CN1394955A - Human cytokine receptor protein, its code sequence, preparation method and application - Google Patents

Abstract

The present invention relates to the field of gene engineering, in particular, said invention provides human cytokine receptor protein, its code sequence, preparation method and application. Sand invented human cytokine receptor h CKR as member of cytokine receptor family can utilize extracellular immunoglobulin-like structure to combine with cytokine, and utilize its intravellular partial phosphoesterase activity to transfer the signal carried by cytokine into cell interior and act on target molecular so as to produce regulation and control action on physiological motion of cell and biosome. These physiological action includes: promoting tissue growth, regulating and controlling growth and multiplication of cell, activating or inhibiting immune cell, stimulating hematopoietic cell, activating inflammation reaction and resisting infection of virus and bacteria, etc.

Description

A kind of human cytokine receptor, its encoding sequence, method for making and purposes

Technical field

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of a kind of human cytokine receptor (hCKR), the invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.

Background technology

Cytokine is produced by various kinds of cell, have the effect of extensive adjusting cell function peptide molecule (Fundamental Immunology, Forth Edition, 1999, pp741-774, Lippincott-RavenPublishers, Philadelphia).Cytokine not only acts on immunity system and hemopoietic system, also extensively acts on nerve, endocrine system, and the proliferation and differentiation and the effector function of pair cell interphase interaction, cell have important regulatory role.Cytokine performance extensively various biological function depends on to combine and pass the signal along to cell interior with the cytokine receptor on target cell membrane surface and realizes (Cell 1989 May19; 57 (4): 521-4; Curr Opin Immunol 1994 Aug; 6 (4): 631-5).

Because the extensive physiological action of cytokine and effect thereof produce the dependency to acceptor, the research of people's pair cell factor acceptor is increasingly extensive and deep.

Cytokine receptor is divided into 3 functional zone: the film outskirt, stride the film district, the film inner region.The film outskirt combines with cytokine; Stride the film district and be rich in hydrophobic amino acid, its effect is that acceptor is fixed on the film; The film inner region then with cell in molecule act on mutually, signal is reached in the film.

At present, according to the homology and the constitutional features of cytokine receptor, it mainly can be divided into four types: immunoglobulin superfamily, hematopoietic cytokine receptor superfamily, trk C superfamily and Chemokine Receptors ( Http:// www.37c.com.cn/literature/library/theory/096/096040301.h tml).

1. immunoglobulin gene superfamily.The cytolemma outskirt of this receptoroid has one or more immunoglobulin like domain.IL-1, IL-6, M-CSF, SCF, PDGF and FGF acceptor all belong to this type of.(gpl30-like monocyte receptor GLM-R) also belongs to (J Biol Chem 2002 May 10 of this family to the gpl30-sample monocyte acceptor of latest find; 277 (19): 16831-6).

2. Hemopoietic factor receptor family.This receptoroid claims erythropoietin receptor family or I cytokines receptor family again, and its after birth outskirt has two discontinuous cysteine residues and WSXWS motif (W representative color propylhomoserin, S represents Serine, X represents any amino acid).IL-2～7, IL-9, IL-11, IL-13, IL-15, GM-CSF and G-CSF acceptor belong to this type of.

3. trk C superfamily.Its after birth of NGFR superfamily member is outer to be rich in the Cys zone by what 3～6 about 40 amino acid were formed.As NGFR, TNF-R (CD120a), TNF-R (CD120b), CD40, CD27, T cell cDNA-41BB coded product, rat T cell antigen OX40 and human medullary cell surface active antigen Fas (CD95).

4. Chemokine Receptors family.The acceptor of this family is the G-protein linked receptor, is made up of seven hydrophobic film districts of striding, and this receptoroid is brought into play biological effect with after corresponding part combines through the coupling gtp binding protein.The Chemokine Receptors kind of having found has IL-8RA, IL-8RB, MIP-1 α/RANTES R, NCP-1R and cell chemotactic factor acceptor.

Summary of the invention

An object of the present invention is to provide a kind of polynucleotide sequence, a kind of human cytokine receptor of this polynucleotide sequence coding ( hUman cYto kIne rEceptor, hCKR).

Another object of the present invention provides a kind of albumen, and this albumen is named as human cytokine receptor (hCKR).

A further object of the present invention provides a kind of recombinant technology that utilizes and produces the proteic method of described hCKR.

The present invention also provides this hCKR nucleotide sequence and proteic application.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people hCKR protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 122-919 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 122-919 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.2.More preferably, this sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 122-919 position.

