CN1152315A - Monocyte chemotactic protein-4 - Google Patents

Abstract

Polynucleotides which encode the polypeptide MCP-4, as well as such polypeptides, antibodies and antagonist/inhibitors against the polypeptide and the use of the polypeptide and antagonist/inhibitors as pharmaceutical for treatment of tumors, wounds, parasitic infection, regulation of hematopoiesis, inflammation, rheumatoid arthritis, lung inflammation, allergies, atherosclerosis and infectious diseases such as tuberculosis.

Description

Monocyte chemotactic protein-4

The present invention relates to the polynucleotide of new identification, the polypeptide of polynucleotide encoding and the purposes and the preparation of these polynucleotide and polypeptide thus.More particularly, polypeptide of the present invention is monocyte chemotactic protein-4 (MCP-4).The invention still further relates to the method for the effect that suppresses these polypeptide.

Triformed unicellular chemotactic protein, that is, and MCP-1, MCP-2, MCP-3.These all albumen have all been done the evaluation on the 26S Proteasome Structure and Function, and have also done clone and expression.MCP-1 and MCP-2 have the ability of attraction white corpuscle (monocyte and white corpuscle), and MCP-3 also has the ability that attracts eosinophil and T lymphocyte (Dahinderi, E. etc., J.Exp.Med., 179:751-756 (1994)).

Originally, person monocytic cell's specificity attracting factor purifying (Matsushima, K. etc., J.Exp.Med., 169:1485-1490 (1989)) from glioma cell line and monocyte clone.This factor is by the neurospongioma deutero-chemokine (GDCF) of initial design such as Matsuhima and monocyte chemotactic and activation factor (MCAF).This factor now is called MCP-1.CDNA with the MCP-1 of rear clone shows that it is extremely similar to mouse JE gene.The JE gene can be induced (Cochran, B.H. etc., Cell 33:939-947 (1983)) in a large number by platelet-derived somatomedin in the mouse fibroma.Mouse JE is extremely similar to MCP-1.It is 62% identical that MCP-1 and mouse JE have in 68 total N-terminal residue districts.JE and MCP-1 are that kind of homologue is accepted widely.Relevant with mouse JE albumen on polypeptide of the present invention-MCP-4 26S Proteasome Structure and Function with MCP-1, referring to Fig. 2.

Use JE/MCP-1 and suppress that neoplastic method is disclosed among the PCT patent application WO-92/20372 in the vertebrates, wherein also disclose and a kind ofly treated the method for local intercurrent disease of malignant tumour and the method that control parasitizes by using JE/MCP-1.Find, the expression inhibiting of JE/MCP-1 albumen in malignant cell cell form the ability of tumour in vivo.

People MCP-1 has 8,700 daltonian 76 amino acid whose basic peptides.MCP-1 is abduction delivering (Leonard, E.J. and Yoshimura, T., Immunol.Today, 11:97-101 (1990)) in monocyte, eosinophil and fibroma cell mainly.Induce this expression the factor be that IL-1, TNF or lipopolysaccharides are handled.

The performance of other of MC-1 comprises the ability that attracts into acquaintance's basophilic granulocyte in the Toxins, pertussis mode strongly.MCP-1 is the cytokine (Bischoff, S.C. etc., J.Exp.Med., 175:1271-1275 (1992)) that can directly induce the histamine that is discharged by basophilic granulocyte.Moreover MCP-1 is by promoting the formation of leukotrienes C4 with interleukin-13, interleukin-15 or granulocyte/pretreated basophilic granulocyte of scavenger cell clone's stimulating factor.MCP-1 inductive basophilic granulocyte amboceptor release meeting plays an important role in the pathology of allergy inflammation and other expression MCP-1.

Clone with nucleotide sequence of coding person monocytic cell's chemotactic and activation factor (MCAF) has disclosed the primary structure (Furtani of the MCAF polypeptide of being made up of the ripe MCAF sequence of the signal peptide sequence of 23 amino-acid residues of inferring and 76 amino-acid residues, Y.H. etc., Biochem.Res.Commu., 159:249-55 (1989)).The complete amino acid sequence of human glioma's deutero-monocyte chemotactic factor (GDCF-2) is also determined.The attractive monocyte of this peptide, but do not attract neutrophil.GDCF-2 comprises 76 amino-acid residues and determines.This peptide chain contains 4 halfcystines, and on 11,12,36 and 52, it produces a pair of ring, is clustered on the disulfide linkage.Moreover the MCP-1 gene has been assigned to (Mehrabian, M.R. etc., Genomics, 9:200-3 (1991)) on the human chromosome 17.The latent effect of some data signal MCP-1 is that the monocyte of mediation arterial wall infiltrates.It seems that monocyte be the center (Nelken, N.A. etc., J.Clin.Invest., 88:1121-7 (1991)) that forms mechanism as the atheroma of the somatomedin propulsion source of the progenitor cell of foam cell and mediation intimal hyperplasia.The replenishing of mononuclear macrophage that the synovia that also has been found that MCP-1 is created between the inflammatory phase relevant with rheumatoid arthritis plays an important role, and the synovial tissue scavenger cell is the leading source of this cytokine.Find, MCP-1 content in from the synovia of rheumatoid arthritis patients apparently higher than from the osteoarthritis patient or from other arthritic's synovia (Koch, A.E. etc., J.Clin.Invest., 90:772-9 (1992)).

MCP-2 and MCP-3 range proinflammatory albumen subtribe, and relevant with MCP-1 on the function, because they attract monocyte specifically, but do not attract neutrophil (VanDamme, J.Exp.Med., 176:59-65 (1992)).MCP-3 demonstrates respectively and 71% and 58% comes from MCP-1 and MCP-2 together.MCP-3 regulates huge inflammatory cytokine of biting function.

One aspect of the present invention provide novel mature polypeptide for MCP-4 with and fragment, analogue and derivative.Polypeptide of the present invention is the people source.

Another aspect of the present invention provides the polynucleotide (DNA or RNA) of these polypeptide of coding.

One side more of the present invention provides a kind of method of producing these polypeptide with recombinant technology.

The method of utilizing the polynucleotide of this peptide species or this peptide species of encoding to come (for example) treatment tumour, promotion wound healing, control parasitiation and regulate hemopoietic for therapeutic purpose that provides on the one hand more of the present invention.

The antibody that provides these polypeptide more on the one hand of the present invention.

