CN1186501A - Human chemokine beta-8, chemokine beta-1 and macrophage inflammatory protein-4 - Google Patents

Abstract

Human Ck'beta'-8, MIP-4 and Ck'beta'-1 and DNA (RNA) encoding such chemokine polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such chemokine polypeptides for the treatment of leukemia, tumors, chronic infections, autoimmune disease, fibrotic disorders, wound healing and psoriasis. Antagonists against such chemokine polypeptides and their use as a therapeutic to treat rheumatoid arthritis, autoimmune and chronic and acute inflammatory and infective diseases, allergic reactions, prostaglandin-independent fever and bone marrow failure are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and for detecting altered concentrations of the polypeptides.

Description

Human chemokine beta-8, chemokine beta-1 and macrophage inflammatory protein-4

This application is the part continuation application of pending application 08/446,881 (May 5 nineteen ninety-five is in United States Patent and Trademark Office's application).

The present invention relates to recently the purposes of identified polynucleotides, polypeptide, these polynucleotide and polypeptide and the generation of these polynucleotide and polypeptide by these polynucleotide encodings.More particularly, polypeptide of the present invention has been pushed and has been accredited as human chemokine beta-8 (Ck β-8), macrophage inflammatory protein-4 (MIP-4) and chemokine beta-1 (Ck β-1) qualitatively.The present invention also relates to suppress the method for the effect of these polypeptide.

Chemokine is also referred to as the intercrine cytokine, is the subfamily of structurally and functionally related cytokine.These bulks of molecule are 8-10kd.In general, chemokine demonstrates 20% to 75% homology on amino acid levels, is feature with four conserved cysteine residue that form two disulfide linkage.In the past two cysteine residues be arranged as the basis, chemokine is divided into two subfamilies, α and β.In the α subfamily, therefore preceding two halfcystines by an amino acid separately are called " C-X-C " subfamily.In the β subfamily, therefore these two halfcystines are called " C-C " subfamily on contiguous position.So far, at least 8 different members of this family in the mankind, have been identified.

The intercrine cytokine demonstrates various functions.A flag sign is the ability that they cause the chemotactic migration of different cell types (comprising monocyte, neutrophil(e) cell, T-lymphocyte, basophilic leukocyte and inoblast).Many chemokines have short scorching active and relevant with a plurality of steps of Inflammatory response.These activity comprise the hormesis that histamine release, lysosomal enzyme and leukotrienes discharge, and increase the target immunocyte and are adsorbed onto on the endotheliocyte, strengthen the combination of complement proteins, induce the expression and the RB of granulocyte adsorbed molecules and complement receptor.Except with inflammation-related, shown that some chemokine demonstrates other activity.For example, macrophage inflammatory protein-1 (MIP-1) can suppress the propagation of hemopoietic stem cell, PF4 (PF-4) is strong endothelial cell growth inhibition, and interleukin-8 (IL-8) promotes Keratinocytic propagation, and GRO is the autocrine growth factor of melanoma cells.

According to diversified biological activity, chemokine is relevant with morbid state (comprising lymphocyte transportation, wound healing, hematopoiesis adjusting and immunologic derangement (as transformation reactions, asthma and sacroiliitis)) with a lot of physiology, this point no wonder.The example of hematopoietic lineage instrumentality is MIP-1.Originally MIP-1 is accredited as the pro-inflammatory cytokine of the endotaxin induction of scavenger cell generation.Afterwards studies show that MIP-1 is different by two kinds, but relevant protein MIP-1 α and MIP-1 β form.MIP-1 α and MIP-1 β are the lymphocytic chemoattractants of scavenger cell, monocyte and T.What is interesting is that the biological chemistry purifying of pluripotent stem cell inhibition (SCI) shows that with ensuing sequential analysis SCI is identical with MIP-1 α.And, shown that MIP-1 β can offset the ability that MIP-1 α suppresses hemopoietic stem cell proliferation.This discovery has caused such hypothesis, i.e. the major physiological effect of MIP-1 is the hematopoiesis of regulating marrow, and the inflammatory function that is proposed is accessory.It is relevant that MIP-1 α blocks the ability of cell cycle as the mode of action of stem cell inhibition and its in the G1/S interphase.As and if the restraining effect of MIP-1 α limited by immature ancester cell, and its ancestors to late period under the situation that rHuGM-CSF (GM-CSP) exists have real hormesis.Cloned several groups of chemokines, they may be the human homologues of MIP-1 α and MIP-1 β.In all cases, from library, separate cDNA at activated T cells RNA preparation.Can detect MIP-1 albumen in the wound inflammatory cell in early days, show that the protein induced wound inoblast of MIP-1 produces IL-1 and IL-6.In addition, when or be subcutaneously injected on the foot pad (footpad) of mouse, when perhaps intracisternal injection is in the celiolymph of rabbit, natural purifying MIP-1 (comprising MIP-1, MIP-1 α and MIP-1 beta polypeptides) causes acute inflammation (Wolpe and Cerami, 1989, FASEB J.3:2565-73).Except these direct or indirect short inflammatory matter of MIP-1, in the experiment mice model that uses the aseptic wound chamber, in the early stage inflammatory stage of wound healing, regain MIP-1 (Fahey, etc., 1990, cytokine, 2:92).For example, PCT application WO92/05198 (by the application of Chiron company) disclose a kind of in yeast as the active dna molecule that produces mammalian macrophage inflammatory protein (MIPS) template.