In another aspect of this invention, provide a kind of isolating hCKR protein polypeptide, it comprises: have polypeptide or its active fragments of SEQID NO.3 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.3 polypeptide of sequence.

In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.

In another aspect of this invention, provide a kind of described carrier transformed host cells.

In another aspect of this invention, the method that provides a kind of generation to have the polypeptide of hCKR protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hCKR protein-active operationally is connected in expression regulation sequence, form the hCKR protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 122-919 position among described nucleotide sequence and the SEQ ID NO.1;

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

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

(d) isolate polypeptide with hCKR protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1704 Nucleotide, and its detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 122-919 position Nucleotide.

In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, term " hCKR albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with HCKR protein-active is as 122-919 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 122-919 position Nucleotide of SEQ ID NO.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in 122-919 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 122-919 position.In addition, this term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 122-919 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.1 sequence of people HCKR identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " hCKR protein polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of hCKR protein-active.This term also comprises having and variant form people hCKR identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of hCKR and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of hCKR DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-hCKR polypeptide to obtain.The present invention also provides other polypeptide, as comprises hCKR polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of hCKR polypeptide.Usually, this fragment have the hCKR peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of hCKR albumen or polypeptide.The difference of these analogues and natural hCKR polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

The present invention also comprises the antisense sequences of hCKR polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of hCKR in the cell.

The present invention also comprises a kind of probe molecule, and this molecule has 8-100 of hCKR polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the hCKR that encodes.

The present invention also comprises the method that detects the hCKR nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the encoding sequence of hCKR polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

On the other hand, the present invention also comprises hCKR DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into hCKR gene product or fragment.Preferably, refer to that those can combine with hCKR gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of hCKR, comprise that also those do not influence the antibody of hCKR protein function.The present invention also comprise those can with modify or without the hCKR gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the hCKR gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing hCKR or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler, Nature256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the hCKR function and the antibody that does not influence the hCKR function.Each antibody-like of the present invention can utilize the fragment or the functional zone of hCKR gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of hCKR gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

In the present invention, the cDNA nucleotide sequence of people hCKR is so to obtain, and is template with people's testis λ gt10cDNA library (available from Clontech company), and be primer with two pairs of oligonucleotide: A1CGAAGCCC CTGAGCTGAA AAG is a forward primer; A2:CACCA TTGGCGATGGGAGGCT is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, size are respectively the purpose fragment of about 900 base pairs.

HCKR albumen of the present invention carries out nucleic acid and albumen homology retrieval with BLAST software in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, found that it and some acceptor have certain homology, and has immunoglobulin domains in its structure, stride film district characteristic sequence (http://smart.embl-heidelberg.de/smart/show_motifs.pl) and phosphoesterase A2 characteristic sequence (http://www2.ebi.ac.uk/servicestmp/47940.html), have cytokine receptor protein family member's constitutional features.

Cytokine receptor is divided into 3 functional zone: the film outskirt, stride the film district, the film inner region.The film outskirt combines with cytokine; Stride the film district and be rich in hydrophobic amino acid, its effect is that acceptor is fixed on the film; The film inner region then with cell in molecule act on mutually, signal is reached in the film.At present, according to the homology and the constitutional features of cytokine receptor, it mainly can be divided into four types: immunoglobulin superfamily, hematopoietic cytokine receptor superfamily, trk C superfamily and Chemokine Receptors.Wherein the film outskirt of this receptoroid of constitutional features of the first kind has one or more immunoglobulin like domain.

The protein sequence of hCKR of the present invention has the type sequence of immunoglobulin like domain: TERYVTLFASIILKCDYTTSAQLQDVVVTWRFKSFCKDPIFDYYSASYQAALSLGQ DPSNDCNDNQREVRIVAQRRGQNEPVLGVDYRQRKITIQNRADLVINEVMWWDHGV YYCTIEAPGDTSGDPDKEVKLIVL (6-142 among the SEQ ID NO 3).The protein sequence of the hCKR of postorder contains strides film district characteristic sequence: LTVIFIILGALLLLLLIGVCWC (144-166 among the SEQ ID NO 3).In addition, the protein sequence of hCKR has been found the conserved sequence of phosphoesterase A2 in also: C-C-x (2)-H-x (2)-C, and wherein, H is an avtive spot, x (2) represents 2 arbitrary amino acids.In hCKR, the sequence that meets this feature is: CCPAHCCC (181-188 among the SEQ ID NO 3).

