and antibodies that do not affect the function of hSTK. Various antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of hSTK gene products. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of hSTK gene products can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (such as glycosylated or phosphorylated proteins or polypeptides), which can be obtained by immunizing animals with gene products produced in eukaryotic cells (eg, yeast or insect cells).

In the present invention, the cDNA nucleotide sequence of human hSTKa is obtained in this way, using the human testis λgt10 cDNA library (purchased from Clontech Company) as a template, and using two pairs of oligonucleotides as primers: A1 CGATGACATCGACGGGGAAGGAC; A2 CAACAA GCTGCTG CAGGACCTG is Forward primer; B1: CAGGTCCTCATC CTCCTGGCTCG; B2 GATGGCCTC GTGGAGGT GACATG is the reverse primer for PCR. The PCR conditions were 93°C for 4 minutes, followed by 35 cycles of 93°C for 1 minute, 64°C for 1 minute and 72°C for 1 minute, and finally 72°C for 5 minutes. The PCR fragments detected by electrophoresis are fragments and impurities of about 1,000 and 1,100 base pairs, respectively. Remove impurities, increase the concentration of the required nucleotide fragments, digest the obtained fragments with PshAI respectively, and connect them, and finally obtain the target fragments.

Using the human testis λgt10 cDNA library (purchased from Clontech) as a template, two pairs of oligonucleotides were used as primers: A1CGATGACATCG ACGGGGAAGGAC; A2 CAACAA GCTGCTG CAGGACCTG as forward primers; B1: CA GGTCCTCATC CTCCTGGCTCG; B2 GCTAGTG TGTCTAAGGCTGCTCG as reverse primers, Perform PCR. The PCR conditions were 93°C for 4 minutes, followed by 35 cycles of 93°C for 1 minute, 64°C for 1 minute and 72°C for 1 minute, and finally 72°C for 5 minutes. The PCR fragments detected by electrophoresis are fragments and impurities of about 1,000 and 1,300 base pairs, respectively. Remove impurities, increase the concentration of the required nucleotide fragments, digest the obtained fragments with PshAI respectively, and connect them, and finally obtain the target fragments.

The hSTKa and hSTKc of the present invention are transcribed and translated from the same DNA, but different proteins are produced due to the different cutting and splicing methods of the transcripts during the transcription process, but they are very similar in sequence and preparation method sex.

Use the hSTKa and hSTKc proteins of the present invention in the Non-redundant GenBank+EMBL+DDBJ+PDB database and the Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database to perform nucleic acid and protein homology searches using BLAST software. It was found that it has certain homology with some identified serine/threonine kinase protein family members (such as CAA64382, JC1446 and CAA07196), and its structure has the characteristic functions of serine/threonine kinase protein family members area.

In summary, hSTKa and hSTKc of the present invention belong to serine/threonine kinase family members, and have the common characteristics of the family members, that is, they can bind cytokines through extracellular immunoglobulin-like structures and pass through their intracellular Part of the phosphatase activity transmits the signal carried by cytokines into the cell and acts on the target molecule, thereby regulating the physiological activities of cells and organisms. These physiological functions include promoting tissue growth, regulating cell growth and proliferation, activating and inhibiting immune cells, stimulating hematopoietic cells, activating inflammatory responses, and preventing virus and bacterial invasion.

Linking hSTKa and hSTKc nucleotide or amino acid sequences of the present invention with pharmaceutically acceptable carriers can be used to make reagents, medicines and kits to prevent, diagnose and treat diseases caused by abnormal expression of hSTKa or hSTKc.

Table 1 is the amino acid sequence homology comparison of hSTKa, hSTKac and CAA07196. Among them, " ^* " indicates that the amino acids at the corresponding positions in the three sequences are the same, ":" indicates that the amino acids at the corresponding positions in the three sequences are different but have roughly the same physical and chemical properties, and "." indicates the corresponding positions in the three sequences The physicochemical properties of the amino acids above have a certain identity, and "-" indicates that there is no corresponding amino acid at the corresponding position in the three sequences.

Detailed ways

Example 1

Cloning and Determination of cDNA Sequence of hSTKa

(1).Primer amplification

Using the human testis λgt10 cDNA library (purchased from Clontech) as a template, two pairs of oligonucleotides were used as primers: A1CGATGACATCG ACGGGGAAGGAC; A2 CAACAA GCTGCTG CAGGACCTG as forward primers; B1: CA GGTCCTCATC CTCCTGGCTCG; B2 GATGGCCTC GTGGAGGTGACATG as reverse primers, Perform PCR. The PCR condition was 93°C for 4 minutes, followed by 35 cycles of 93°C for 1 minute, 64°C for 1 minute and 72°C for 1 minute, and finally 72°C for 5 minutes. The PCR fragments detected by electrophoresis are fragments and impurities of about 1,000 and 1,100 base pairs, respectively. Remove impurities, increase the concentration of the required nucleotide fragments, digest the obtained fragments with PshAI respectively, and connect them, and finally obtain the target fragments.

(2).Sequencing of PCR products

The PCR amplified product was ligated with the pGEM-T ^TM vector (Promega), transformed into Escherichia coli JM103, the plasmid was extracted with the QIAprepPlasmid kit (QIAGEN), and the inserted fragment was directional and serially deleted with the double-stranded nested deletion kit (Pharmacia) , and then quickly identify and sort the deletions by PCR. The sequentially truncated deletions were sequenced with the SequiTherm EXCEL ^TM DNA Sequencing Kit (Epicentre Technologies), and finally the full-length cDNA sequence was obtained, with a total of 2023 bp. The detailed sequence is shown in SEQ ID NO.1, where the open reading frame is located at positions 3-2009 Nucleotides.

The amino acid sequence of hSTKa was deduced according to the obtained full-length cDNA sequence, with a total of 668 amino acid residues, and its amino acid sequence is detailed in SEQ ID NO.2.

Example 2

Expression of hSTKa in Escherichia coli

In this example, the cDNA sequence encoding hSTKa was amplified with PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence to obtain hSTKa cDNA as an insert fragment.

The 5' oligonucleotide primer sequence used in the PCR reaction is:

5'-CCCCGGATCCATGACA TCGACGGGGA AGG, the primer contains the restriction endonuclease restriction endonuclease site of BamHI, after the restriction site is the partial coding sequence of hSTKa beginning with the initiation codon;

The 3' end primer sequence is:

5'TGACAAGCTTACTTTTCG GGATAATTC, the primer contains the restriction endonuclease restriction site of HindIII, translation terminator and partial coding sequence of hSTKa.

The restriction endonuclease cutting sites on the primers correspond to the restriction endonuclease cutting sites on the bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA), which encodes antibiotic resistance ( Amp ^r ), a bacterial origin of replication (ori), an IPTG-regulated promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His ) and restriction enzyme cloning sites.

The pQE-9 vector and insert were digested with BamHI and HindIII, and the insert was subsequently ligated into the pQE-9 vector keeping the open reading frame at the beginning of the bacterial RBS. The ligation mixture was subsequently used to transform an E. coli strain purchased from Qiagen under the tradename M15/rep4 containing multiple copies of the plasmid pREP4 expressing the lacI repressor and carrying kanamycin resistance (Kan ^r ). The transformants were screened on the LB culture dish containing Amp and Kan, the plasmid was extracted, the size and direction of the inserted fragment were identified by digestion with PshAI, and the cDNA fragment of hSTK was correctly inserted into the vector by sequencing.

Positive transformant clones containing the desired constructs were cultured overnight (O/N) in liquid LB medium supplemented with Amp (100 μg/ml) and Kan (25 μg/ml). The overnight (O/N) culture was diluted at a dilution rate of 1:100-1:250, and then inoculated into a large volume of culture medium. When the cultured cells grew to 600 optical density (OD ₆₀₀ ) of 0.4-0.6, IPTG ( "Isopropylthio-β-D-galactoside") to a final concentration of 1 mM. By inactivating the lacI repressor, IPTG-induced initiation of P/O leads to increased gene expression levels. Cells were incubated for an additional 3-4 hours followed by centrifugation (6000 xg, 20 minutes). Inclusion bodies were lysed by sonication, cells were collected and the cell pellet was dissolved in 6M guanidine hydrochloride. After clarification, the solubilized hSTK was purified from solution by nickel-chelate column chromatography under conditions that allow tight binding of the 6-His tag-containing protein. hSTK was eluted from the column with 6M guanidine hydrochloride (pH 5.0). Several methods can be used to denature and precipitate proteins from guanidine hydrochloride. Either use a dialysis step to remove the guanidine hydrochloride, or isolate the purified protein from the nickel-chelation column, and the purified protein can be bound to a second column with a decreasing linear guanidine hydrochloride gradient. Proteins were denatured upon binding to the column and subsequently eluted with guanidine hydrochloride (pH 5.0). Finally, the soluble protein was dialyzed against PBS, and the protein was then stored in a stock solution with a final concentration of 10% (w/v) glycerol.