Stimulant/the inhibitor that provides these polypeptide more on the one hand of the present invention.For therapeutic purpose can be used to suppress the effect of these polypeptide, for example, treatment rheumatoid arthritis, pneumonia, allergy, communicable disease and be used for preventing inflammation and atherosclerosis.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Following accompanying drawing is an explanation specific embodiments of the present invention, rather than is intended to limit by the included scope of the present invention of claims.

Fig. 1 describes the aminoacid sequence of the deduction of cDNA sequence and corresponding M CP-4.119 shown aminoacid sequences are full-length proteins, and roughly preceding 22 amino acid are represented leader sequence, and 97 amino acid longs are proteic mature forms.Adopt the abbreviation of standard amino acid single-letter.

Fig. 2 illustrates the homology of MCP-4 and the proteic cDNA sequence of mouse JE.In each three pulsating top sequence are MCP-4, and the bottom sequence is a mouse JE albumen.

Fig. 3 shows the Northern engram analysis result of the mRNA transcript of MCP-4 in people's cell.

The banding pattern of MCP-4 behind expression of Fig. 4 showed cell and the purifying.

Fig. 5 is the schematic illustrations of pQE-9 carrier.

One aspect of the present invention provides a kind of nucleotides (polynucleotides) of separation, its tool of encoding Have the mature polypeptide of amino acid sequence of deduction shown in Figure 1 or on March 10th, 1994 with ATCC The mature polypeptide of the clone's of preserving number 75703 preservations cDNA coding.

Polynucleotides of the present invention are to find from the monocyte cDNA library of activation. It contains volume Code roughly 119 amino acid lengths and wherein front 22 amino acid residues are the targeting sequencings of inferring The ORF of albumen. Therefore maturation protein is expected to be 97 amino acid lengths. Single with mouse on its structure (MCP-1 and JE) is relevant for the nucleus chemotactic protein, show 27% identical and 56% similar in appearance to Whole people's CCL2 sequence. Described polypeptide contains in all chemotactic factor (CF)s that come across the feature motif All four kinds of cysteine residues. MCP-1/JE compares with mouse, the interval between these cysteines Obtain keeping, show that strongly this new gene is chemotactic factor (CF).

Polypeptide of the present invention can be rna form or dna form, and DNA comprises cDNA, base Because of group DNA and synthetic DNA. DNA can be two strands or strand, and if strand is single Chain can be coding strand and non-coding (antisense) chain. The coded sequence of encoding mature polypeptide can with Fig. 1 Shown in identical or identical with the clone of preservation of coded sequence, maybe can be different code sequences Row, this different coded sequence (because the repetition of gene-code or result of degeneracy), the maturation of coding is many Coded identical of the DNA of peptide and Fig. 1 or the cDNA of preservation.

The polynucleotides of the mature polypeptide of the cDNA coding mature polypeptide of code pattern 1 or preservation can To comprise: the coded sequence that is mature polypeptide; The coded sequence of mature polypeptide and additional code sequence (such as targeting sequencing or secretion sequence or front protein sequence); The coded sequence of maturation protein (with can not have The additional code sequence that can not have) and non-coding sequence (such as 5 of mature polypeptide ' and/or 3 ' coded sequence Introne or non-coding sequence).

Term " polynucleotides of coded polypeptide " only comprise for the polynucleotides of the coded sequence of polypeptide with And comprise the polynucleotides of additional code sequence and/or non-coding sequence.

The invention still further relates to the variant class of above-described polynucleotides, these variant codings have The amino acid sequence of deduction shown in Figure 1 or by the fragment of the polypeptide of the cDNA of preservation coding, similar Thing and derivative. The variant of polynucleotides can be the allelic variant of the polynucleotides of natural generation Or the variant of the described polynucleotides of non-natural generation.

So, the present invention includes polynucleotides and variant thereof. Described polynucleotide encoding such as Fig. 1 Shown mature polypeptide or the identical mature polypeptide coded with the clone's of preservation cDNA. Described Polynucleotides variant coding polypeptide shown in Figure 1 or many by the clone's of preservation cDNA coding The fragment of peptide, derivative or analog. These nucleotide derivatives comprise the deletion mutation body, replace change Allosome and interpolation or insertion variant.

As noted above, polynucleotides can have a coded sequence, and it is coding shown in Figure 1 The allelic variant of the natural generation of the clone's of sequence or preservation coded sequence. Institute is public as this area Know that allelic variant is the changeable-shaped of polynucleotide sequence, it can replace, lacks or add One or more nucleotides, and basically do not change the function of coded polypeptide.

The present invention also comprises polynucleotide, wherein the encoding sequence of mature polypeptide can be integrated into help from the polynucleotide sequence of host expresses and secrete polypeptide with identical frame, described sequence for example, a kind of leader sequence that works at the control polypeptide as secretion sequence by host's transhipment.Polypeptide with leader sequence is preceding albumen (preprotein) and can has the leader sequence that is formed the mature form of polypeptide by host's cracking.Polynucleotide also can be encoded to the former albumen that mature polypeptide adds additional 5 ' amino-acid residue.Maturation protein with former sequence is former albumen (proprotein) and is this proteic inactivation form.In case former sequence is cleaved, generation be activated protein.

Like this, for example, the ripe albumen of polynucleotide codified of the present invention, or have the maturation protein of presequence or have former sequence and the albumen of presequence (leader sequence).

Polynucleotide of the present invention also can have with frame and are blended in encoding sequence in the flag sequence that can make peptide purification of the present invention.Flag sequence can be a flag sequence of being supplied with six histidine mark things by the pQE-9 carrier, this marker is provided at the mature polypeptide in the mark of being blended in of purifying under the condition of host bacterium, or, for example, when adopting Mammals for example during the COS-7 cell, flag sequence can be hemagglutinin (HA) marker.The HA marker is corresponding to influenza hemagglutinin protein deutero-epi-position (Winson, I. etc., Cell, 37:767 (1984)).

The invention still further relates to like this some polynucleotide, they hybridize on the sequence recited above (as have between infructescence at least 50% with preferably 70% identical).The present invention be more particularly directed at the polynucleotide that hybridize under the condition of strictness on the above-mentioned polynucleotide.Herein, term " strict condition " meaning is only at least 95% and preferably 97% when identical, hybridize and just can take place between sequence.In some preferred embodiments, hybridize to polynucleotide encoding on the top described polynucleotide keep basically with by the cDNA of Fig. 1 or the identical biological function or the active polypeptide of cDNA encoded polypeptide of preservation.