The basic structure of immunoglobulin (Ig) is the symmetrical tetrapeptide chain structure that is formed by connecting by interchain disulfide bond by two identical long-chains (heavy chain) and two identical short chains (light chain), every long-chain chain and short chain all are divided into aminoterminal (N end) and carboxyl terminal (C end), arrange likeness in form " Y " molecule.At the N of four peptide chains of Ig end, promptly 1/2 of light chain and 1/4 district of heavy chain, the amino acid kind, putting in order changes greatlyyer with configuration with antibodies specific different, be called the variable region, and this is the position in conjunction with extracellular signaling molecule.At the C of four peptide chains of Ig end, promptly 1/2 of light chain and 3/4 district of heavy chain, the amino acid kind, put in order and configuration relatively stable, be called the stable region ( Http:// www.yctvu.com.cn/html/KFJY/6.htm).Immunoglobulin like domain often relates to protein-protein, albumen-ligand interaction.

Phospholipase A2 (PA2) is the enzyme from the second carbon-based group release fat acid of glycerine.PA2 is a small-sized albumen of being made up of 120 amino-acid residues.Contain four to seven disulfide linkage in its space structure, so structure is firmer.A required calcium ion of PA2 and lipid acid combines two conserved residues in its structure---and a Histidine and an aspartic acid participate in the catalysis networking.

In sum, hCKR of the present invention is as the cytokine receptor family member, the immunoglobulin (Ig) spline structure outer by born of the same parents engages with cytokine, and be passed in the born of the same parents by phosphate esterase active the signal that cytokine is carried of part in its born of the same parents, act on target molecule, thereby the physiological activity of pair cell and even organism plays regulating and controlling effect.These physiological actions comprise the promotion tissue growth, and the growth of regulating cell and propagation activate, suppress immunocyte, and the hemopoietic cell activates inflammatory reaction, prevents virus, bacterial invasion etc.

HCKR nucleic acid of the present invention, its antisense strand and fragment are made probe, can detect the hCKR gene unconventionality; With hCKR protein sequence generate a reagent box or medicine, adjustable ganglion cell's growing multiplication promotes the wound healing after scalds and burns, cutting tissue and even organ are extractd, and prevents wound infection.With hCKR nucleic acid antisense strand of the present invention hCKR antibody generate a reagent box can be used for alleviating or assisting therapy owing to the too high disease that causes of hCKR expression amount, also can assist the diagnosis of tumour and cancer clinically, prevention and treatment.In addition, hCKR of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen, for example the N end of hCKR of the present invention and the N end of mouse CKR can be exchanged, to produce the albumen that new activity is higher or have new features.At the antibody of hCKR of the present invention, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment

Embodiment 1

The clone and the mensuration of the cDNA sequence of hCKR

1. primer amplification

With people's testis λ gt10cDNA library (available from Clontech company) is template, is primer with two pairs of oligonucleotide: A1 CGAAGCCC CTGAGCTGAA AAG is a forward primer; A2:CACCATTGGCGATGG GAGGCT is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, size are respectively the purpose fragment of about 900 base pairs.

2.PCR the order-checking of product

With pcr amplification product and pGEM-T ^TMCarrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, obtains full length cDNA sequence at last, is total to 1704bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 122-919 position Nucleotide.

Derive the aminoacid sequence of hCKR according to the full length cDNA sequence that obtains, totally 265 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

The expression of hCKR in intestinal bacteria

In this embodiment,, use PCR Oligonucleolide primers to increase, obtain hCKR cDNA as inserting fragment corresponding to 5 of this dna sequence dna ' and 3 ' end with the cDNA sequence of coding hCKR.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5 ' AGGGGGATTC ATGGCATGG CCCAAACTGC, this primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence of the hCKR that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-GTCTAAGCTTTTGGCA TCTGCGGGAG GCT, this primer contain the part encoding sequence of restriction enzyme site, translation termination and the hCKR of HindIII restriction enzyme.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Ampr), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With BamHI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid cuts with the ApaBI enzyme and to identify and insert clip size and direction, and the cDNA fragment of sequence verification hCKR has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/mI).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved hCKR from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out hCKR from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps use the dialysis step to remove Guanidinium hydrochloride, perhaps isolate purifying protein from nickel-chelate column, the albumen behind the purifying can be incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed to containing PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.3 with ordinary method.The difference of this sequence and SEQ ID NO.2 is that it does not contain 1-24 amino acids among the SEQ ID NO.2.This is that this signal peptide sequence is cut in the expression process because 1-24 amino acid is the signal peptide sequence of hCKR among the SEQ ID NO.2.