In addition, the N-terminal and C-terminal 10 amino acid lengths of the expressed protein were sequenced by conventional methods, and it was found to be consistent with the sequence of SEQ ID NO.2.

Example 3

Expression of hSTKa in eukaryotic cells (CHO cell lines)

In this example, the cDNA sequence encoding hSTKa was amplified with the following oligonucleotide primers to obtain hSTKacDNA as an insert.

The 5' oligonucleotide primer sequence used in the PCR reaction is:

5’-CCCCAAGCTTATGACA TCGACGGGGA AGG

The primer contains a HindIII restriction endonuclease enzyme cutting site, followed by the partial coding sequence of hSTKa starting from the start codon after the enzyme cutting site;

The 3' end primer sequence is:

5’-TGACGGATCCACTTTTCG GGATAATTC

The primer contains the cutting site of BamHI restriction endonuclease, a translation terminator and the partial coding sequence of hSTKa.

The restriction endonuclease cutting sites on the primers correspond to the restriction endonuclease cutting sites on the CHO cell expression vector pcDNA3, which encodes antibiotic resistance (Amp ^r and Neo ^r ), a phage replication Origin (f1 ori), a viral origin of replication (SV40 ori), a T7 promoter, a viral promoter (P-CMV), a Sp6 promoter, an SV40 promoter, an SV40 tailing signal and the corresponding polyA sequence , a BGH tailed signal and the corresponding polyA sequence.

The pcDNA3 vector and insert were digested with HindIII and BamHI, and then the insert was ligated into the pcDNA3 vector. The E. coli DH5α strain was subsequently transformed with the ligation mixture. Transformants were screened on LB dishes containing Amp, and clones containing the desired construct were cultured overnight (O/N) in LB liquid medium supplemented with Amp (100 μg/ml). The plasmid was extracted, digested with PshAI to identify the size and direction of the inserted fragment, and sequenced to verify that the cDNA fragment of hSTKa had been correctly inserted into the vector.

Plasmid transfection into CHO cells was performed by lipofection with Lipofectin (GiBco Life). After 48 hours of transfection, after 2-3 weeks of continuous G418 pressurized selection, the cells were collected and the supernatant of the cells was assayed for protease activity. G418 was removed and subcultured continuously; for extreme dilution of mixed clone cells, select cell subclones with higher protein activity. The above-mentioned positive subclones were cultured in large quantities according to conventional methods. After 48 hours, the cells and supernatant were collected, and the cells were disrupted by ultrasonic lysis. Using 50 mM Tris·HCl (pH 7.6) solution containing 0.05% Triton as the balance and eluent, the activity peaks of the above proteins were collected with a pre-balanced SuperdexG-75 column. A DEAE-Sepharose column equilibrated with 50mM Tris HCl (pH8.0) was used to carry out gradient elution with 50mM Tris HCl (pH8.0) solution containing 0-1M NaCl as the eluent, and the activity peak of the above protein was collected. Then, the expressed protein solution was dialyzed with PBS (pH7.4) as the dialysate. Finally freeze-dried and stored.

In addition, the N-terminal and C-terminal 10 amino acid lengths of the expressed protein were sequenced by conventional methods, and it was found to be consistent with the sequence of SEQ ID NO.2.

Example 4

Cloning and Determination of cDNA Sequence of hSTKc

1. Primer amplification

Using the human testis λgt10 cDNA library (purchased from Clontech) as a template, two pairs of oligonucleotides were used as primers: A1CGATGACATCG ACGGGGAAGGAC; A2 CAACAA GCTGCTG CAGGACCTG as forward primers; B1: CA GGTCCTCATC CTCCTGGCTCG; B2 GCTAGTG TGTCTAAGGCTGCTCG as reverse primers, Perform PCR. The PCR conditions were 93°C for 4 minutes, followed by 35 cycles of 93°C for 1 minute, 64°C for 1 minute and 72°C for 1 minute, and finally 72°C for 5 minutes. The PCR fragments detected by electrophoresis are fragments and impurities of about 1,000 and 1,300 base pairs, respectively. Remove impurities, increase the concentration of the required nucleotide fragments, digest the obtained fragments with PshAI respectively, and connect them, and finally obtain the target fragments.

2. Sequencing of PCR products

The PCR amplified product was ligated with the pGEM-T ^TM vector (Promega), transformed into Escherichia coli JM103, the plasmid was extracted with the QIAprepPlasmid kit (QIAGEN), and the inserted fragment was directional and serially deleted with the double-stranded nested deletion kit (Pharmacia) , and then quickly identify and sort the deletions by PCR. The sequentially truncated deletions were sequenced with SequiTherm EXCEL ^TM DNA Sequencing Kit (Epicentre Technologies), and finally the full-length cDNA sequence was obtained, with a total of 2223 bp. The detailed sequence is shown in SEQ ID NO.3, where the open reading frame is located at position 3-2009 Nucleotides.

The amino acid sequence of hSTKc was deduced according to the obtained full-length cDNA sequence, with a total of 736 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.4.

Example 5

Expression of hSTKc in Escherichia coli

In this example, the cDNA sequence encoding hSTKc was amplified with PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence to obtain hSTKc cDNA as an insert.

The 5' oligonucleotide primer sequence used in the PCR reaction is:

5'-CCCCGGATCCATGACA TCGACGGGGA AGG, the primer contains the restriction endonuclease restriction site of BamHI, after the restriction site is the partial coding sequence of hSTKc beginning with the initiation codon;

The 3' end primer sequence is:

5'-AGTGAAGCTTAG GCTGCTCGCG GCGGGCG, the primer contains the restriction endonuclease restriction site of HindIII, translation terminator and partial coding sequence of hSTKc.

The restriction endonuclease cutting sites on the primers correspond to the restriction endonuclease cutting sites on the bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA), which encodes antibiotic resistance ( Amp ^r ), a bacterial origin of replication (ori), an IPTG-regulated promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His ) and restriction enzyme cloning sites.

The pQE-9 vector and insert were digested with BamHI and HindIII, and the insert was subsequently ligated into the pQE-9 vector keeping the open reading frame at the beginning of the bacterial RBS. The ligation mixture was subsequently used to transform an E. coli strain purchased from Qiagen under the tradename M15/rep4 containing multiple copies of the plasmid pREP4 expressing the lacI repressor and carrying kanamycin resistance (Kan ^r ). The transformants were screened on the LB culture dish containing Amp and Kan, the plasmid was extracted, the size and direction of the inserted fragment were identified by digestion with PshAI, and the cDNA fragment of hSTKc was correctly inserted into the vector by sequencing.

Positive transformant clones containing the desired constructs were cultured overnight (O/N) in liquid LB medium supplemented with Amp (100 μg/ml) and Kan (25 μg/ml). The overnight (O/N) culture was diluted at a dilution rate of 1:100-1:250, and then inoculated into a large volume of culture medium. When the cultured cells grew to 600 optical density (OD ₆₀₀ ) of 0.4-0.6, IPTG ( "Isopropylthio-β-D-galactoside") to a final concentration of 1 mM. By inactivating the lacI repressor, IPTG-induced initiation of P/O leads to increased gene expression levels. Cells were incubated for an additional 3-4 hours followed by centrifugation (6000 xg, 20 minutes). Inclusion bodies were lysed by sonication, cells were collected and the cell pellet was dissolved in 6M guanidine hydrochloride. After clarification, the solubilized hSTK was purified from solution by nickel-chelate column chromatography under conditions that allow tight binding of the 6-His tag-containing protein. hSTK was eluted from the column with 6M guanidine hydrochloride (pH 5.0). Several methods can be used to denature and precipitate proteins from guanidine hydrochloride. Either use a dialysis step to remove the guanidine hydrochloride, or isolate the purified protein from the nickel-chelation column, and the purified protein can be bound to a second column with a decreasing linear guanidine hydrochloride gradient. Proteins were denatured upon binding to the column and subsequently eluted with guanidine hydrochloride (pH 5.0). Finally, the soluble protein was dialyzed against PBS, and the protein was then stored in a stock solution with a final concentration of 10% (w/v) glycerol.