Mouse MIP-1 α is different but very relevant cytokine with MIP-1 β.These two kinds proteinic partial purification mixtures influence the neutrophilia function and cause partial inflammation and heating.In yeast cell, express MIP-1 α and be purified into homogeneity.Structural analysis has confirmed that MIP-1 α has similar secondary and tertiary structure to PF-4 and IL-8, and has the sequence homology that limits with their.Proved that also MIP-1 α is active in vivo, the stem cell of protection mouse prevents that it killed at external thymidine by tritiate afterwards.Show that also MIP-1 α promotes more typing ancestors granular leukocyte macrophage colony to form the propagation (Clemens.J.M. etc., cytokine, 4:76-82 (1992)) of cell at the response rHuGM-CSF.

The polypeptide of the present invention (Ck β-1) that originally is called MIP-1 γ in the parent patent application, it is the newcomer of β chemokine family based on the homology of aminoacid sequence.Ck β-8 polypeptide that originally is called MIP-3 in the parent patent application also is the newcomer of β chemokine family based on the homology of aminoacid sequence.

According to one aspect of the present invention, it provides new mature polypeptide, and they are that human Ck β-8, human MIP-4 and human Ck β-1 and their have biologic activity and diagnosis or treatment go up useful fragment, analogue and derivative.

According to another aspect of the present invention, the isolated nucleic acid molecule that it provides these polypeptide of encoding, what comprise that mRNA, DNA, cDNA, genomic dna and they have a biologic activity goes up useful fragment, analogue and derivative with diagnosis or treatment.

According to another aspect of the present invention, it provides the method that produces these polypeptide by recombinant technology, this method is included in to cultivate under the condition that promotes said protein expression and comprises the reorganization prokaryotic organism and/or the Eukaryotic host cell of nucleotide sequence, and reclaims said protein.

According to another aspect of the present invention; it provides the polynucleotide of these polypeptide or these polypeptide of encoding to be used for the treatment of the method for purpose; for example protect bone marrow stem cell to prevent that it is subjected to the infringement of chemical treatment reagent at chemotherapeutic period; remove the leukemia cell; immune response stimulating is regulated hematopoiesis and lymphocytic transportation, treatment psoriasis, solid tumor; promote the host at the defence of resistive chronic and acute infection with stimulate wound healing.

According to another aspect of the present invention, it provides the antibody of anti-these polypeptide.

According to another aspect of the present invention, it provides the antagonist of these polypeptide, and they can be used for suppressing the effect of these polypeptide, for example suppresses the generation of IL-1 and TNF-α, treatment aplastic anemia, myelodysplastic syndrome, asthma, sacroiliitis.

According to another aspect of the present invention, it provides the nucleic acid probe of the nucleic acid molecule that comprises sufficient length, so as specifically with Ck β-8, Ck β-1 and MIP-4 nucleic acid array hybridizing.

According to another aspect of the present invention, it provides the diagnostic measurement method of the sudden change of the nucleotide sequence that detects not enough with these expression of polypeptides in overexpression diseases associated and detection coding these polypeptide.

According to another aspect of the present invention, it provides therapeutical agent and the diagnostic reagent this purpose that is used for the treatment of human diseases in order to develop, and the polynucleotide of these polypeptide or these polypeptide of encoding are made relevant in vitro study compositions and methods as synthesizing with dna vector with scientific research, DNA.

By the instruction of this paper, it will be apparent to those skilled in the art these and other aspect of the present invention.