Preservation thing herein can be according to the preservation that requires of " budapest treaty of the microbial preservation that is used for patented procedure of international recognition ".These preservations for for those skilled in the art provide convenience, are not the desired preservations of 35 U.S.C.$112. clauses only.In the preserved material contained polynucleotide sequence and thus the aminoacid sequence of encoded polypeptides and be used to solve and the relevant contradiction of any sequence description herein in the lump as the reference of this paper.The material of manufacturing, use or sale preservation should not give this permission herein through permission.

The invention still further relates to and have the aminoacid sequence shown in Figure 1 of inferring or by the MCP-4 polypeptide of the cDNA amino acid sequence coded of preservation, and the fragment of these polypeptide, analogue and derivative.

Term " fragment ", " derivative " and " analogue ", when polypeptide that refers to Fig. 1 or cDNA encoded polypeptides, the meaning is to have kept biological function identical with this peptide species or active polypeptide basically.Thus, analogue comprises a kind of former albumen, and this former albumen can generate active mature polypeptide and activates by cutting former albumen.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides or synthetic polypeptide, preferred recombinant polypeptide.

The fragment of the cDNA encoded polypeptides of polypeptide shown in Figure 1 or preservation, derivative or analogue can be (i) polypeptide that wherein one or more amino-acid residues are guarded or nonconservative amino-acid residue (preferred conservative amino-acid residue) replaces, and the amino-acid residue of this replacement can yes or no by the genetic code coding, or (ii) wherein one or more amino-acid residues comprise substituent polypeptide, the (iii) polypeptide that merges of mature polypeptide and another compound wherein, as increase the compound (for example polyoxyethylene glycol) of the transformation period on the polypeptide, or (iv) wherein additional amino acid is fused to the polypeptide on the mature polypeptide, as leader sequence or secretion sequence or adopt purifying mature polypeptide or former proteic sequence.Because the instruction of this paper, these fragments, derivative and analogue are in those skilled in the art's ken.

Polypeptide of the present invention and polynucleotide preferably provide with isolating form, and preferably are purified to homogeneous.

Term " isolating " meaning is meant that material shifts out from its former environment (for example, if its physical environment is natural existence).For example, the naturally occurring polynucleotide or the polypeptide that are present in the living animal are unsegregated, but identical polynucleotide or the polypeptide separated in part from natural system and the whole coexisting substances are isolating.These polynucleotide can be that the part of carrier and/or these polynucleotide or polypeptide can be the parts of composition, and are still isolatingly, and like this, this carrier or composition just are not the parts of its environment.

Host cell that carries carrier of the present invention that the present invention also relates to comprise the carrier of polynucleotide of the present invention, produces by genetic engineering and the method for producing polypeptide of the present invention with recombinant technology.

It can be for example cloning vector or expression vector that host cell is with carrier of the present invention, carrier of the present invention by genetically engineered (transduction or conversion or transfection).Carrier can be for example plasmid form, virion, phage etc.The engineering host cell can be cultivated in the nutritional medium of the improved routine that is suitable for activating promotor, screening transformant or amplification MCP-4 gene.Culture condition, for example, temperature, pH or the like, be the choosing host cell of expressing adopt in advance those, and they are conspicuous for those of ordinary skill.

Polynucleotide of the present invention can be used for producing polypeptide through recombinant technology.Thus, for example, polynucleotide can be included among multiple expression vector arbitrary of express polypeptide.These carriers comprise chromosomal, achromosomal and the synthetic DNA sequence, for example, and the derivative of SV40; Bacterial plasmid; Phage DNA; Baculovirus; Yeast plasmid; Make up the deutero-carrier by plasmid and phage DNA, viral DNA (as vaccinia virus, adenovirus, fowlpox virus and pseudorabies virus).Yet as long as carrier is reproducible and stable in the host, any carrier all can use.

Suitable dna sequence dna can insert carrier by several different methods.Usually, dna sequence dna is to insert suitable limited restriction enzyme site by the known method of prior art.These methods and other method are in those skilled in the art's ken.

Dna sequence dna in the expression vector is gone up with guiding mRNA synthetic through being operationally connected to suitable expression control sequenc (promotor).What the representative example of these promotors can be mentioned has: LTR or SV40 promotor, other promotor that the known control in the gene of E.coli.lac or trp, phage PL promotor and prokaryotic cell prokaryocyte and eukaryotic cell or their virus is expressed.Expression vector also can contain the ribosome bind site and the transcription terminator of translation initiation.Carrier also can comprise the sequence that suitable amplification is expressed.

In addition, expression vector preferably contains one or more marker gene, so that the phenotypic characteristic in order to the screening transfection host cell to be provided, as the neomycin resistance (or as tsiklomitsin among the E.coli or amicillin resistance) of Tetrahydrofolate dehydrogenase or eukaryotic cell cultivation.

The carrier that contains suitable dna sequence dna as described above, and suitable promotor or control sequence can be used for transforming suitable host, make this host expresses albumen.

What suitable host's representative example can be mentioned has: bacterial cell, as E.coli, streptomycete, typhimurium encoded plasminogen activator; The fungal cell is as yeast; Insect cell is as Drosophila and sf9; Zooblast is as CHO, COS or Bowes melanoma; Vegetable cell, etc.Because the instruction of this paper selects suitable host in those skilled in the art's ken.

More particularly, the present invention also comprises the recombinant precursor of one or more sequences of broadly describing above comprising.These constructs comprise carrier, as plasmid and virus vector, in these carriers just or in the other direction to insert sequence of the present invention.Aspect this embodiment preferred, this construct also comprises regulates sequence (comprising that (for example) can be operatively connected the promotor on sequence).A large amount of suitable carrier and promotors are well known by persons skilled in the art, and can buy.Following carrier provides in the mode of example.Bacterium: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluscript SK, pbsks, pNH8A, pNH16a, pNH18A, pNh46A (Stratagene); Ptrc99a, pkk223-3, pkk233-3, pDR540, pRIT5 (Pharmacia).Eukaryotic cell: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).Yet as long as they are reproducible and stable in the host, other plasmid or carrier also can adopt.

Adopt CAT (CAT) carrier or other carrier that has selective marker promoter region can be selected from any required gene.Two suitable carriers are PKK232-8 and PCM7.The bacterium promotor that specifically provides name comprises lacI, lacZ, T3, T7, gbt, λ PR, PL and trp.Eukaryotic promoter comprises that CMV is early stage immediately, HSV thymidine kinase, early stage and late period SV40, be derived from retroviral LTRs and mouse metallothionein(MT)-I.Selecting suitable carrier and promotor is in those of ordinary skills' ken.