In addition, the N-terminal and C-terminal 10 amino acid lengths of the expressed protein were sequenced by conventional methods, and it was found to be consistent with the sequence of SEQ ID NO.4.

Example 6

Expression of hSTKc in eukaryotic cells (CHO cell lines)

In this example, the cDNA sequence encoding hSTKc was amplified with the following oligonucleotide primers to obtain hSTKccDNA as an insert.

The 5' oligonucleotide primer sequence used in the PCR reaction is:

5’-CCCCAAGCTTATGACA TCGACGGGGA AGG

The primer contains a HindIII restriction endonuclease enzyme cutting site, followed by a partial coding sequence of hSTKc beginning with a start codon after the enzyme cutting site;

The 3' end primer sequence is:

5’-AG TGGGATCCAG GCTGCTCGCG GCGGGCG

The primer contains the cutting site of BamHI restriction endonuclease, a translation terminator and the partial coding sequence of hSTKc.

The restriction endonuclease cutting sites on the primers correspond to the restriction endonuclease cutting sites on the CHO cell expression vector pcDNA3, which encodes antibiotic resistance (Amp ^r and Neo ^r ), a phage replication origin (f1 ori), a viral origin of replication (SV40ori), a T7 promoter, a viral promoter (P-CMV), a Sp6 promoter, an SV40 promoter, an SV40 tailing signal and the corresponding polyA sequence, A BGH tailing signal and corresponding polyA sequence.

The pcDNA3 vector and insert were digested with HindIII and BamHI, and then the insert was ligated into the pcDNA3 vector. The E. coli DH5α strain was subsequently transformed with the ligation mix. Transformants were screened on LB dishes containing Amp, and clones containing the desired construct were cultured overnight (O/N) in LB liquid medium supplemented with Amp (100 μg/ml). The plasmid was extracted, digested with PshAI to identify the size and direction of the inserted fragment, and sequenced to verify that the cDNA fragment of hSTKc had been correctly inserted into the vector.

Plasmid transfection into CHO cells was performed by lipofection with Lipofectin (GiBco Life). After 48 hours of transfection, after 2-3 weeks of continuous G418 pressurized selection, the cells were collected and the supernatant of the cells was assayed for protease activity. G418 was removed and subcultured continuously; for extreme dilution of mixed clone cells, select cell subclones with higher protein activity. The above-mentioned positive subclones were cultured in large quantities according to conventional methods. After 48 hours, the cells and supernatant were collected, and the cells were disrupted by ultrasonic lysis. Using 50 mM Tris·HCl (pH 7.6) solution containing 0.05% Triton as the balance and eluent, the activity peaks of the above proteins were collected with a pre-balanced SuperdexG-75 column. Then use a DEAE-Sepharose column equilibrated with 50mM Tris HCl (pH8.0) to carry out gradient elution with 50mM Tris HCl (pH8.0) solution containing 0-1M NaCl as the eluent, and collect the activity peak of the above protein. Then, the expressed protein solution was dialyzed with PBS (pH7.4) as the dialysate. Finally freeze-dried and stored.

In addition, the N-terminal and C-terminal 10 amino acid lengths of the expressed protein were sequenced by conventional methods, and it was found to be consistent with the sequence of SEQ ID NO.4.

Example 7

homologous comparison

Use hSTKa of the present invention and hSTKc albumen (SEQ ID NO.2 and SEQ ID NO.4) in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database with BLAST The software performs nucleic acid and protein homology searches. It was found that it has certain homology with some identified serine/threonine kinase protein family members (such as CAA64382, JC1446 and CAA07196), and its structure has the structural characteristics of serine/threonine kinase protein family members .

The structural features of serine/threonine kinase protein family members are [LIVMFYC]-x-[HY]-x-D-[LIVMFY]-K-x(2)-N-[LIVMFYCT](3). Among them, [LIVMFYC] means Choose one of the amino acids from LIVMFYC, x is any amino acid, and x(2) is any 2 amino acids. The proteins of the present invention belong to this structure as follows:

hSTKa：RLEKTLGKGQTGLVKLGVHCVTCQKVAIKIVNREKLSESVLMKVEREIAILKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLTPKEARKFFRQIISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPHYACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPHFIPPDCQSLLRGMSEVDAARRLTLEHIQKHIWY(SEQ ID NO.2中19-270)；

hSTKc：RLEKTLGKGQTGLVKLGVHCVTCQKVAIKIVNREKLSESVLMKVEREIAILKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLTPKEARKFFRQIISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPHYACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPHFIPPDCQSLLRGMSEVDAARRLTLEHIQKHIWY(SEQ ID NO.4中19-270)( htttp：//smart.embl-heidelberg.de/smart/show motifs.pl )

In summary, hSTKa and hSTKc of the present invention belong to serine/threonine kinase family members, and have the common characteristics of the family members, that is, they can bind cytokines through extracellular immunoglobulin-like structures and pass through their intracellular Part of the phosphatase activity transmits the signal carried by cytokines into the cell and acts on the target molecule, thereby regulating the physiological activities of cells and organisms. These physiological functions include promoting tissue growth, regulating cell growth and proliferation, activating and inhibiting immune cells, stimulating hematopoietic cells, activating inflammatory responses, and preventing virus and bacterial invasion.

The hSTKa and hSTKc of the present invention can be used as members of the family for further functional research, and can also be used to produce fusion proteins with other proteins, such as immunoglobulins to produce fusion proteins. In addition, hSTKa and hSTKc of the present invention can also be fused or exchanged fragments with other members of the family to produce new proteins, for example, the N-terminal of hSTKa and hSTKc of the present invention can be exchanged with the N-terminal of mouse STK to produce new proteins Proteins with higher activity or new properties.

Antibodies against hSTKa and hSTKc of the present invention are used for screening other members of the family, or for affinity purification of related proteins (such as other members of the family).

Example 8

Antibody preparation

The hSTKa and hSTKc recombinant proteins of the present invention are used to immunize animals to produce antibodies, as follows. The recombinant molecules are separated by chromatography for further use. It can also be separated by SDS-PAGE gel electrophoresis, and the electrophoresis bands are excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Mice were injected intraperitoneally with 50-100 [mu]g/0.2 ml emulsified protein. Fourteen days later, mice were boosted by intraperitoneal injection of the same antigen emulsified with incomplete Freund's adjuvant at a dose of 50-100 µg/0.2 ml. Give booster immunizations at least three times every 14 days. The specific reactivity of the obtained antiserum was assessed by its ability to precipitate the hSTK gene translation product in vitro.

Example 9

as a substrate for microarrays

Microarray, also known as DNA chip. By using some well-known biological software (such as LASERGENESOFTWARE (DNASTAR), select hSTK full-length cDNA, EST, or gene fragments as the substrate sample of the microarray. Then by using inkjet technology and a series of chemical methods such as radiation, chemical Fix the sample on the carrier, such as glass, to obtain a chip (Schena, M. et al. (1995) Science 270: 467-470; and Shalon, D. et al. (1996) Genome Res. 6:639-645.), the formed elements are point-like, strip-shaped, etc. A typical chip usually contains a certain number of elements. After the hybridization reaction, unbound probes are washed away, and then a scanner is used to detect hybridization The components of the reaction and the degree of the reaction. The complementarity and binding amount of the probe to each chip component can be judged by analyzing the scanned images.

Hybridization results can be used to detect hSTKa and hSTKc gene variation, mutation and polymorphism, understand the molecular basis of diseases caused by abnormal expression of hSTKa and hSTKc, diagnose diseases, and improve and monitor the activity of related drugs.