Following accompanying drawing is used for illustrating embodiment of the present invention, and has no intention to be used for limiting the scope of the present invention that claim limits.

Fig. 1 is the cDNA sequence of coding Ck β-8 and the aminoacid sequence of inferring accordingly.21 leader sequences that amino acid represent is inferred of beginning.All signal sequences are determined by the proteinic N-terminal peptide sequencing of baculovirus expression.

Fig. 2 is the cDNA sequence of coding Ck β-1 and the aminoacid sequence of inferring accordingly.19 amino acid represent leader sequences of beginning.

Fig. 3 is the cDNA sequence of coding MIP-4 and the aminoacid sequence of inferring accordingly.20 amino acid represent leader sequences of beginning.

Fig. 4 has shown the amino acid identity of Ck β-8 (top) and human MIP-1 α (following).Four halfcystine features that shown all chemokines.

Fig. 5 is two aminoacid sequences, and wherein Shang Mian sequence is human MIP-4 aminoacid sequence, and following sequences is human MIP-1 α (a human lymphocytes in tonsil LD78 β amyloid protein precursor).

Fig. 6 has shown the aminoacid sequence contrast of Ck β-1 (top) and human MIP-1 α (following).

Fig. 7 is a gel photograph, and the Ck β-1 of HA mark carries out electrophoresis thereon with Ck β-1 after expressing in the COS cell.

Fig. 8 is the SDS-PAGE gel photograph of Ck β-1 behind baculovirus expression system expression and purifying.

Fig. 9 is the SDS-PAGE gel photograph of Ck β-8 behind baculovirus expression system expression and three step purifying.

Figure 10. use the microchamber device (Neuro Probe company) in 48 holes to measure the chemoattractant activity of having determined Ck β-8 by chemotaxis.Experimental technique such as manufacturers's handbook are described.For Ck β-8 concentration of every kind of experiment, in 5 high power fields (field), checked migration.The result of gained represents the mean value of twice independent experiment.Shown chemoattractant activity to THP-1 cell (A) and PBMCs (B).

Figure 11. the variation of cellular calcium concentration of response Ck β-8 of having used Hitachi F-2000 fluorescent spectrophotometer assay.The Ck β-8 of bacterial expression is joined in the THP-1 cell of Indo-1 load to ultimate density be 50nM, and level in the born of the same parents of monitoring calcium concn.

Figure 12. handled monocytic series THP-116 hour with LPS (0.1-10ng/ml) or Ck β-8 (to 50ng/ml).The supernatant liquor of tissue culture is carried out the secretion of elisa assay with quantitative TNF-α.

Figure 13. handle human peripheral blood mononuclear cells with the ever-increasing Ck β-8 of consumption (producing) by the elutriation analysis purifying by baculovirus.The supernatant liquor of tissue culture is carried out the secretion of elisa assay with quantitative TNF-α, IL-6, IL-1, GM-CSF and granulocyte colony-stimulating factor (G-CSF).

Figure 14. with low-density bone marrow cells in mice colony inoculation (1,500 cell/plates) containing agar, contain or do not contain indication chemokine (100ng/ml), but contain in the substratum of IL-3 (5ng/ml), SCF (100ng/ml), IL-1 α (10ng/ml) and M-CSF (5ng/ml).Shown data representation is from mean value that twice independent experiment obtained (repetition is all carried out in each experiment).At postvaccinal 14 days counting colonies.The average percentage of the colony number that the colony numerical table that produces when containing chemokine produces when being shown the chemokine that does not contain any adding.

Figure 15 has illustrated the effect that Ck β-8 and the Ck β-1 pair of mouse bone marrow cells colony form through HPP-CFC (A) and LPP-CFC (B).

Figure 16 has illustrated the effect that new isolating medullary cell colony that the Ck β-1 of baculovirus expression and Ck β-8 couple M-CFS and SCF stimulate forms.

Figure 17 has illustrated the increment of lin colony of the medullary cell that Ck β-8 and Ck β-1 couple IL-3 and SCF stimulate and the effect of differentiation.

The effect that the GR-1 of the lin colony of Figure 18 .Ck β-8 and Ck β-1 pair medullary cell and Mac-1 (surface markers) positive cell colony produce.The lin cell (b) or in the growth medium of Ck β-1 (50ng/ml) is cultivated having added IL-3 (5ng/ml) and SCF (100ng/ml) (separately (a)) and Ck β-8 (50ng/ml).Analyze then with the monoclonal antibody dyeing of cell, and by FACScan with anti-marrow differentiation GR.1, Mac-1, Sca-1 and CD45R surface antigen.The positive cell of shown data accounts for the percentage ratio of (A) and little (B) cell colony greatly.