Embodiment 3

The expression of hCKR in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, the following Oligonucleolide primers of cDNA sequence of coding hCKR is increased, obtain hCKR cDNA as inserting fragment.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5’-AGGGAAGCTT?ATGGCATGG?CCCAAACTGC

This primer contains the restriction enzyme site of HindIII restriction enzyme, is the part encoding sequence of the hCKR that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-GTCTGGATCCTTGGCA?TCTGCGGGAG?GCT

This primer contains the part encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and hCKR.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (fl ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII and BamHI digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid cuts evaluation with the ApaBI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification hCKR has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to use lipofection, carries out with Lipofectin (GiBco Life), and transfection is after 48 hours, and through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (pH7.6) solution that contains 0.05% Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mMTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.3 with ordinary method.The difference of this sequence and SEQ ID NO.2 is that it does not contain 1-24 amino acids among the SEQ ID NO.2.This is that this signal peptide sequence is cut in the expression process because 1-24 amino acid is the signal peptide sequence of hCKR among the SEQ ID NO.2.

Embodiment 4

Homology relatively

The hCKR albumen (SEQ ID NO.3) that obtains with embodiment 2 and embodiment 3 carries out nucleic acid and albumen homology retrieval with BLAST software in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDSranslations+PDB+SwissProt+Spupdate+PIR database.Found that it and some acceptor have certain homology, and have immunoglobulin domains in its structure, stride film district characteristic sequence (http://smart.embl-heidelberg.de/smart/show_motifs.pl) and phosphoesterase A2 characteristic sequence (http://www2.ebi.ac.uk/servicestmp/47940.html), have cytokine receptor protein family member's constitutional features.

Cytokine receptor is divided into 3 functional zone: the film outskirt, stride the film district, the film inner region.The film outskirt combines with cytokine; Stride the film district and be rich in hydrophobic amino acid, its effect is that acceptor is fixed on the film; The film inner region then with cell in molecule act on mutually, signal is reached in the film.At present, according to the homology and the constitutional features of cytokine receptor, it mainly can be divided into four types: immunoglobulin superfamily, hematopoietic cytokine receptor superfamily, trk C superfamily and Chemokine Receptors.Wherein the film outskirt of this receptoroid of constitutional features of the first kind has one or more immunoglobulin like domain.

The protein sequence of hCKR of the present invention has the type sequence of immunoglobulin like domain: TERYVTLFASIILKCDYTTSAQLQDVVVTWRFKSFCKDPIFDYYSASYQAALSLGQ DPSNDCNDNQREVRIVAQRRGQNEPVLGVDYRQRKITIQNRADLVINEVMWWDHGV YYCTIEAPGDTSGDPDKEVKLIVL (6-142 among the SEQ ID NO 3).The protein sequence of the hCKR of postorder contains strides film district characteristic sequence: LTVIFIILGALLLLLLIGVCWC (144-166 among the SEQ ID NO 3).In addition, the protein sequence of hCKR has been found the conserved sequence of phosphoesterase A2 in also: C-C-x (2)-H-x (2)-C, and wherein, H is an avtive spot, x (2) represents 2 arbitrary amino acids.In hCKR, the sequence that meets this feature is: CCPAHCCC (181-188 among the SEQ ID NO 3).

The basic structure of immunoglobulin (Ig) is the symmetrical tetrapeptide chain structure that is formed by connecting by interchain disulfide bond by two identical long-chains (heavy chain) and two identical short chains (light chain), every long-chain chain and short chain all are divided into aminoterminal (N end) and carboxyl terminal (C end), arrange likeness in form " Y " molecule.At the N of four peptide chains of Ig end, promptly 1/2 of light chain and 1/4 district of heavy chain, the amino acid kind, putting in order changes greatlyyer with configuration with antibodies specific different, be called the variable region, and this is the position in conjunction with extracellular signaling molecule.At the C of four peptide chains of Ig end, promptly 1/2 of light chain and 3/4 district of heavy chain, the amino acid kind, put in order and configuration relatively stable, be called the stable region.( Http:// www.yctvu.com.cn/html/KFJY/6.htm) immunoglobulin like domain often relates to albumen one albumen, albumen one ligand interaction.

Phospholipase A2 (PA2) is the enzyme from the second carbon-based group release fat acid of glycerine.PA2 is a small-sized albumen of being made up of 120 amino-acid residues.Contain four to seven disulfide linkage in its space structure, so structure is firmer.A required calcium ion of PA2 and lipid acid combines.Two conserved residues in its structure---a Histidine and an aspartic acid participate in the catalysis networking.