Example 10:

Kit preparation

Kit 1:

It contains (1) primer pairs and instructions for specific amplification of hSTKa and hSTKc. The kit may or may not contain the reagents required to reverse transcribe mRNA into cDNA. Wherein, the sequence of the specific primer is derived from the sequence of SEQ ID NO:1. The reverse transcribed cDNA is specifically amplified to detect whether the sample contains the nucleic acid sequences of hSTKa and hSTKc. The kit can also contain other reagents required for performing PCR reactions, such as buffers and the like.

Furthermore, it is preferred that at least one of the primers used spans two exons, since then the genomic sequences corresponding to hSTKa and hSTKc will not give rise to amplification products.

Kit 2:

The kit contains specific antibodies against hSTKa and hSTKc (such as polyclonal antibodies prepared in Example 5, or monoclonal antibodies against hSTKa and hSTKc produced by standard hybridoma technology), and instructions for use. This kit is used to directly detect the presence or absence of hSTKa and hSTKc proteins in samples. Immune complexes are first formed by specific immune reactions, and then detected by conventional techniques.

Kit 3:

The kit contains probes that specifically hybridize to hSTKa and hSTKc mRNA. It may or may not also contain a hybridization buffer. The kit detects whether there are nucleic acid molecules of hSTKa and hSTKc in the sample through the hybridization reaction between the nucleic acid molecules and hSTKa and hSTKc specific probes.

The kit can also be used to detect gene variation, mutation and polymorphism, and detect a series of genes related to the hSTK of the present invention, thereby playing an auxiliary role in the diagnosis and treatment of related diseases.

Example 11:

made into medicine

The hSTKa and hSTKc proteins of the present invention and their antibodies, inhibitors, antagonists or receptors can be used as medicines, which can promote wound healing after burns, tissue resection and even organ removal, and prevent wound infection. The hSTKa and hSTKc nucleic acid antisense chains or hSTKa and hSTKc antibodies of the present invention are made into kits, which can be used to alleviate or assist in the treatment of diseases caused by excessive expression of hSTKa and hSTKc, and can also assist in the clinical diagnosis of tumors and cancers. Prevention and treatment.

When the protein of the present invention and its antibody, inhibitor, antagonist, or receptor are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

In addition, hSTKa and hSTKc nucleic acid (coding sequence or antisense sequence) of the present invention can be directly introduced into cells to increase the expression level of hSTKa and hSTKc or inhibit the overexpression of hSTKa and hSTKc. The hSTKa and hSTKc proteins or their active polypeptide fragments of the present invention can be administered to patients to treat or alleviate related diseases caused by hSTKa and hSTKc deletion, non-function or abnormality. In addition, the nucleic acid sequence or antibody based on the present invention can also be used for relevant diagnosis or prognosis.

When the hSTKa and hSTKc protein polypeptides of the present invention are used as medicines, the therapeutically effective dose is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is about 10 mg/kg body weight. µg/kg body weight - about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

SEQUENCE LISTING <210>1<211>2032<212>nucleotide<213>human<220><221>coding sequence<222>(3)..(2009)<223><400>1cg atg aca tcg acg ggg aag gac ggc ggc gcg cag cac gcg cag tat         47Met Thr Ser Thr Gly Lys Asp Gly Gly Ala Gln His Ala Gln Tyr1               5                   10                  15gtt ggg ccc tac cgg ctg gag aag acg ctg ggc aag ggg cag aca ggt        95Val Gly Pro Tyr Arg Leu Glu Lys Thr Leu Gly Lys Gly Gln Thr Gly

20 25 30CTG GTG AAG CTG GGG GGG GTT CAC GTC GTC ACC TGC CAG GCC ATC 143leu Val Lys Leu Gly Val Val THR CYS Val Ala Ile

35 40 45AAG ATC GAC CGT GAG AAG CTC GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG CTG AAG GTG 191lys Ile Val Asn ARG GLU LEU Ser Val Leu Met Lys Val Val Val

50 55 60gag CGG GAG ATC GCG ATC CTG CTC CTC ATT GAG CAC CAC CAC CAC CAC CTC

65                  70                  75aag ctg cac gac gtt tat gaa aac aaa aaa tat ttg tac ctg gtg cta       287Lys Leu His Asp Val Tyr Glu Asn Lys Lys Tyr Leu Tyr Leu Val Leu80                  85                  90                  95gaa cac gtg tca ggt ggt gag ctc ttc gac tac ctg gtg aag aag ggg 335Glu His Val Ser Gly Gly Glu Leu Phe Asp Tyr Leu Val Lys Lys Gly

100 105 110AGG CTG ACG CCT AAG GAG GCT CGG AAG TTC TTC CGG CAG ATC ATC ATC TCT 383ARG Leu Thr Pro Lys Glu ARG LYS PHE PHE GLN ILE ILN iLe Ile iLn iLe PLN iLE PLN iLE PLU -

115 120 125GCG CTG GAC TTC TGC CAC CAC CAC CAC ATA TGC CAC AGG GAT CTG AAA 431ALA Leu ASP PHE CYS Serle Cys

135 135 140cct GAA AAC CTC CTG CTG GAC GAG AAC AAC AAC ATC CGC ATC GCA GAC 479Pro Glu Leu Leu ASN Asn Ile ARG Ile Ala Ala Asp

145                 150                 155ttt ggc atg gcg tcc ctg cag gtt ggc gac agc ctg ttg gag acc agc      527Phe Gly Met Ala Ser Leu Gln Val Gly Asp Ser Leu Leu Glu Thr Ser160                 165                 170                 175tgt ggg tcc ccc cac tac gcc tgc ccc gag gtg atc cgg ggg gag aag 575Cys Gly Ser Pro His Tyr Ala Cys Pro Glu Val Ile Arg Gly Glu Lys

180 185 190TAT GAC GGC CGG AAG GCG GAC GAC GAC GTG AGC TGC GGC GGC GGC ATC CTG TTC 623TYR ARG LYS Ala Ala Ala's TrS Ge Leu Phes

195 200 205GCC TTG CTG GGG GGG GGG GCT CTG CCC GAC GAC GAC GAC GAC AAC TTG CGA CGA 671ALA Leu Val Gly Ala PHE ASP ASN Leu ARG Gln Leu ARG Gln

210 215 220CTG CTG GAG AAG GTG AAG CGG GGC GGC GTG TTC CAC CAC CCG CAC TTT ATC 719leu Leu Lys Val Val Phe His Met Pro His PHE Ile

225                 230                 235ccg ccc gac tgc cag agt ctg cta cgg ggc atg agc gag gtg gac gcc      767Pro Pro Asp Cys Gln Ser Leu Leu Arg Gly Met Ser Glu Val Asp Ala240                 245                 250                 255gca cgc cgc ctc acg cta gag cac att cag aaa cac ata tgg tat ata 815Ala Arg Arg Leu Thr Leu Glu His Ile Gln Lys His Ile Trp Tyr Ile

260 265 270GGG GGC AAG AAT GAG CCC GAA CCA GAG CAG CAG CAG CCC AAG GTG 863GLY GLY LYS ASN GLU PRO GLU Gln Pro Ile Pro Arg Lys Val

275 280 285CAG ATC CGC TCG CTG CCC AGC CTG GAC GAC GAC GAC CCC GAC GAC GAC GTG 911GLN Ile ARG Sero Seru Glo ASP PRO ASP Val Leuu

290 295 300GAC AGC ATG CAC TCA CTG GGC TGC TGC CGA GAC CGC AAC AAG CTG 959asp Serou Gly Cys PHE ARG ARG Asn Lys Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's Lu Le Lu Lu Lu Lu Lu -Aws that Cla's that

305                 310                 315cag gac ctg ctg tcc gag gag gag aac cag gag aag atg att tac ttc     1007Gln Asp Leu Leu Ser Glu Glu Glu Asn Gln Glu Lys Met Ile Tyr Phe320                 325                 330                 335ctc ctc ctg gac cgg aaa gaa agg tac ccg agc cag gag gat gag gac 1055Leu Leu Leu Asp Arg Lys Glu Arg Tyr Pro Ser Gln Glu Asp Glu Asp

340 345 350CTG CCC CCC CCC CCC CGG AAC GAG ATA GAC CCC CCC CGG AAG CGT GAC TCC 1103leu Pro Pro ARG Asn Glu Pro Pro ARG LYS ARG Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val ASP Ser