Figure 19 has illustrated that the existence of Ck β-8 (+) has suppressed the colony formation of medullary cell response IL-3, M-CSF and GM-CSF.

Figure 20. the Ck β-8 of dose response suppresses the formation of medullary cell colony.Separate and the processing cell as Figure 19.In based on the agar colony forming assay,, exist and be with or without under the situation of Ck β-8 (1,10,50 and 100ng/ml) at IL-3, GM-CSF or M-CSF (5ng/ml) and inoculate the cell handled density with 1,000 cell/plate.The per-cent of the colony number that shown data form during with the specificity factor Individual existence is represented the formation of colony.Said data are respectively to repeat the mean value of plate, and error bars shows standard deviation.

Figure 21. hemopoieticgrowth factor exist or non-existent situation under, the inducing of Ck β-8 and Ck β-1 pair apoptosis.Flushing (flush) bone marrow cells in mice from femur and shin bone with ficol density gradient separation bone marrow cells in mice, is removed monocyte by plastics absorption.With the cell colony of gained based on the interpolation of MEM IL-3 (5ng/ml), GM-CSF (5ng/ml), M-CSF (10ng/ml) and G-CSF (10ng/ml), add or do not add incubated overnight in the substratum of Ck β-8 (50ng/ml) or Ck β-1 (250ng/ml) in addition.In addition, culturing cell in the substratum that contains or do not contain Ck β-8 and Ck β-1 separately.After 24 hours, harvested cell uses boehringermannheim necrocytosis ELISA test kit inducing apoptosis.Data are illustrated in the per-cent that increases on the background basis, and the background of being considered is the amount that apoptosis takes place in the culture of cultivating under the situation that each somatomedin exists.

Figure 22. the expression of the RNA of coding Ck β-8 among the human monocyte.From the monocyte of new elutriation, separate total RNA, and through 100U/ml hu rIFN-g or 100ng/ml LPS or both processing.The RNA of each processing of electrophoretic separation on 1.2% sepharose (8 μ g), and transfer on the nylon membrane.By warp ³²The quantitative Ck β-8 of the detection of the cDNA of P mark mRNA is by photodensitometer quantitative said band on the radioautogram that obtains.

According to one aspect of the present invention, it provides the nucleic acid (polynucleotides) that separates, and these nucleic acid are compiled Code has Fig. 1, the mature polypeptide of 2 and 3 amino acid sequences of inferring (be respectively SEQ ID NO.2, 4 and 6), perhaps coding with the clone's of ATCC 75676 (preservation on February 9 in 1994) preservation cDNA Ck β-8 polypeptide of coding and coding are with ATCC 75675 (preservation on February 9 in 1994) preservation The ripe MIP-4 polypeptide of clone's cDNA coding and coding were with ATCC 75572 (1993 10 Moon preservation on the 13rd) Ck β-1 polypeptide of the clone's of preservation cDNA coding.

On the polynucleotides structure of code book invention polypeptide with belong to cell factor or chemotactic factor (CF) family Short scorching supergene " intercrine " is relevant. Ck β-8 and MIP-4 are the homologues of MIP-1, and And MIP-1 α is compared more homology of MIP-1 β. The polynucleotides of coding Ck β-8 derive from the sustainer The cDNA library of skin, and contain one the coding 120 amino acid residue polypeptide ORF, this Peptide species demonstrates obvious homology to many chemotactic factor (CF)s. The top coupling is to human macrophage The coupling of inflammatory protein 1 α demonstrates 36% identity property and 66% similitude (Fig. 4).

The polynucleotides of coding MIP-4 derive from human ripe lungs cDNA library, and contain one The ORF of 89 amino acid residue polypeptide of coding, this peptide species demonstrates bright to many chemotactic factor (CF)s Aobvious homology. The top coupling is the coupling to human lymphocytes in tonsil LD78 β albumen, shows Go out 60% identity property and 89% similitude (Fig. 5). And, in all chemotactic factor (CF)s with the feature primitive Four cysteine residues that exist are all guarded in two clones. In this gene, the first two The fact of cysteine residues on the adjacent position is divided into " C-C " or the inferior family of β of chemotactic factor (CF) with them Family. In other subfamily (" CXC " or α subfamily), the first two cysteine residues is by an ammonia The sour residue of base separately.