In another embodiment, the present invention relates to contain the host cell of above-mentioned construct.Host cell can be senior eukaryotic cell (as mammalian cell) or rudimentary eukaryotic cell (as yeast cell), or host cell can be prokaryotic cell prokaryocyte (as a bacterial cell).Construct is imported host cell can implement (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)) by the transfection or the electroporation of calcium phosphate transfection, DEAE-Dextran mediation.

Construct in the host cell can use with the method for routine, produces the gene product by the recombination sequence coding.In addition, polypeptide of the present invention can be synthetic by conventional Peptide synthesizer.

Maturation protein can be expressed in Mammals, yeast, bacterium or other cell under suitable promotor control.The cell free translation system also can adopt to use by DNA construct derived RNA of the present invention produces these albumen.Be used for prokaryotic cell prokaryocyte and eukaryotic suitable clone and expression vector and be described in, Sambrook etc., Molecular Cloning:A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).Disclosing of this literary composition incorporated this paper into as a reference.

Senior eukaryotic cell is transcribed by enhancer sequence being inserted in the carrier and is increased the DNA's of code book invention polypeptide.Enhanser is the DNA cis acting factor, about usually 10-300bp, and it acts on promotor, increases transcribing of promotor.Example comprises the SV40 enhanser (the sub-enhanser of a kind of cytomegalovirus early promoter) that is positioned at replication orgin rear side bp100-270, at the polyoma enhanser and the adenovirus enhanser of replication orgin rear side.

Usually, the selective marker that recombined protein carrier will comprise replication orgin and allow host's transfection (for example, the ampicillin resistance gene of E.coli and S.cerevisiae TRP1 gene) and by the senior expressing gene deutero-promotor of just transcribing of downstream configurations sequence.This promotor can be derived sugared ferment enzyme such as glycerol 3-phosphate acid kinase (PGK), α-factor, acid phosphatase or heat shock protein etc. by the operon of the sugared ferment enzyme of coding.Allos structure sequence and translation initiation and end sequence (and albumen that preferably can directed secretion translation is gone into the leader sequence of periplasmic space or the outer medium of born of the same parents) are assembled in mutually suitable.Randomly, heterologous sequence can be encoded and be comprised the fusion rotein of the terminal identification polypeptide of N-of giving institute's phase feature, and institute's phase feature is as stability or simplify purifying express recombinant product.

The structural DNA sequence of the useful expression vector that bacterium is used by the required expressing protein of will encoding and translation initiation that is fit to and end signal are inserted into together to have handling of functional promotor and makes up in reading mutually.Carrier should comprise one or more Phenotypic Selection marker and replication orgin, is provided at the amplification among the host with maintenance and (if hope) of guaranteeing carrier.The prokaryotic hosts that is fit to of transfection comprises the kind of E.coli, subtilis, typhimurium encoded plasminogen activator and various false monospore Bacillaceae and streptomyces, the host that also can select to adopt other.

As representativeness but the example of indefiniteness, the useful expression vector that bacterium is used comprises the replication orgin of selective marker and bacterium, and the replication orgin of this selective marker and bacterium comes from the plasmid of the commercially available gene element that comprises the cloning vector pBR322 (ATCC 37017) that knows.These merchants sell carrier and comprise, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA).With these pBR322 " skeleton " part and the suitable promotor and the structure sequence combination of desire expression.

After the host strain transfection that is fit to and this host strain grew to suitable cell density, the mode (for example, temperature transition or chemical induction) of selecting promotor to pass through to suit was induced and cell is cultivated one period again.

Cell is typically by (mode with physics or chemistry is broken) centrifugal collection, and the crude extract of reservation gained is done repurity.

Adopt the microorganism cells of expressing protein to break with any method easily, comprise freeze-thaw cycle, sonication, mechanical disruption or use the molten born of the same parents' agent of cell, these methods are well-known to those skilled in the art.

Various mammalian cell culture systems also can be used for express recombinant protein.The example of Mammals expression system comprise the COS-7 system (be described in Gluzman, Cell is among the 23:175 (1981)) of monkey renal fibroma and other can the expression matching carrier clone, for example, C127,3T3, CHO, HeLa and bhk cell system.Mammalian expression vector comprise replication orgin, suitable promotor and enhanser and any needs ribosome bind site polyadenylation site, donor splicing site and acceptor site, transcribe end sequence and 5 ' flank non-transcribed sequence.Can be used to provide required non-transcribed gene element by SV40 splice site and polyadenylation site deutero-dna sequence dna.

Polypeptide can reclaim and purifying from the culture of reconstitution cell by the following method: ammonium sulfate or ethanol precipitation, acid extraction method, negatively charged ion or cation-exchange chromatography, phosphinylidyne cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography method and Yelkin TTS chromatography.Preferably during purifying, there is the calcium ion of (the roughly 0.15-5mM) of lower concentration to exist (Price etc., J.Biol.Chem., 244:917 (1969)).If desired, can use the protein refolding step to improve the configuration of maturation protein.At last, can adopt high performance liquid chromatography to carry out final purification step.

Polypeptide of the present invention can be natural purified product, or the product of chemical synthesis process, or produce from protokaryon or eucaryon host (for example, with bacterium, yeast, higher plant, insect and mammalian cell in the substratum) with recombinant technology.According to the host who is used in the recombinant method for production, polypeptide of the present invention can be glycosylated or nonglycosylated.Polypeptide of the present invention also can comprise the initial methionine amino-acid residue.

Specifically with regard to MCP-4, polypeptide of the present invention can be used for promoting the healing of wound.Because MCP-4 is a chemokine, it is the chemoattractant of white corpuscle (as unicellular, T lymphocyte, basophilic granulocyte etc.); Therefore, it causes the target immunocyte to infiltrate wound area.

The MCP-4 polypeptide also can ooze out (plural effusions) or ascites as anti-tumor therapeutic agent with as the local complication such as the side of treatment malignant tumour.MCP-4 is injected the anatomical position that involves to cause local unicellular accumulation and activation.

The existence of MCP is accompanied by the part increase that eosinophil exists in the live body.Eosinophil has the distinguished function of killing the Parasite larvae (as the Parasite larvae in schistosomicide, defect and ascariasis) of invading tissue.

MCP-4 can work on the adjusting hemopoietic by regulating various hemopoietic progenitor cell activation and differentiation.

According to the present invention, polypeptide also can use by express these polypeptide at live body, this so-called " gene therapy ".