355 360 365CCG ATG CTG AAC CGG CAC GGC AAG CGG CGG CCA GAA CGC AAA TCC ATG 1151PRO MET Leu Asn ARG HS ARG Pro Glu ARG LYS MET MET

370 375 380GAG GTG CTC AGC GTG GAC GGC GGC GGC GGC GGC CCG CCG CCG CGG CGG 1199Glu Val Val THR AS PRO Val Pro Ala ARA ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG

385                 390                 395gcc att gag atg gcc cag cac ggc cag agg tct cgg tcc atc agc ggt     1247Ala Ile Glu Met Ala Gln His Gly Gln Arg Ser Arg Ser Ile Ser Gly400                 405                 410                 415gcc tcc tca ggc ctt tcc acc agc cca ctc agc agc ccc cgg gtg acc 1295Ala Ser Ser Gly Leu Ser Thr Ser Pro Leu Ser Ser Pro Arg Val Thr

420 425 430CCT CAC CCC TCA CCA AGG GGC AGT CCC CCC CCC CCC AAG GGG ACA 1343PRO HIS PRO ARG GLY GLY Serg

435 440 445CCT GTC CAC ACG CCA AAG GAG GAG AGC CCG GCC ACG CCC CCC CCC ACG 1391Pro Val His THR Pro LYS GLY THRY THR Pro ASN PRO Asn

450 455 460ccc ccg tcc agc ccc agc gtc gga ggg gtg ccc tgg agg gcg cgg ctc 1439Pro Ar Val u Pro Ser A la Ser p Tr g Pro Val Gly

465                 470                 475aac tcc atc aag aac agc ttt ctg ggc tca ccc cgc ttc cac cgc cga     1487Asn Ser Ile Lys Asn Ser Phe Leu Gly Ser Pro Arg Phe His Arg Arg480                 485                 490                 495aaa ctg caa gtt ccg acg ccg gag gag atg tcc aac ctg aca cca gag 1535Lys Leu Gln Val Pro Thr Pro Glu Glu Met Ser Asn Leu Thr Pro Glu

500 505 510TCG TCA GAG CTG GCG AAG AAG AAG TCC TGG TGG AAC TTC AGC AGC 1583ser Seru Leu Ala Lys Sern Phe Gly Asn Phe Ile Ser

515 520 525CTG Gag Aag Gag Gag Gag CAG ATC GTG GTC ATC AAA GAC AAA CCT CTG 1631leu LYS Glu Gln Ile Lysre Lysp Lysp Lou

530 535 540AGC TCC AAG GCT GCT GAC ATC GCC GCC TTC CTG AGT CCC AGT 1679ser Serle Lysp Ile Val His Ala Phe Leu Serle Pro Ser

545                 550                 555ctc agc cac agc gtc atc tcc caa acg agc ttc cgg gcc gag tac aag     1727Leu Ser His Ser Val Ile Ser Gln Thr Ser Phe Arg Ala Glu Tyr Lys560                 565                 570                 575gcc acg ggg ggg cca gcc gtg ttc cag aag ccg gtc aag ttc cag gtt 1775Ala Thr Gly Gly Pro Ala Val Phe Gln Lys Pro Val Lys Phe Gln Val

580 585 590GAT ATC ACC TAC ACG GGT GGT GGG GCG GCG GCG CAG AAG GAG AAC GGC ATC 1823asp Ile Thr Tyr Ty GLY GLY Gln Lysn Glys GLU Asn GLY ile

595 6005TAC TCC GTC ACC TTC ACC CTC CTC CTC CCC CCC CGC TTC TTC AAG 1871Tyr Seru Thr Leu Serg Phe Phe Lys

610 615 620AGG GAG GAG GAG ACC ATC CAG GCC CAG CTG CTG AGC ACA CAC GAC CCG 1919arg Val Val Gln Ala Gln Leu Leu Seru His ASP PRO Pro

625                 630                 635cct gcg gcc cag cac ttg tca gac acc act aac tgt atg gaa atg atg     1967Pro Ala Ala Gln His Leu Ser Asp Thr Thr Asn Cys Met Glu Met Met640                 645                 650                 655acg ggg cgg ctt tcc aaa tgt gga att atc ccg aaa agt taa 2009Thr Gly Arg Leu Ser Lys Cys Gly Ile Ile Pro Lys Ser

660 665CATGTCACCT CCACGAGCC ATC 2032 <210> 2 <211> 668 <212> Amino acid <213> Human <400> 2MET THR SER GLY LYS ASP GLY GLN HIS Ala Gln Tyr Valr Tyr ARG LEU GLRG LEU GLL ARG Leu Gl Leu Gly Lys Gly Gln Thr Gly Leu

20 25 30Val Lys Leu Gly Val His Cys Val Thr Cys Gln Lys Val Ala Ile Lys

35 40 45Ile Val Asn Arg Glu Lys Leu Ser Glu Ser Val Leu Met Lys Val Glu

50 55 60arg Glu iLe Ale Leu Lys Leu Ile Glu His Prou His Val Leu Lys65 70 80leu His ASP Val Tyr Glu Asn Lys tyr Leu Val Leu Glu Glu Glu Glu Glu Glu

85 90 95His Val Ser Gly Gly Glu Leu Phe Asp Tyr Leu Val Lys Lys Gly Arg

100 105 110Leu Thr Pro Lys Glu Ala Arg Lys Phe Phe Arg Gln Ile Ile Ser Ala

115 120 125Leu Asp Phe Cys His Ser His Ser Ile Cys His Arg Asp Leu Lys Pro

130 135 140GLU ASN Leu Leu Leu asp Glu Lysn Asn Ile Ala Ala ALA ALA ASP PHE145 155 160GLY MET ALA Seru Gln Val GLY ASER Leu Leu THR Ser CYS CYS CYS CYS CYS CYS CYS CYs

165 170 175Gly Ser Pro His Tyr Ala Cys Pro Glu Val Ile Arg Gly Glu Lys Tyr

180 185 190Asp Gly Arg Lys Ala Asp Val Trp Ser Cys Gly Val Ile Leu Phe Ala

195 200 205 Leu Leu Val Gly Ala Leu Pro Phe Asp Asp Asn Leu Arg Gln Leu

210 215 220leu Lys Val Val Lys ARG GLY VAL PHE HIS MET Pro His PHE Ile Pro2225 230 235 240pro ASP CYS GLN Seru Leu ARG GLY MET Serg Ala Ala Ala Ala Ala Ala Ala Ala Ala

245 250 255Arg Arg Leu Thr Leu Glu His Ile Gln Lys His Ile Trp Tyr Ile Gly

260 265 270Gly Lys Asn Glu Pro Glu Pro Glu Gln Pro Ile Pro Arg Lys Val Gln

275 280 285Ile Arg Ser Leu Pro Ser Leu Glu Asp Ile Asp Pro Asp Val Leu Asp

290 295 300ser Met His Seru GLY CYS PHE ARG ARG Asn LEU Leu Leu GLN30 310 320ASP Leu Leu Leu Glu Gln Gln Gln Gln Met Ile Tyr Phe Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's Met Ile Tyr Leu's Ges that theanity

325 330 335Leu Leu Asp Arg Lys Glu Arg Tyr Pro Ser Gln Glu Asp Glu Asp Leu

340 345 350Pro Pro Arg Asn Glu Ile Asp Pro Pro Arg Lys Arg Val Asp Ser Pro

355 360 365 Met Leu Asn Arg His Gly Lys Arg Arg Pro Glu Arg Lys Ser Met Glu

370 375 380val Leu Ser Val THR ASP GLY GLY GLE VAL PRO ALA ARG ARG ALA385 395 400ILE GLU MET ALA GLN HIS GLN ARG Serg Serg Serg Serge Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

405 410 415Ser Ser Gly Leu Ser Thr Ser Pro Leu Ser Ser Pro Arg Val Thr Pro

420 425 430His Pro Ser Pro Arg Gly Ser Pro Leu Pro Thr Pro Lys Gly Thr Pro

435 440 445Val His Thr Pro Lys Glu Ser Pro Ala Gly Thr Pro Asn Pro Thr Pro

450 455 460Pro Ser Ser Val Gly Gly Val Pro TRP ARG ARG Leu asn465 475 480SER ILE LYS Asn Ser, Leu ARG PHE HIS ARG ARG LYS LYS