The polynucleotides of coding Ck β-1 contain the ORF of 93 amino acid polypeptides of encoding, this polypeptide Front 19 amino acid are targeting sequencings, so said mature polypeptide contains 74 amino acid residues. Ck β-1 couple of human macrophage inflammatory protein α demonstrates obvious homology, and be amino acid whose at 80 In one section sequence, have 48% identity property and 72% similitude. And, comprise four of feature primitive Individual cysteine residues is guarded.

Polynucleotides of the present invention can be rna form or dna form, wherein DNA comprise cDNA, Genomic DNA and synthetic DNA. This DNA can be two strands or strand, if strand, can It is coding strand or non-coding (antisense) chain. The coded sequence of this encoding mature polypeptide can and Fig. 1,2 Identical with the clone's of (the SEQ ID NO.1,3 and 5) shown in 3 or preservation coded sequence; Owing to lose Pass Feng Yu or the degeneracy of password, this coded sequence also can be a kind of different coded sequence, It can and Fig. 1,2 with the cDNA coding of 3 DNA (SEQ ID NO.1,3 and 5) or preservation mutually Mature polypeptide together.

The mature polypeptide of code pattern 1,2 and 3 (SEQ ID NO.2,4 and 6) or the cDNA of coding preservation The polynucleotides of the mature polypeptide of coding can comprise: the coded sequence that only is the encoding mature polypeptide; The coded sequence of encoding mature polypeptide and additional coded sequence are such as leading or secretion sequence or front albumen Sequence; The coded sequence of encoding mature polypeptide (with having or not having an additional coded sequence) and non-coding order Row are such as 5 of the coded sequence of introne or encoding mature polypeptide ' and/or the non-coding sequence of 3 ' end.

Like this, " polynucleotides of coded polypeptide " this term comprises and only contains the many of polypeptid coding sequence Nucleotides and also contain additional coding and/or the polynucleotides of non-coding sequence.

The invention still further relates to above-described polynucleotides variant, this variant coding has Fig. 1,2 Hes The polypeptide of the amino acid sequence of inferring of 3 (SEQ ID NO.2,4 and 6) or by the clone's of preservation cDNA Fragment, analog and the derivative of the polypeptide of coding. This polynucleotides variant can be natural generation The allelic variant of polynucleotides or the polynucleotides variant that produces of non-natural.

Like this, the present invention includes coding becoming shown in Fig. 1,2 and 3 (SEQ ID NO.2,4 and 6) Ripe polypeptide or by the polynucleotides of the mature polypeptide of the clone's of preservation cDNA coding, and coding as figure 1, the mature polypeptide shown in 2 and 3 (the SEQ ID NO.2,4 and 6) or by the clone's of preservation cDNA The polynucleotides variant of fragment, analog and the derivative of the mature polypeptide of coding. These nucleotides become Body comprises disappearance variant, replacement variant, interpolation or inserts variant.

As above indicated, this polynucleotides can have Fig. 1,2 and 3 (SEQ ID NO.1, The code sequence of the allelic variant of the natural generation of clone's 3 and 5) or preservation coded sequence Row. Allelic variant is the another kind of form of polynucleotide sequence as you know in this area, and is wherein many Nucleotide sequence can have replacement, disappearance or the interpolation of one or more nucleotides, and this can be from reality Change the function of coded polypeptide on the matter.

The present invention also comprises such polynucleotides, wherein the sequence of said encoding mature polypeptide can Be integrated into polynucleotide sequence in the identical reading frame, this polynucleotide sequence helps the host thin The expression and secretion of polypeptide among the born of the same parents, such as have in the targeting sequencing control cell of secretion sequence function The transhipment of polypeptide. Polypeptide with targeting sequencing is front albumen (preprotein), the host cell excision This targeting sequencing forms ripe polypeptide form. This polynucleotides former albumen of also encoding (proprotein), this former albumen is the maturation protein that 5 ' end has additional amino acid residue. Have The maturation protein of former sequence (prosequence) is former albumen, is a kind of albumen form of non-activity. Cut Except former sequence, what stay is activated maturation protein.

Like this, can encode a kind of maturation protein or the albumen of former sequence is arranged or the albumen that existing former sequence has presequence (presequence) (leader sequence) again of polynucleotide of the present invention.