For example, patient's cell can carry out the genetically engineered operation at the encode polynucleotide (DNA or RNA) of MCP-4 polypeptide of external use, and the cell with through engineering approaches offers the patient that desire is treated with this polypeptide then.These methods are well known in the art.For example, cell can contain the retrovirus of the RNA of code book invention polypeptide by use, with gene engineering method operation known in the art.

Equally, cell can be handled through genetic engineering technique in vivo, expresses the MCP-4 polypeptide by for example methods known in the art in vivo.As known in the art, produce the production cell of the retroviral particle of the RNA contain code book invention polypeptide, can be applied in patient's body, produce engineering cell in the body, and express polypeptide in vivo.Use these and other method of polypeptide of the present invention, because instruction of the present invention will be readily apparent to persons skilled in the art.For example, the expression vector of engineering cell can not be a retrovirus, for example, can be with suitable transport carrier combinations after, be used for producing in vivo the adenovirus of engineering cell.

Polypeptide of the present invention can be used in combination with suitable pharmaceutical carrier.This composition comprises polypeptide and the medicinal acceptable carrier or the excipient for the treatment of significant quantity.Such carrier includes but not limited to salt solution, buffer saline, glucose, water, glycol, ethanol and combination thereof.These preparations should be suitable for method of application.

The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, the prompting by the indication form of making, using or selling the government authorities of medicine or biological products to provide can be arranged, this prompting reflects productions, use or the government authorities of sale permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.

Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the approach of intracutaneous.MCP-4 comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that deliver medicine to patient's MCP-4 will depend on many factors, as administering mode, the natural condition of desiring the curer and diagnostician's judgement.Usually, MCP-4 will be with the amount administration at least about 10 μ g/Kg body weight, and in most of the cases dosage is no more than approximately 8mg/Kg body weight every day.Consider route of administration, symptom etc., in most of the cases, dosage is that pact μ g/Kg every days 10 is to about 1mg/Kg body weight.

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence-specific ground is target with the particular location of single human chromosome, and karyomit(e) hybridization therewith.And, need to identify the concrete site on karyomit(e) at present.Now, seldom there is chromosome marking reagent to can be used for the marker chromosomes position based on actual sequence data (repetition polytypism).According to the present invention, DNA is plotted on the karyomit(e), be with the important the first step of these sequences with the gene-correlation connection that is relevant to disease.

Briefly, by prepare PCR primer (preferred 15-25bp) by cDNA, can be with sequence to chromosome mapping.Promptly select not stride across the primer of an exon of genomic dna with the Computer Analysis of cDNA, complicated thus amplification program.Then, these primers are used for the somatic cell hybrid that the PCR screening contains single human chromosome.Have only these hybrids that contain corresponding to the people's gene of primer can produce the fragment of amplification.

The PCR mapping of somatic cell hybrid is that concrete DNA is assigned to concrete chromosomal quick method.Use has the present invention of identical Oligonucleolide primers, can use from specific chromosomal segmental panel (panel) or a large amount of genomic clone aggregates of similar fashion and obtain inferior location (sublocalization).Can similarly be used for being plotted on its chromosomal other mapping strategy comprise in situ hybridization, with the karyomit(e) prescreen of the airflow classification of mark with by hybridizing to the prescreen in the specific cDNA of construct karyomit(e) storehouse.

Can be used for accurate chromosomal localization in a step to the cDNA clone's of the Metaphase Chromosome of diffusion fluorescence in situ hybridization (FISH).This technology can be used in the cDNA that is short to 500 or 600 bases; Yet greater than 2, the clone of 000bp more trends towards being attached to the specific staining body position of simply detected enough strength of signal.FISH need use by EST deutero-clone, and the clone is the bigger the better.For example, 2,000bp just can, 4,000 is better, and perhaps be not that to obtain good result's (rational per-cent on the time) needed greater than 4,000.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manual of Basic Techniques, Pergamon Press, New York (1988).

The chromosome position accurately in case sequence is mapped, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data are found in, for example, V.Mckusick, Mendelian Inheritance in Man (by with online can the acquisition of Johns Hopkins University WelchMedical Library).By association analysis, determine gene and the relation of the disease of some chromosomal regions of having mapped already then.

Then, need to measure the different of ill and not cDNA between diseased individuals or genome sequence.If observe sudden change in some or all of diseased individuals, and do not observe in any normal individual, then sudden change may be the reason of disease.

With the resolving power of present physical mapping and gene mapping technology, accurately be positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (supposing 1 megabasse mapping resolving power and gene of every 20kb) between 50 to 500 potential Disease-causing genes.

More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as stretching visible from karyomit(e) or using based on detectable disappearance of the PCR of cDNA sequence or transposition.At last, the gene complete of several individualities ordering is that conclusive evidence sudden change existence and sudden change and many types of difference are needed.

Polypeptide, its fragment or other derivative or its analogue or express their cell can be used as immunogen, are used for producing its antibody.These antibody can be for example polyclonal antibody or monoclonal antibody.The present invention also comprises chimeric chain, single-chain antibody, and the humanized antibodies, and the Fab fragment, or the product of Fab expression library.Can produce these antibody and fragment with the various known methods in this area.

Antibody at producing corresponding to polypeptide of sequence of the present invention can obtain by directly polypeptide being injected into animal (preferably inhuman).The antibody of Huo Deing can combine with its polypeptide then like this.In this mode, in addition coding only the fragments sequence of a polypeptide also can be used for producing antibody in conjunction with whole natural polypeptides.Then, these antibody can be used for polypeptide is separated from the tissue of expressing this polypeptide.

Be the preparation monoclonal antibody, can use any technology that the antibody of producing by the continuous cell line culture is provided, the example of these technology comprises hybridoma technology (Kohler and Milstein, 1975, Nature, 256:495-497), trioma technology, human B cell hybridoma technology (Kozbor etc., 1983, Immuolgy Today 4:72), with the EBV hybridoma technology (Cole etc. that produce human monoclonal antibodies, 1985, Monoclonal Antibodies and Cancer Terapy, Alan R.Liss, Inc., pp77-96).

For the technology (USP 4,946,778) that the production single-chain antibody is described can be improved the single-chain antibody that is used for producing immune peptide product of the present invention.

The invention still further relates to the diagnostic testing process of quantitative and qualitative detection MCP-4 level.These tests are known in the art, and comprise that ELISH measures and radioimmunoassay.The MCP-4 level that detects in the test can be with laying down a definition the importance of MCP-4 in various diseases and be used to the disease of diagnosing MCP-4 to work.