485 490 495Leu Gln Val Pro Thr Pro Glu Glu Met Ser Asn Leu Thr Pro Glu Ser

500 505 510Ser Pro Glu Leu Ala Lys Lys Ser Trp Phe Gly Asn Phe Ile Ser Leu

515 520 525Glu Lys Glu Glu Gln Ile Phe Val Val Ile Lys Asp Lys Pro Leu Ser

530 535 540Ser Ile La Ala asp Ile Val His Ala Phe Leu Serle Pro Seru545 550 560Ser His Sering Ile Serite ARG Ala Glu Tyr Lys Ala

565 570 575Thr Gly Gly Pro Ala Val Phe Gln Lys Pro Val Lys Phe Gln Val Asp

580 585 590Ile Thr Tyr Thr Glu Gly Gly Glu Ala Gln Lys Glu Asn Gly Ile Tyr

595 600 605Ser Val Thr Phe Thr Leu Leu Ser Gly Pro Ser Arg Arg Phe Lys Arg

610                 615                 620Val Val Glu Thr Ile Gln Ala Gln Leu Leu Ser Thr His Asp Pro Pro625                 630                 635                 640Ala Ala Gln His Leu Ser Asp Thr Thr Asn Cys Met Glu Met Met Thr

645 650 655Gly Arg Leu Ser Lys Cys Gly Ile Ile Pro Lys Ser

660 665<210>3<211>2223<212>nucleotide<213>human<220><221>coding sequence<222>(3)..(2213)<223><400>3cg atg aca tcg acg ggg aag gac ggc ggc gcg cag cac gcg cag tat        47Met Thr Ser Thr Gly Lys Asp Gly Gly Ala Gln His Ala Gln Tyr1               5                   10                  15gtt ggg ccc tac cgg ctg gag aag acg ctg ggc aag ggg cag aca ggt       95Val Gly Pro Tyr Arg Leu Glu Lys Thr Leu Gly Lys Gly Gln Thr Gly

20 25 30CTG GTG AAG CTG GGG GGG GTT CAC GTC GTC ACC TGC CAG GCC ATC 143leu Val Lys Leu Gly Val Val THR CYS Val Ala Ile

35 40 45AAG ATC GAC CGT GAG AAG CTC GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG CTG AAG GTG 191lys Ile Val Asn ARG GLU LEU Ser Val Leu Met Lys Val Val Val

50 55 60gag CGG GAG ATC GCG ATC CTG CTC CTC ATT GAG CAC CAC CAC CAC CAC CTC

65                  70                  75aag ctg cac gac gtt tat gaa aac aaa aaa tat ttg tac ctg gtg cta      287Lys Leu His Asp Val Tyr Glu Asn Lys Lys Tyr Leu Tyr Leu Val Leu80                  85                  90                  95gaa cac gtg tca ggt ggt gag ctc ttc gac tac ctg gtg aag aag ggg 335Glu His Val Ser Gly Gly Glu Leu Phe Asp Tyr Leu Val Lys Lys Gly

100 105 110AGG CTG ACG CCT AAG GAG GCT CGG AAG TTC TTC CGG CAG ATC ATC ATC TCT 383ARG Leu Thr Pro Lys Glu ARG LYS PHE PHE GLN ILE ILN iLe Ile iLn iLe PLN iLE PLN iLE PLU -

115 120 125GCG CTG GAC TTC TGC CAC CAC CAC CAC ATA TGC CAC AGG GAT CTG AAA 431ALA Leu ASP PHE CYS Serle Cys

135 135 140cct GAA AAC CTC CTG CTG GAC GAG AAC AAC AAC ATC CGC ATC GCA GAC 479Pro Glu Leu Leu ASN Asn Ile ARG Ile Ala Ala Asp

145                 150                 155ttt ggc atg gcg tcc ctg cag gtt ggc gac agc ctg ttg gag acc agc      527Phe Gly Met Ala Ser Leu Gln Val Gly Asp Ser Leu Leu Glu Thr Ser160                 165                 170                 175tgt ggg tcc ccc cac tac gcc tgc ccc gag gtg atc cgg ggg gag aag 575Cys Gly Ser Pro His Tyr Ala Cys Pro Glu Val Ile Arg Gly Glu Lys

180 185 190TAT GAC GGC CGG AAG GCG GAC GAC GAC GTG AGC TGC GGC GGC GGC ATC CTG TTC 623TYR ARG LYS Ala Ala Ala's TrS Ge Leu Phes

195 200 205GCC TTG CTG GGG GGG GGG GCT CTG CCC GAC GAC GAC GAC GAC AAC TTG CGA CGA 671ALA Leu Val Gly Ala PHE ASP ASN Leu ARG Gln Leu ARG Gln

210 215 220CTG CTG GAG AAG GTG AAG CGG GGC GGC GTG TTC CAC CAC CCG CAC TTT ATC 719leu Leu Lys Val Val Phe His Met Pro His PHE Ile

225                 230                 235ccg ccc gac tgc cag agt ctg cta cgg ggc atg agc gag gtg gac gcc      767Pro Pro Asp Cys Gln Ser Leu Leu Arg Gly Met Ser Glu Val Asp Ala240                 245                 250                 255gca cgc cgc ctc acg cta gag cac att cag aaa cac ata tgg tat ata 815Ala Arg Arg Leu Thr Leu Glu His Ile Gln Lys His Ile Trp Tyr Ile

260 265 270GGG GGC AAG AAT GAG CCC GAA CCA GAG CAG CAG CAG CCC AAG GTG 863GLY GLY LYS ASN GLU PRO GLU Gln Pro Ile Pro Arg Lys Val

275 280 285CAG ATC CGC TCG CTG CCC AGC CTG GAC GAC GAC GAC CCC GAC GAC GAC GTG 911GLN Ile ARG Sero Seru Glo ASP PRO ASP Val Leuu

290 295 300GAC AGC ATG CAC TCA CTG GGC TGC TGC CGA GAC CGC AAC AAG CTG 959asp Serou Gly Cys PHE ARG ARG Asn Lys Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's Lu Le Lu Lu Lu Lu Lu -Aws that Cla's that

305                 310                 315cag gac ctg ctg tcc gag gag gag aac cag gag aag atg att tac ttc     1007Gln Asp Leu Leu Ser Glu Glu Glu Asn Gln Glu Lys Met Ile Tyr Phe320                 325                 330                 335ctc ctc ctg gac cgg aaa gaa agg tac ccg agc cag gag gat gag gac 1055Leu Leu Leu Asp Arg Lys Glu Arg Tyr Pro Ser Gln Glu Asp Glu Asp

340 345 350CTG CCC CCC CCC CCC CGG AAC GAG ATA GAC CCC CCC CGG AAG CGT GAC TCC 1103leu Pro Pro ARG Asn Glu Pro Pro ARG LYS ARG Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val ASP Ser

355 360 365CCG ATG CTG AAC CGG CAC GGC AAG CGG CGG CCA GAA CGC AAA TCC ATG 1151PRO MET Leu Asn ARG HS ARG Pro Glu ARG LYS MET MET

370 375 380GAG GTG CTC AGC GTG GAC GGC GGC GGC GGC GGC CCG CCG CCG CGG CGG 1199Glu Val Val THR AS PRO Val Pro Ala ARA ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG

385                 390                 395gcc att gag atg gcc cag cac ggc cag agg tct cgg tcc atc agc ggt     1247Ala Ile Glu Met Ala Gln His Gly Gln Arg Ser Arg Ser Ile Ser Gly400                 405                 410                 415gcc tcc tca ggc ctt tcc acc agc cca ctc agc agc ccc cgg gtg acc 1295Ala Ser Ser Gly Leu Ser Thr Ser Pro Leu Ser Ser Pro Arg Val Thr

420 425 430CCT CAC CCC TCA CCA AGG GGC AGT CCC CCC CCC CCC AAG GGG ACA 1343PRO HIS PRO ARG GLY GLY Serg

435 440 445CCT GTC CAC ACG CCA AAG GAG GAG AGC CCG GCC ACG CCC CCC CCC ACG 1391Pro Val His THR Pro LYS GLY THRY THR Pro ASN PRO Asn