In sum, hCKR of the present invention is as the cytokine receptor family member, the immunoglobulin (Ig) spline structure outer by born of the same parents engages with cytokine, and be passed in the born of the same parents by phosphate esterase active the signal that cytokine is carried of part in its born of the same parents, act on target molecule, thereby the physiological activity of pair cell and even organism is mentioned regulating and controlling effect.These physiological actions comprise the promotion tissue growth, and the growth of regulating cell and propagation activate, suppress immunocyte, and the hemopoietic cell activates inflammatory reaction, prevents virus, bacterial invasion etc.

HCKR of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, hCKR of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen, for example the N end of hCKR of the present invention and the N end of mouse CKR can be exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of hCKR of the present invention, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment 5

Preparation antibody

Embodiment 2 or 3 recombinant proteins that obtain are used for immune animal to produce antibody, specific as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation hCKR gene translation product with it.

Example 6

Substrate as micromatrix

Micromatrix is called the DNA chip again.(, select by using some famous biosoftwares hCKR full-length cDNA, EST or gene fragment substrate samples as micromatrix as LASERGENESOFTWARE (DNASTAR).By using ink-jet technology and a series of chemical process such as ray, chemistry, thermodynamics, mechanical means etc. that sample is fixed on the carrier,, obtain chip (Schena, M.et al. (1995) Science 270:467-470 then as on glass; And Shalon, D.et al. (1996) Genome Res.6:639-645.), the element of formation has point-like, bar shaped or the like.A typical chip contains the element of some amount usually.Behind the hybridization, the unconjugated probe of flush away detects the element of generation hybridization and the degree of reaction with scanner then.The complementarity of probe and each chip component and binding capacity all can be judged by the analysis to the image that scans.

Results of hybridization can be used for detecting hCKR genovariation, sudden change and polymorphism, understands because hCKR expresses the molecular basis of the undesired disease that causes, diagnoses the illness, and can improve and monitor the activity of related agents.

Embodiment 7:

The preparation of test kit

Test kit 1:

The primer that it contains (1) specific amplification hCKR to and operation instruction.This test kit also can contain or not contain the required reagent that the mRNA reverse transcription is become cDNA.Wherein, the sequence of this Auele Specific Primer is derived from the sequence of SEQ ID NO:1.The cDNA that reverse transcription is become carries out specific amplification, whether to contain the nucleotide sequence of hDGF7 in the test sample.This test kit also can contain and carries out other required reagent of PCR reaction, for example damping fluid etc.

In addition, preferably, at least one used primer has been crossed over two exons, because will not produce amplified production corresponding to the genome sequence of hCKR this moment.

Test kit 2:

This test kit contains the specific antibody (for example polyclonal antibody of preparation among the embodiment 5, the perhaps monoclonal antibody of the anti-hCKR of the hybridoma technology generation of usefulness standard) at hCKR, and operation instruction.Whether this test kit is used for direct test sample and exists or lack hCKR albumen.Form immunocomplex by specific immune response earlier, detect immunocomplex with routine techniques then.

Test kit 3:

This test kit contains can be specifically and the probe of the mRNA hybridization of hCKR.It also can contain or not contain hybridization buffer.This test kit comes whether to exist in the test sample nucleic acid molecule of hCKR by the hybridization between nucleic acid molecule and the hCKR specific probe.

Test kit also can be used for detecting genovariation, sudden change and polymorphism, and detects the relevant gene of hCKR a series of and of the present invention, thereby diagnosis, the treatment of relative disease helped out.

Example 8:

Make medicine

HCKR albumen of the present invention and antibody, inhibitor, antagonist or acceptor etc. can be used as medicine, can promote the wound healing after scalds and burns, cutting tissue and even organ are extractd, and prevent wound infection.With hCKR nucleic acid antisense strand of the present invention hCKR antibody generate a reagent box can be used for alleviating or assisting therapy owing to the too high disease that causes of hCKR expression amount, also can assist the diagnosis of tumour and cancer clinically, prevention and treatment.

Albumen of the present invention and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is generally about 5-8, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.