The invention provides the method for identification of M CP-4 acceptor.The gene of coding MCP-4 acceptor can be identified by cloning by expression.In brief, polyadenylation RNA by the cell preparation of corresponding MCP-4, and will be divided into aggregate from the cDNA library that RNA produces, be used for rotaring redyeing COS cell or other not respond the cell of MCP-4.The transfectional cell that is grown on the glass swash plate is exposed under the MCP-4 of mark.MCP-4 can be with known variety of way mark, and described mode comprises the iodate or the eliminating of the recognition site of site-specific protein kinase.Fixing and cultivate after, swash plate is carried out radioautographic analysis.Identify positive aggregate, prepare inferior aggregate, with repeating inferior aggregate transfection and screening more again, final produce the mono-clonal that coding is inferred acceptor.As the other method that acceptor is identified, the MCP-4 of mark can be the photoaffinity that links to each other with the extraction prepared product of cytolemma or expression MCP-4 acceptor molecule.Cross-linked material splits by PAGE, and is exposed to X-ray film.Contain the MCP-4 acceptor the marker ligand compound can excise, split into peptide fragment, and carry out the little ordering of albumen.From the aminoacid sequence that little ordering obtains, can be used to design one group and produce oligonucleotide probe, with screening cDNA storehouse, identification code is inferred the gene of acceptor.

The present invention also provides screening of medicaments to identify the method that improves (stimulant) or check the medicament of (antagonist) MCP-4 and its acceptor interaction.Stimulant improves the biological function of MCP-4, and antagonist reduces or eliminate this class function.For example, the film preparation of mammalian cell or expression MCP-4 can be cultivated by the MCP-4 with mark in the presence of medicine.Measure the medicine raising then or check this interactional ability.In addition, after MCP-4 and its acceptor interaction, measure the response of known second messenger's system, medicine is existed or do not exist make comparisons.This class second messenger system includes but not limited to cAMP guanylate cyclase, ionic channel or phosphoinositide hydrolysis.

The present invention also relates to the antagonist/inhibitor molecules of polypeptide of the present invention.Antagonist comprises the negative advantage mutant of MCP-4.MCP-4 is four poly-polypeptide, and one of them muton can cause whole polypeptide not have function.The negative advantage mutant of MCP-4 is attached on the MCP-4 acceptor, but can not activate its bonded cell (white corpuscle and monocyte).The test that detects the negative advantage mutant of MCP-4 is external chemotaxis test, wherein use many wells chemotactic chamber of the polycarbonate membrane that has no polyvinylpyrrolidone, in the existence of strong antagonist/inhibitor or doping molecule with not, measure MCP-4 to leukocytic chemoattractant ability.

The example of inhibitor is antisense DNA or RNA construct.Can be used for controlling gene by the formation of triple helical body or antisense DNA or RNA (two methods all are to be combined into the basis with polynucleotide and DNA or RNA) antisense technology expresses.For example, 5 ' encoding part of the polynucleotide sequence of code book invention mature polypeptide, can be used for designing about 10-40 base pair length antisense rna oligonucleotide.The design dna oligonucleotide, be complementary to relate to the gene region of transcribing (the triple helical body--referring to Lee etc., Nucl.Acids Res., 6:3073 (1979); Cooney etc., Science, 241:456 (1988); With Dervan etc., Science, 251:1360 (1991), prevent to transcribe generation thus with MCP-4.Antisense rna oligonucleotide and mRNA are hybridized in vivo, and check the mRNA molecule and translate (antisense--Okano, J.Neurochem., 56:560 (1991) among the MCP-4; Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, FL (1988)).In addition, sense-rna and DNA can transporte to cells, and like this, they are expressed in vivo, to suppress the generation of MCP-4.

Another example of antagonist is the peptide derivant of MCP-4, and this derivative is the natural of MCP-4 or synthetic modification analogue, this analogue lose biological function but still identification and be incorporated into acceptor, check acceptor thus effectively.

Cytophilicly compile relevantly because acute and chronic inflammation lung disease and the monokaryon in the lung are huge, therefore, by preventing to attract monocyte to wound or site of injury, antagonist/inhibitor can be used for treating inflammation, and the adjusting normal lung is huge has a liking for cell quantity.They also can be used for treatment of arthritis, because finding the amount of MCP obviously increases in the synovia of rheumatoid arthritis patients, illustrate that the MCP that synovia is produced attracts monocyte, monocytic inflow and attraction are important on the pathology of sex change and inflammatory arthritis.

Because MCP mediation monocyte infiltrates arterial wall, and monocyte infiltration arterial wall causes atheronecrosis, therefore antagonist/inhibitor also can be used for treating atheronecrosis, and directly induce basophilic granulocyte to discharge histamine owing to shown MCP already, so antagonist/inhibitor can be used for preventing allergy.

Because tuberculosis target cell (normally monocyte) causes monocyte and discharges MCP, and MCP attracts more monocyte to lung, causes serious inflammation, so antagonist/inhibitor also can be used to treat communicate illness such as tuberculosis.Antagonist/inhibitor also can be used in the composition of (for example as described above) pharmaceutically acceptable carrier.

The invention still further relates to the test method that mediates the potential antagonist/inhibitor that is specific to MCP-4.The example of this test, under suitable condition, with the MCP-4 acceptor and have film in conjunction with potential antagonist/inhibitor of MCP-4 acceptor or reorganization MCP-4 acceptor in conjunction with being at war with inhibition test.MCP-4 can be mark (as using radioactivity), can be measured the validity of potential antagonist/inhibitor like this by the quantity of the MCP-4 that is attached to acceptor.

Further describe the present invention with reference to the following example; Yet, should be appreciated that to the invention is not restricted to these embodiment.All parts or amount all by weight, unless otherwise.

For the ease of understanding the following example, will describe some normal method and/or terms that occurs below.

" plasmid " can be abbreviated to the p on the lower position, connects capitalization and/or numeral before or after it.The initial plasmid of this paper can be commercially available, nonrestrictive public Ke De's, or can be according to disclosed method by commercially available plasmid construction.In addition, the plasmid that is equal to described plasmid is known in the art and is conspicuous for those of ordinary skill.