450 455 460ccc ccg tcc agc ccc agc gtc gga ggg gtg ccc tgg agg gcg cgg ctc 1439Pro Ar Val u Pro Ser A la Ser p Tr g Pro Val Gly

465                 470                 475aac tcc atc aag aac agc ttt ctg ggc tca ccc cgc ttc cac cgc cga     1487Asn Ser Ile Lys Asn Ser Phe Leu Gly Ser Pro Arg Phe His Arg Arg480                 485                 490                 495aaa ctg caa gtt ccg acg ccg gag gag atg tcc aac ctg aca cca gag 1535Lys Leu Gln Val Pro Thr Pro Glu Glu Met Ser Asn Leu Thr Pro Glu

500 505 510TCG TCA GAG CTG GCG AAG AAG AAG TCC TGG TGG AAC TTC AGC AGC 1583ser Seru Leu Ala Lys Sern Phe Gly Asn Phe Ile Ser

515 520 525CTG Gag Aag Gag Gag Gag CAG ATC GTG GTC ATC AAA GAC AAA CCT CTG 1631leu LYS Glu Gln Ile Lysre Lysp Lysp Lou

530 535 540AGC TCC AAG GCT GCT GAC ATC GCC GCC TTC CTG AGT CCC AGT 1679ser Serle Lysp Ile Val His Ala Phe Leu Serle Pro Ser

545                 550                 555ctc agc cac agc gtc atc tcc caa acg agc ttc cgg gcc gag tac aag     1727Leu Ser His Ser Val Ile Ser Gln Thr Ser Phe Arg Ala Glu Tyr Lys560                 565                 570                 575gcc acg ggg ggg cca gcc gtg ttc cag aag ccg gtc aag ttc cag gtt 1775Ala Thr Gly Gly Pro Ala Val Phe Gln Lys Pro Val Lys Phe Gln Val

580 585 590GAT ATC ACC TAC ACG GGT GGT GGG GCG GCG GCG CAG AAG GAG AAC GGC ATC 1823asp Ile Thr Tyr Ty GLY GLY Gln Lysn Glys GLU Asn GLY ile

595 6005TAC TCC GTC ACC TTC ACC CTC CTC CTC CCC CCC CGC TTC TTC AAG 1871Tyr Seru Thr Leu Serg Phe Phe Lys

610 615 620AGG GAG GAG GAG ACC ATC CAG GCC CAG CTG CTG AGC ACA CAC GAC CCG 1919arg Val Val Gln Ala Gln Leu Leu Seru His ASP PRO Pro

625                 630                 635cct gcg gcc cag cac ttg tca gac acc act aac tgt atg gaa atg atg     1967Pro Ala Ala Gln His Leu Ser Asp Thr Thr Asn Cys Met Glu Met Met640                 645                 650                 655acg ggg cgg ctt tcc aaa tgt ggc agc cca ttg agt aac ttc ttt gac 2015Thr Gly Arg Leu Ser Lys Cys Gly Ser Pro Leu Ser Asn Phe Phe Asp

660 665 670GTA AAA CAA CAA CTT TTT TCA GAC GAG AAC GGG CAG GCG GCC CAG 2063VAL ILE LYS GLN Leu PHE SERU LYSN ALASLN AL -S AS AL -S's

675 685GCC CCC AGC AGC ACG CCC GCC AAG CGG AGT GCC CAC CCA CCA CTC GGT GAC 211ALA PRO ALA LYS Ala His Gly Pro Gly Prou Gly ASP

690 695 700TCC GCC GCC GCT GGC CCC CCC GGG GGG GCC GCC GCC GAG TAG TAG TAG TAG TAG CCA ALA Ala Gly Pro GLY GLY GLY GLY ALA GLU TYYR PRO GEA

705                 710                 715ggc aag gac acg gcc aag atg ggc ccg ccc acc gcc cgc cgc gag cag     2207Gly Lys Asp Thr Ala Lys Met Gly Pro Pro Thr Ala Arg Arg Glu Gln720                 725                 730                 735cct tag acacactagc                                                  2223Pro<210>4<211>736<212 >氨基酸<213>人类<400>4Met Thr Ser Thr Gly Lys Asp Gly Gly Ala Gln His Ala Gln Tyr Val1               5                   10                  15Gly Pro Tyr Arg Leu Glu Lys Thr Leu Gly Lys Gly Gln Thr Gly Leu

20 25 30Val Lys Leu Gly Val His Cys Val Thr Cys Gln Lys Val Ala Ile Lys

35 40 45Ile Val Asn Arg Glu Lys Leu Ser Glu Ser Val Leu Met Lys Val Glu

50 55 60arg Glu iLe Ale Leu Lys Leu Ile Glu His Prou His Val Leu Lys65 70 80leu His ASP Val Tyr Glu Asn Lys tyr Leu Val Leu Glu Glu Glu Glu Glu Glu

85 90 95His Val Ser Gly Gly Glu Leu Phe Asp Tyr Leu Val Lys Lys Gly Arg

100 105 110Leu Thr Pro Lys Glu Ala Arg Lys Phe Phe Arg Gln Ile Ile Ser Ala

115 120 125Leu Asp Phe Cys His Ser His Ser Ile Cys His Arg Asp Leu Lys Pro

130 135 140GLU ASN Leu Leu Leu asp Glu Lysn Asn Ile Ala Ala ALA ALA ASP PHE145 155 160GLY MET ALA Seru Gln Val GLY ASER Leu Leu THR Ser CYS CYS CYS CYS CYS CYS CYS CYs

165 170 175Gly Ser Pro His Tyr Ala Cys Pro Glu Val Ile Arg Gly Glu Lys Tyr

180 185 190Asp Gly Arg Lys Ala Asp Val Trp Ser Cys Gly Val Ile Leu Phe Ala

195 200 205 Leu Leu Val Gly Ala Leu Pro Phe Asp Asp Asn Leu Arg Gln Leu

210 215 220leu Lys Val Val Lys ARG GLY VAL PHE HIS MET Pro His PHE Ile Pro2225 230 235 240pro ASP CYS GLN Seru Leu ARG GLY MET Serg Ala Ala Ala Ala Ala Ala Ala Ala Ala

245 250 255Arg Arg Leu Thr Leu Glu His Ile Gln Lys His Ile Trp Tyr Ile Gly

260 265 270Gly Lys Asn Glu Pro Glu Pro Glu Gln Pro Ile Pro Arg Lys Val Gln

275 280 285Ile Arg Ser Leu Pro Ser Leu Glu Asp Ile Asp Pro Asp Val Leu Asp

290 295 300ser Met His Seru GLY CYS PHE ARG ARG Asn LEU Leu Leu GLN30 310 320ASP Leu Leu Leu Glu Gln Gln Gln Gln Met Ile Tyr Phe Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's Met Ile Tyr Leu's Ges that theanity

325 330 335Leu Leu Asp Arg Lys Glu Arg Tyr Pro Ser Gln Glu Asp Glu Asp Leu

340 345 350Pro Pro Arg Asn Glu Ile Asp Pro Pro Arg Lys Arg Val Asp Ser Pro

355 360 365 Met Leu Asn Arg His Gly Lys Arg Arg Pro Glu Arg Lys Ser Met Glu

370 375 380val Leu Ser Val THR ASP GLY GLY GLE VAL PRO ALA ARG ARG ALA385 395 400ILE GLU MET ALA GLN HIS GLN ARG Serg Serg Serg Serge Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

405 410 415Ser Ser Gly Leu Ser Thr Ser Pro Leu Ser Ser Pro Arg Val Thr Pro

420 425 430His Pro Ser Pro Arg Gly Ser Pro Leu Pro Thr Pro Lys Gly Thr Pro

435 440 445Val His Thr Pro Lys Glu Ser Pro Ala Gly Thr Pro Asn Pro Thr Pro

450 455 460Pro Ser Ser Val Gly Gly Val Pro TRP ARG ARG Leu asn465 475 480SER ILE LYS Asn Ser, Leu ARG PHE HIS ARG ARG LYS LYS