In addition, hCKR nucleic acid of the present invention (encoding sequence or antisense sequences) can directly be introduced cell, with expression level that improves hCKR or the overexpression that suppresses hCKR.HCKR albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of hCKR disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

When hCKR protein polypeptide of the present invention is used as medicine, the treatment effective dose is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight one about 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Sequence table＜210〉1＜211〉1704＜212〉nucleotides＜213〉human＜220〉＜221〉coded sequence＜222〉(122) .. (919)＜223〉＜400〉1cgagggaggg gaaacccgag gggttccttg gagaaggtgg tgcgtcctgg ggcggcagct 60gaggaagaaa gacgcagtgc cccgaagccc ctgagctgaa aagggccaga aagggggcgg 120c atg gca tgg ccc aaa ctg ccc gca cct tgg ctg ctg ctc tgc acc tgg, 169 Met Ala Trp Pro Lys Leu Pro Ala Pro Trp Leu Leu Leu Cys Thr Trp, 15 10 15ctc cca gca ggg tgc ctg tcc ttg ctt gtg acg gtc cag cac aca gaa 217Leu Pro Ala Gly Cys Leu Ser Leu Leu Val Thr Val Gln His Thr Glu

20 25 30cgc?tat?gtc?acc?ctg?ttt?gcc?tct?atc?atc?ctc?aaa?tgt?gac?tac?acc 265Arg?Tyr?Val?Thr?Leu?Phe?Ala?Ser?Ile?Ile?Leu?Lys?Cys?Asp?Tyr?Thr

35 40 45acc?tct?gcc?cag?ctc?cag?gac?gtg?gtg?gtg?aca?tgg?cgc?ttc?aag?tcc 313Thr?Ser?Ala?Gln?Leu?Gln?Asp?Val?Val?Val?Thr?Trp?Arg?Phe?Lys?Ser

50 55 60ttc?tgc?aag?gac?cct?atc?ttt?gac?tac?tac?tca?gcg?tca?tac?cag?gca 361Phc?Cys?Lys?Asp?Pro?Ile?Phe?Asp?Tyr?Tyr?Ser?Ala?Ser?Tyr?Gln?Ala65 70 75 80gct?tta?tcc?ctg?ggc?cag?gac?cca?tcc?aat?gac?tgc?aac?gac?aac?cag 409Ala?Leu?Ser?Leu?Gly?Gln?Asp?Pro?Ser?Asn?Asp?Cys?Asn?Asp?Asn?Gln

85 90 95cgg?gaa?gtt?cgc?ata?gtg?gcc?cag?cgg?cgg?ggg?cag?aat?gag?ccc?gtg 457Arg?Glu?Val?Arg?Ile?Val?Ala?Gln?Arg?Arg?Gly?Gln?Asn?Glu?Pro?Val

100 105 110ctg?ggg?gta?gat?tac?cgg?cag?cgc?aag?atc?acc?arc?cag?aac?cga?gca 505Leu?Gly?Val?Asp?Tyr?Arg?Gln?Arg?Lys?Ile?Thr?Ile?Gln?Asn?Arg?Ala

115 120 125gat?ctc?gtg?ata?aat?gaa?gtg?atg?tgg?tgg?gac?cat?gga?gtg?tat?tac 553Asp?Leu?Val?Ile?Asn?Glu?Val?Met?Trp?Trp?Asp?His?Gly?Val?Tyr?Tyr

130 135 140tgc?acc?att?gag?gct?cca?ggg?gac?aca?tca?gga?gac?ccc?gat?aag?gaa 601Cys?Thr?Ile?Glu?Ala?Pro?Gly?Asp?Thr?Ser?Gly?Asp?Pro?Asp?Lys?Glu145 150 155 160gta?aag?ctc?atc?gtc?cta?cac?tgg?ctg?aca?gtg?atc?ttc?atc?atc?ctg 649Val?Lys?Leu?Ile?Val?Leu?His?Trp?Leu?Thr?Val?Ile?Phe?Ile?Ile?Leu

165 170 175gga?gcc?ctc?ctc?ctc?ctg?ctg?ctg?att?gga?gtg?tgc?tgg?tgc?cag?tgc 697Gly?Ala?Leu?Leu?Leu?Leu?Leu?Leu?Ile?Gly?Val?Cys?Trp?Cys?Gln?Cys

180 185 190tgt?cct?cag?tat?tgc?tgc?tgc?tat?atc?cgc?tgt?ccc?tgc?tgt?cct?gcc 745Cys?Pro?Gln?Tyr?Cys?Cys?Cys?Tyr?Ile?Arg?Cys?Pro?Cys?Cys?Pro?Ala