" digestion " of DNA is meant with only acting on the restriction enzyme of some sequence of DNA with the DNA catalyze cleavage.Various restriction enzyme used herein is commercially available, and its reaction, cofactor and other requirement are that those of ordinary skills are known.Be the purpose of analyzing, typically, 1 μ g plasmid or dna fragmentation use the enzyme of about 2 units in 20 μ l damping fluids.Carry out the purpose of plasmid construction for DNA isolation, typically, be used in the DNA of the enzymic digestion 5-50 μ g of the 20-250 unit in the more volume.The suitable damping fluid and the amount of substrate of concrete restriction enzyme are described in detail by producer, and what adopt usually is to cultivate 1 hour at 37 ℃, but can change according to supplier's indication.After the digestion, reactant is directly moved on polyacrylamide gel, separate 345Xelectr (required fragment).

The size separation of cutting fragment is to use 8% polyacrylamide gel of being described by (Goeddel, D. etc., Nucleic Acids Res., 8:4057 (1980)) to carry out.

" oligonucleotide " be meant can chemosynthesis the many thymus nucleic acids of strand or many thymus nucleic acids of two complementary strands.These synthetic oligonucleotide do not have 5 ' phosphoric acid, therefore if do not add the phosphoric acid of band ATP in the presence of kinases, then are not connected with another oligonucleotide.Synthetic oligonucleotide can be connected with the fragment of dephosphorylation not.

" connection " be meant two double stranded nucleic acid fragments form the process of phosphinylidyne diester linkages (Maniatia, T. etc., ID., p.16).Unless otherwise, use known damping fluid and condition, the desire of the roughly equimolar amount of per 0.5 μ g connects the T4 dna ligase (" ligase enzyme ") of dna fragmentation with 10 units, can finish connection.

Unless otherwise, conversion is according to Graham, F. and Van der Eb, and A., the method for describing among the Virology, 52:456-457 (1973) is implemented.The bacterial expression of embodiment 1MCP-4 and purifying

The dna sequence dna (ATCC#75703) of coding MCP-4 use corresponding to the MCP-4 of processing proteic 5 ' and 3 ' the initial amplification of PCR Oligonucleolide primers of 3 ' sequence (subtraction signal peptide sequence) and carrier sequence MCP-4 gene.Corresponding to the additional nucleotide of MCP-4 be added to 5 respectively ' and 3 ' sequence on.5 ' Oligonucleolide primers have sequence 5 '-TCAGGATCCCCTACGGGCTCGTGGTC-3 ', this sequence contains Bam H1 restriction endonuclease sites, then is 18 Nucleotide of the MCP-4 encoding sequence that begins of the supposition end amino acid of albumen codon from processing.3 ' sequence 3 '-CGCTCTAGAGTTAAAACGACGGCCAGT-5 ' contains the XbaI site and is positioned at the complementary sequence of the pBluescript SK carrier sequence of MCP-4 DNA inset 3 '.Restriction endonuclease sites is corresponding to the restriction endonuclease sites on fibrocyte expression vector (Qiagen, Inc.9259 Eton Avenue, Chatsworth, CA, 91311).PQE-9 encoding antibody resistance (Amp '), the bacterium of duplicating former (ori), IPTG can regulate promotor operon (P/O), ribosome bind site (RBS), 6-His marker and restriction endonuclease sites.Then with BamH1 and Xab I digestion pQE-9.Extension increasing sequence is connected into pQE-9, and insert in the frame of the sequence that has coding histamine marker and RBS.Fig. 5 is the synoptic diagram of this arrangement.(can obtain by following method Transformed E .coli bacterial strain m15/rep4 with connecting mixture then from Qiagen, trade name M15/rep4): Sambrook, J. etc., Molecular Cloning:A LaboratoryManual, Cold Spring Laboratory Press, 1989.M15/rep4 contains a plurality of copies of expressing the lacI repressor and giving the plasmid pREP4 of kalamycin resistance (Kan ').By the identification of the energy for growth on LB plate transformant, and filter out penbritin/kalamycin resistance bacterium colony.Plasmid DNA is separated by restriction analysis and is determined.To contain grow overnight (O/N) in the liquid nutrient medium in the LB medium that being cloned in of required construction be supplemented with Amp (100ug/ml) and Kan (25ug/ml).With the O/N culture with 1: 100-1: 250 ratio is inoculated a large amount of cultures.It is between 0.4 and 0.6 that cell is grown into optical density (OD) 600 (O.D.600).Then IPTG (sec.-propyl-B-D-sulfo-semi-lactosi pyrans glycosides) is added to final concentration 1mM.Induce removing P/O by inactivation lacI repressor, IPTG, cause the genetic expression that increases.With long 3 to 4 hours of cell regeneration.Cell precipitation dissolves with 6 mole hydrochloride guanidine chaotropic agents.After the clarification, contain under the proteic condition of 6-His marker allowing to combine closely, by chromatogram on the Nickel-Chelate post, with dissolved MCP-4 from then in the solution purifying come out.Hochli, E. etc., J.Chromatography 41l:177-184 (1984).With MCP-4 (purity 95%) wash-out from the post, and is the purpose of renaturation with 6 moles Guanidinium hydrochloride pH5.0, is adjusted to 3 mole hydrochloride guanidines, 100mM sodium phosphate, 10mM Triptide (reduction) and 2 moles of Triptides (oxidation).Incubation was dialysed albumen after 12 hours to the 10mM sodium phosphate in this solution.Fig. 4.The phraseology of MCP-4 in embodiment 2 people's cells

Carry out the Northern engram analysis, measure the expression level of MCP-4 in people's cell.Separate total cell RNA sample with RNAzolTM B system (Houston, TX 77033 for Biotecx Laboratories, Inc.6023 South Loop East).Total RNA from the about 10 μ g of each concrete people's separate tissue separates on 1% sepharose, and trace (Sambrool, fritsch and Maniatis, M olecular Cloning, Cold Spring HarborPress, (1989)) to NF.According to Stratagene Prime-It test kit with the reaction that makes marks of 50ngDNA fragment.The DNA of mark Select-G-50 column purification.(5?Prime-3?prime，Inc.5603Arapahoe?Road，Boulder，CO?80303)。Then, filter is with radiolabeled total length MCP-4 gene, and under 65 ℃, with 1,000, the speed of 000cpm/ml is hybridized in 0.5MNaPO4, pH 7.4 and 7%SDS and spent the night.At room temperature wash twice and after twice of 60 ℃ of flushing, filter is exposed under-70 ℃ with strengthening window of tube with 0.5 * SSC, 0.1%SDS.The messenger RNA(mRNA) of MCP-4 is rich in T cell, monocyte and the T clone of activation and inactivation.