485 490 495Leu Gln Val Pro Thr Pro Glu Glu Met Ser Asn Leu Thr Pro Glu Ser

500 505 510Ser Pro Glu Leu Ala Lys Lys Ser Trp Phe Gly Asn Phe Ile Ser Leu

515 520 525Glu Lys Glu Glu Gln Ile Phe Val Val Ile Lys Asp Lys Pro Leu Ser

530 535 540Ser Ile La Ala asp Ile Val His Ala Phe Leu Serle Pro Seru545 550 560Ser His Sering Ile Serite ARG Ala Glu Tyr Lys Ala

565 570 575Thr Gly Gly Pro Ala Val Phe Gln Lys Pro Val Lys Phe Gln Val Asp

580 585 590Ile Thr Tyr Thr Glu Gly Gly Glu Ala Gln Lys Glu Asn Gly Ile Tyr

595 600 605Ser Val Thr Phe Thr Leu Leu Ser Gly Pro Ser Arg Arg Phe Lys Arg

610                 615                 620Val Val Glu Thr Ile Gln Ala Gln Leu Leu Ser Thr His Asp Pro Pro625                 630                 635                 640Ala Ala Gln His Leu Ser Asp Thr Thr Asn Cys Met Glu Met Met Thr

645 650 655Gly Arg Leu Ser Lys Cys Gly Ser Pro Leu Ser Asn Phe Phe Asp Val

660 665 670Ile Lys Gln Leu Phe Ser Asp Glu Lys Asn Gly Gln Ala Ala Gln Ala

675 680 685Pro Ser Thr Pro Ala Lys Arg Ser Ala His Gly Pro Leu Gly Asp Ser

690 695 700ALA Ala Gly Pro Gly Pro Gly Gly Gly Gly Ala Glu Tyr Pro Thr Gly705 715 720lys Asp THR ALA LYS MET GLY Pro THR ARG Gln Pro Gln Pro

725 730 735

Table 1hSTKa MTSTGKDGGAQHAQYVGPYRLEKTLGKGQTGLVKLGVHCVTCGKVAIKIVNREKLSESVLhSTKc MTSTGKDGGAQHAQYVGPYRLEKTLGKGQTGLVKLGVHCVTCQKVAIKIVNRE-------------------------19 --------------hSTKa           MKVEREIAILKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLTPKEARKhSTKc           MKVEREIAILKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLTPKEARKCAA07196        -----------LIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLTPKEARK

***************************************************hSTKa FFRQIISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPHhSTKc FFRQIISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPHCAA07196 GSLLETSCGSPHCAA07196 FFRQIISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFG

***************************************************** **********hSTKa           YACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPHFIPhSTKc           YACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPHFIPCAA07196        YACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPHFIP

***************************************************** **********hSTKa           PDCQSLLRGMSEVDAARRLTLEHIQKHIWYIGGKNEPEPEQPIPRKVQIRSLPSLEDIDPhSTKc           PDCQSLLRGMSEVDAARRLTLEHIQKHIWYIGGKNEPEPEQPIPRKVQIRSLPSLEDIDPCAA07196        PDCQSLLRGMSEVDAARRLTLEHIQKHIWYIGGKNEPEPEQPIPRKVQIRSLPSLEDIDP

***************************************************** **********hSTKa           DVLDSMHSLGCFRDRNKLLGDLLSEEENQEKMIYFLLLDRKERYPSQEDEDLPPRNEIDPhSTKc           DVLDSMHSLGCFRDRNKLLQDLLSEEENQEKMIYFLLLDRKERYPSQEDEDLPPRNEIDPCAA07196        DVLDSMHSLGCFRDRNKLLQDLLSEEENQEKMIYFLLLDRKERYPSQEDEDLPPRNEIDP

***************************************************** **********hSTKa           PRKRVDSPMLNRHGKRRPERKSMEVLSVTDGGSPVPARRAIEMAQHGQRSRSISGASSGLhSTKc           PRKRVDSPMLNRHGKRRPERKSMEVLSVTDGGSPVPARRAIEMAQHGQRSRSISGASSGLCAA07196        PRKRVDSPMLNRHGKRRPERKSMEVLSVTDGGSPVPARRAIEMAQHGQRSRSISGASSGL

***************************************************** **********hSTKa           STSPLSSPRVTPHPSPRGSPLPTPKGTPVHTPKESPAGTPNPTPPSSPSVGGVPWRARLNhSTKc           STSPLSSPRVTPHPSPRGSPLPTPKGTPVHTPKESPAGTPNPTPPSSPSVGGVPWRARLNCAA07196        STSPLSSPRVTPHPSPRGSPLPTPKGTPVHTPKESPAGTPNPTPPSSPSVGGVPWRARLN

***************************************************** **********hSTKa           SIKNSFLGSPRFHRRKLQVPTPEEMSNLTPESSPELAKKSWFGNFISLEKEEQIFVVIKDhSTKc           SIKNSFLGSPRFHRRKLQVPTPEEMSNLTPESSPELAKKSWFGNFISLEKEEQIFVVIKDCAA07196        SIKNSFLGSPRFHRRKLQVPTPEEMSNLTPESSPELAKKSWFGNFISLEKEEQIFVVIKD

***************************************************** **********hSTKa           KPLSSIKADIVHAFLSIPSLSHSVISQTSFRAEYKATGGPAVFQKPVKFQVDITYTEGGEhSTKc           KPLSSIKADIVHAFLSIPSLSHSVISQTSFRAEYKATGGPAVFQKPVKFQVDITYTEGGECAA07196        KPLSSIKADIVHAFLSIPSLSHSVISQTSFRAEYKATGGPAVFQKPVKFQVDITYTEGGE

***************************************************** **********hSTKa           AQKENGIYSVTFTLLSGPSRRFKRVVETIQAQLLSTHDPPAAQHLSDTTNCMEMMTGRLShSTKc           AQKENGIYSVTFTLLSGPSRRFKRVVETIQAQLLSTHDPPAAQHLSDTTNCMEMMTGRLSCAA07196        AQKENGIYSVTFTLLSGPSRRFKRVVETIQAQLLSTHDPPAAQHLSEPPPPAPGLSWGAG

*************************************************:.. : :hSTKa KCG-------IIPKS------------------------------------- ------hSTKc KCGSPLSNFFDVIKQLFSDEKNGQAAQAPSTPAKRSAHGPLGDSAAAGPGPGGDAEYPTGCAA07196 LKG-------QKVATSYESSL--------------------------------- ------

* *:hSTKa ----------------hSTKc KDTAKMGPPTARREQPCAA07196 ----------------

Claims (18)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of hSTK protein kinase polypeptide, and nucleotide sequence has at least 70% homology among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can with nucleotide sequence hybridization among the SEQ ID NO.1.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.2.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence is a nucleotide sequence among the SEQ ID NO.1.

4. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.4.

5. dna molecular as claimed in claim 1 is characterized in that, this sequence is a nucleotide sequence among the SEQ ID NO.3.

6. the hSTK protein polypeptide that separation obtains is characterized in that, it is with SEQ ID NO.2 aminoacid sequence at least 95% homology to be arranged.

7. the hSTK protein polypeptide that separation obtains is characterized in that, it is polypeptide, its active fragments or its reactive derivative with SEQ ID NO.2 aminoacid sequence.

8. polypeptide as claimed in claim 7 is characterized in that, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

9. polypeptide as claimed in claim 6 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

10. a carrier is characterized in that, it contains the described DNA of claim 1.

11. one kind with the described carrier transformed host cells of claim 10.

12. host cell as claimed in claim 11 is characterized in that, this cell is intestinal bacteria.

13. host cell as claimed in claim 11 is characterized in that, this cell is an eukaryotic cell.

14. a generation has the method for the polypeptide of hSTK protein-active, it is characterized in that, this method comprises:

(a) will be connected in expression regulation sequence as the described nucleotide sequence of claim 1 to 5, form the hSTK protein expression vector;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of hSTK;

(c) be fit to express under the condition of hSTK protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hSTK protein-active.

15. energy and the described hSTK protein polypeptide of claim 6 specificity bonded antibody.

16. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

17. the application of the described dna molecular of claim 1 is characterized in that, it is used for nucleic acid amplification reaction as primer or with making gene chip; Perhaps be used to prepare medicine.

18. the application of the described protein polypeptide of claim 6 is characterized in that, it is applied to screen the agonist or the inhibitor of human serine/threonine kinase; Perhaps be used to prepare medicine.