195 200 205cac?tgc?tgc?tgt?cct?gag?gaa?gcc?ctg?gcc?cgc?cac?cgc?tac?atg?aag 793His?Cys?Cys?Cys?Pro?Glu?Glu?Ala?Leu?Ala?Arg?His?Arg?Tyr?Met?Lys210 215 220cag?gcc?cag?gcc?cta?ggt?cct?cag?atg?atg?gga?aaa?ccc?ctg?tac?tgg 841Gln?Ala?Gln?Ala?Leu?Gly?Pro?Gln?Met?Met?Gly?Lys?Pro?Leu?Tyr?Trp225 230 235 240ggg?gcg?gac?agg?agc?tcc?cag?gtt?tca?tct?tat?cca?atg?cac?ccg?ctg 889Gly?Ala?Asp?Arg?Ser?Ser?Gln?Val?Ser?Ser?Tyr?Pro?Met?His?Pro?Leu

245 250 255ctg?cag?cga?gcc?tcc?cgc?aga?tgc?caa?tga?cccagaccac?caatcagcct 939Leu?Gln?Arg?Ala?Ser?Arg?Arg?Cys?Gln

260 265cccatcgcca atggtgtcct ggagtatttg gagaaagaac tgcggaacct caacctggcc 999cagcctctgc cccctgacct caaaggcaga tttggccatc cctgcagcat gctgtcctcc 1059ctgggctctg aggtcgtgga acgcagaatc atccacctgc ccccactgat cagagacctg 1119tcatcctcaa ggaggaccag tgactccctg caccagcagt ggctcacccc aattccctcc 1179aggccctggg atctgaggga ggggagaagc caccaccatt accctgattt ccaccaggag 1239ctccaggacc gggggccaaa gtcttgggca ttggaaagaa gggagttgga cccatcgtgg 1299agtggaaggc accgtagctc taggctgaat gggtcaccca tacactggtc agacagggac 1359agcctaagcg atgtcccctc atccagtgag gcacgctggc ggccgagcca ccctcctttc 1419aggagccgct gtcaggagag gccccgcagg cccagccccc gggagagcac tcagaggcac 1479gggagacgac gcaggcaccg cagctactct cctcccttgc cctccggcct cagttcctgg 1539agctctgaag aggacaagga gaggcagccc cagagctggc gggcccaccg ccgcggctcg 1599cactccccac actggcccga ggagaagccg cctagctacc gctcacttga tatcactcca 1659ggcaagaata gcaggaaaaa agggagtgtg gagaggcgct cggag 1704＜210〉2＜211〉265＜212〉＜213〉＜400〉2Met Ala Trp Pro Lys Leu Pro Ala Pro Trp Leu Leu Leu Cys Thr Trp1 5 10 15Leu Pro Ala Gly Cys Leu Ser Leu Leu Val Thr Val Gln His Thr Glu

20 25 30Arg?Tyr?Val?Thr?Leu?Phe?Ala?Ser?Ile?Ile?Leu?Lys?Cys?Asp?Tyr?Thr

35 40 45Thr?Ser?Ala?Gln?Leu?Gln?Asp?Val?Val?Val?Thr?Trp?Arg?Phe?Lys?Ser

50 55 60Phe?Cys?Lys?Asp?Pro?Ile?Phe?Asp?Tyr?Tyr?Ser?Ala?Ser?Tyr?Gln?Ala65 70 75 80Ala?Leu?Ser?Leu?Gly?Gln?Asp?Pro?Ser?Asn?Asp?Cys?Asn?Asp?Asn?Gln

85 90 95Arg?Glu?Val?Arg?Ile?Val?Ala?Gln?Arg?Arg?Gly?Gln?Asn?Glu?Pro?Val

100 105 110Leu?Gly?Val?Asp?Tyr?Arg?Gln?Arg?Lys?Ile?Thr?Ile?Gln?Asn?Arg?Ala

115 120 125Asp?Leu?Val?Ile?Asn?Glu?Val?Met?Trp?Trp?Asp?His?Gly?Val?Tyr?Tyr

130 135 140Cys?Thr?Ile?Glu?Ala?Pro?Gly?Asp?Thr?Ser?Gly?Asp?Pro?Asp?Lys?Glu145 150 155 160Val?Lys?Leu?Ile?Val?Leu?His?Trp?Leu?Thr?Val?Ile?Phe?Ile?Ile?Leu