In view of top instruction, many changes of the present invention and variation are possible, and in the scope of appended claim, except specifically described, the present invention also can implement.

Sequence table (1) general information:(i) applicant:LI, ET AL. is denomination of invention (ii): monocyte chemotactic protein-4 is sequence number (iii): 2 (iv) contact addresses:(A) contact person:CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART ﹠ amp; OLSTEIN ( B ) street: 6 BECKER FARM ROAD ( C ) city: ROSELAND ( D ) states: New Jersey ( E ) country: the U.S. ( F ) postcode: 07068 ( V ) computer-reader form: ( A ) medium type: 3.5 inches disks ( B ) computer: IBM PS/2 ( C ) operating system: MS-DOS ( D ) software: the current application materials of WORD PERFECT 5.1 ( vi ) : ( A ) application number: ( B ) applying date: ( C ) classification number: at excellent application materials ( A ) application number: ( B ) applying date: ( viii ) agent/agency's information: ( A ) name: FERRARO； GREGORY D. ( B ) ：36,134 ( C ) ：325800-160 ( ix ) ： ( A ) ：201-994-1700 ( B ) ：201-994-1744 ( 2 ) SEQ ID NO：1： ( i ) ： ( A ) ：360 ( B ) ： ( C ) ：D ) ： ( ii ) ：cDNA ( Xi ) ：SEQ ID NO：1：ATGGCAGGCC TGATGACCAT AGTAACCAGC CTTCTGTTCC TTGGTGTCTG TGCCCACCAC60ATCATCCCTA CGGGCTCTGT GGTCATACCC TCTCCCTGCT GCATGTTCTT TGTTTCCAAG120AGAATTCCTG AGAACCGAGT GGTCAGCTAC CAGCTGTCCA GCAGGAGCAC ATGCCTCAAG180GGAGGAGTGA TCTTCACCAC CAAGAAGGGC CAGCAGTTCT GTGGCGACCC CAAGCAGGAG240TGGGTCCAGA GGTACATGAA GAACCTGGAC GCCAAGCAGA AGAAGGCTTC CCCTAGGGCC300AGGGCAGTGG CTGTCAAGGG CCCTGTCCAG AGATATCCTG GCAACCAAAC CACCTGCTAA360 ( 2 ) SEQ ID NO：2： ( i ) ： ( A ) ：119 ( B ) ： ( C ) ： ( D ) ： ( ii ) ：： ( Xi ) ：SEQ ID NO：2：Met Ala Gly Leu Met Thr Ile Val Thr Ser Leu Leu Phe LeuGly Val

-20 -15 -10Cys?Ala?His?His?Ile?Ile?Pro?Thr?Gly?Ser?Val?Val?Ile?ProSer?Pro

-5 1 5?10Cys?Cys?Met?Phe?Phe?Val?Ser?Lys?Arg?Ile?Pro?Glu?Asn?ArgVal?Val

15 2025Ser?Tyr?Gln?Lys?Ser?Ser?Arg?Ser?Thr?Cys?Leu?Lys?Gly?GlyVal?Ile

30 35 40Phe?Thr?Thr?Lys?Lys?Gly?Gln?Gln?Phe?Cys?Gln?Asp?Pro?LysGln?Glu

45 50 55Trp?Val?Gln?Arg?Tyr?Met?Lys?Asn?Leu?Asp?Ala?Lys?Gln?LysLys?Ala

60 65 70Ser?Pro?Arg?Ala?Arg?Ala?Val?Ala?Val?Lys?Gly?Pro?Val?GlnArg?Tyr75 80 85 90Pro?Gly?Asn?Gln?Thr?Thr?Cys

95

Claims (22)

1. isolating polynucleotide, it is selected from the group of being made up of following material:

(a) coding has the polynucleotide of fragment, analogue or the derivative of the MCP-4 polypeptide of the aminoacid sequence of inferring shown in Figure 1 or described polypeptide;

(b) coding has the polynucleotide by fragment, analogue or the derivative of MCP-4 polypeptide that is included in the cDNA amino acid sequence coded among the ATCC 75703 or described polypeptide.

2. the polynucleotide of claim 1, wherein said polynucleotide are DNA.

3. the polynucleotide of claim 1, wherein said polynucleotide are RNA.

4. the polynucleotide of claim 1, wherein said polynucleotide are genomic dnas.

5. the polynucleotide of claim 2, wherein said polynucleotide encoding has the MCP-4 of the aminoacid sequence of inferring shown in Figure 1.

6. the polynucleotide of claim 2, wherein said polynucleotide encoding is by the MCP-4 polypeptide of the cDNA coding of ATCC 75703.

7. the polynucleotide of claim 1, it has the encoding sequence of MCP-4 as shown in fig. 1.

8. sharp 2 the polynucleotide that require, it has the encoding sequence with the MCP-4 of ATCC 75703 preservations.

9. carrier, it contains the DNA of claim 2.

10. the engineered host cell that has the carrier of claim 9.

11. a method of producing polypeptide, it comprises by the host cell expression of claim 10 polypeptide by described dna encoding.

12. the method for the cell that a production can express polypeptide, it comprises the cell that makes up the carrier with claim 9 with genetic engineering technique.

13. a separated DNA, it can hybridize on the DNA of claim 2, and coding has the active polypeptide of MCP-4.

14. a peptide species, it is selected from the group of being made up of following material:

(i) have MCP-4 polypeptide or its fragment, analogue or the derivative of putative amino acid sequence shown in Figure 1; (ii) have fragment, analogue or derivative by MCP-4 polypeptide that is included in the cDNA amino acid sequence coded among the ATCC 75703 or described polypeptide.

15. the polypeptide of claim 14, wherein said polypeptide are the MCP-4 with aminoacid sequence of inferring shown in Figure 1.

16. the antibody of the polypeptide of claim 14.

17. the stimulant of the polypeptide of claim 14.

18. the antagonist/inhibitor of the polypeptide of claim 14.

19. a treatment needs the patient's of MCP-4 method, it comprises: to the polypeptide of the claim 14 of patient's administering therapeutic significant quantity.

20. a method for the treatment of the patient who need to suppress MCP-4, it comprises: to the antagonist/inhibitor of the claim 18 of patient's administering therapeutic significant quantity.

21. a pharmaceutical composition, it comprises the polypeptide and the pharmaceutically acceptable carrier of claim 14.

22. the method for claim 19, wherein said treatment significant quantity polypeptide are that coding said polypeptide and the DNA that expresses described polypeptide use in vivo by offering the patient.

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