165 170 175Gly?Ala?Leu?Leu?Leu?Leu?Leu?Leu?Ile?Gly?Val?Cys?Trp?Cys?Gln?Cys

180 185 190Cys?Pro?Gln?Tyr?Cys?Cys?Cys?Tyr?lle?Arg?Cys?Pro?Cys?Cys?Pro?Ala

195 200 205His?Cys?Cys?Cys?Pro?Glu?Glu?Ala?Leu?Ala?Arg?His?Arg?Tyr?Met?Lys

210 215 220Gln?Ala?Gln?Ala?Leu?Gly?Pro?Gln?Met?Met?Gly?Lys?Pro?Leu?Tyr?Trp225 230 235 240Gly?Ala?Asp?Arg?Ser?Ser?Gln?Val?Ser?Ser?Tyr?Pro?Met?His?Pro?Leu

245 250 255Leu?Gln?Arg?Ala?Ser?Arg?Arg?Cys?Gln

260 265＜210〉3＜211〉241＜212〉amino acid＜213〉mankind＜400〉3Leu Val Thr Val Gln His Thr Glu Arg Tyr Val Thr Leu Phe Ala Ser1 5 10 15Ile Ile Leu Lys Cys Asp Tyr Thr Thr Ser Ala Gln Leu Gln Asp Val

20 25 30Val?Val?Thr?Trp?Arg?Phe?Lys?Ser?Phe?Cys?Lys?Asp?Pro?Ile?Phe?Asp

35 40 45Tyr?Tyr?Ser?Ala?Ser?Tyr?Gln?Ala?Ala?Leu?Ser?Leu?Gly?Gln?Asp?Pro

50 55 60Ser?Ash?Asp?Cys?Asn?Asp?Asn?Gln?Arg?Glu?Val?Arg?Ile?Val?Ala?Gln65 70 75 80Arg?Arg?Gly?Gln?Asn?Glu?Pro?Val?Leu?Gly?Val?Asp?Tyr?Arg?Gln?Arg

85 90 95Lys?Ile?Thr?Ile?Gln?Asn?Arg?Ala?Asp?Leu?Val?Ile?Asn?Glu?Val?Met

100 105 110Trp?Trp?Asp?His?Gly?Val?Tyr?Tyr?Cys?Thr?Ile?Glu?Ala?Pro?Gly?Asp

115 120 125Thr?Ser?Gly?Asp?Pro?Asp?Lys?Glu?Val?Lys?Leu?Ile?Val?Leu?His?Trp

130 135 140Leu?Thr?Val?Ile?Phe?Ile?Ile?Leu?Gly?Ala?Leu?Leu?Leu?Leu?Leu?Leu145 150 155 160Ile?Gly?Val?Cys?Trp?Cys?Gln?Cys?Cys?Pro?Gln?Tyr?Cys?Cys?Cys?Tyr

165 170 175Ile?Arg?Cys?Pro?Cys?Cys?Pro?Ala?His?Cys?Cys?Cys?Pro?Glu?Glu?Ala

180 185 190Leu?Ala?Arg?His?Arg?Tyr?Met?Lys?Gln?Ala?Gln?Ala?Leu?Gly?Pro?Gln

195 200 205Met?Met?Gly?Lys?Pro?Leu?Tyr?Trp?Gly?Ala?Asp?Arg?Ser?Ser?Gln?Val

210 215 220Ser?Ser?Tyr?Pro?Met?His?Pro?Leu?Leu?Gln?Arg?Ala?Ser?Arg?Arg?Cys225 230 235 240Gln

Claims (18)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people hCKR protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 122-919 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 122-919 position.

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 the sequence of Nucleotide 122-919 position among the SEQ ID NO.1.

4. isolating hCKR protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.3 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.3 polypeptide of sequence.

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

7. one kind with the described carrier transformed host cells of claim 6.

8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

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

(a) nucleotide sequence that coding is had a polypeptide of hCKR protein-active operationally is connected in expression regulation sequence, form the hCKR protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 122-919 position among described nucleotide sequence and the SEQ ID NO.1;

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

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

(d) isolate polypeptide with hCKR protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 122-919 position among the SEQ ID NO.1.

12. energy and the described hCKR protein polypeptide of claim 4 specificity bonded antibody.

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

14. a probe molecule is characterized in that, it contains 8-100 continuous nucleotide in the described dna molecular of claim 1

15. a test kit is characterized in that, it contains the described dna molecular of claim 1.

16. a test kit is characterized in that, it contains the described antibody of claim 12.

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 the disease that medicine causes owing to human cell factor acceptor encoding gene unusually with prevention, diagnosis or treatment.

18. the application of the described protein polypeptide of claim 4 is characterized in that, it uses agonist or inhibitor with screening human cell factor acceptor; Perhaps be used to prepare the disease that medicine causes owing to the human cell factor expression of receptor unusually with prevention, diagnosis or treatment.