Polynucleotide of the present invention also can have the encoding sequence that is integrated into flag sequence in the frame, and described flag sequence makes can purifying polypeptide of the present invention.Under the situation of host bacterium, described flag sequence can be six histidine marks that provided by the pQE-9 carrier, it makes that the mature polypeptide be integrated into mark can purifying, perhaps for example when using mammalian hosts (as the COS-7 cell), flag sequence can be hemagglutinin (HA).Said hemagglutination mark corresponding to come from the proteinic epi-position of influenza hemagglutinin (Wilson, I., etc., cell, 37:767 (1984)).

The invention still further relates to and the polynucleotide (, preferably having 70% identity property) of sequence hybridization described above if having at least 50% between two sequences.The present invention be more particularly directed under stringent condition polynucleotide with above-described multi-nucleotide hybrid.As used herein, term " stringent condition " refers to only have at least 95% between sequence, and hybridization just can take place when preferably having 97% homology.In a preferred embodiment, hybridize to the such peptide species of polynucleotide encoding on the above-described polynucleotide, it has kept identical biological function or activity with mature polypeptide by the cDNA coding of the cDNA of Fig. 1,2 and 3 (SEQ ID NO.1,3 and 5) or preservation in fact.

In addition, said polynucleotide can be to have at least 20 bases, preferably 30 bases, at least 50 bases more preferably, these polynucleotide and multi-nucleotide hybrid of the present invention, they and polynucleotide of the present invention as indicated above have identity property, but do not have activity.For example, these polynucleotide can be used as the probe of SEQ ID NOS:1,3 and 5 polynucleotide, are used to reclaim said polynucleotide, perhaps as diagnostic probe or as the PCR primer.

The mentioned preservation thing of this paper will be preserved according to the regulation of the microbial preservation budapest treaty of the international recognition that is used for patented procedure.These preservation things only are in order to provide convenience to those skilled in the art, are not 112 required preservations of 35 U.S.C ζ.Be included in the described preserved material polynucleotide sequence and by aminoacid sequence this paper reference in the lump of its encoded polypeptides, and be used to solve any contradiction on this paper sequence description.To any manufacturing of preserved material, using or selling needs not give any such permission through permission at this.

The invention still further relates to have Fig. 1, the aminoacid sequence of inferring of 2 and 3 (SEQ ID NO.2,4 and 6) or have Ck β-8, MIP-4 and Ck β-1 polypeptide, and the fragment of these polypeptide, analogue and derivative by the cDNA amino acid sequence coded of preservation.

Term " fragment ", " derivative " and " analogue " during when the polypeptide of mentioning Fig. 1,2 and 3 (SEQ ID NO.2,4 and 6) or by the cDNA encoded polypeptides of preservation, refer to the biological function or the active polypeptide that keep identical with such polypeptide in fact.Like this, analogue comprises former albumen, and this albumen produces activated mature polypeptide and activates by the former protein part of excision.

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

Said Fig. 1,2 and 3 (SEQ ID NO.2,4 and 6) or by the fragment of the cDNA encoded polypeptides of preservation, derivative or analogue can be: (i) a kind of like this, wherein one or more amino-acid residues can be also can not encoded by genetic code by the amino-acid residue that conservative or non-conservative amino acid residues (preferably conservative amino acid residues) replace and replace, perhaps (ii) a kind of like this, wherein one or more amino-acid residues comprise substituting group, perhaps (iii) a kind of like this, wherein mature polypeptide and another kind of compound merge, described compound is as increasing the compound (for example polyoxyethylene glycol) of polypeptide transformation period, perhaps (iv) a kind of like this, wherein additional amino acid is integrated into mature polypeptide, as leading or secretion sequence, perhaps be used for the sequence of purifying mature polypeptide, perhaps former protein sequence.By the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.

Preferably provide polypeptide of the present invention with isolating form, and preferably peptide purification to homogeneous.

Term " gene " or " cistron " refer to and produce the relevant dna fragmentation of polypeptide chain; It comprises the sequence (leader and tailer sequence) of front and back, coding region and the intervening sequence (intron) between each encode fragment (exon).

Term " isolating " means described material and has broken away from its primal environment (for example, natural surroundings is if it is natural generation).For example, a kind of polynucleotide or polypeptide that is present in the natural generation in the animal alive is not isolating, but with natural system in the identical polynucleotide that separate of the material of some or all coexistence or DNA or polypeptide be isolating.Such polynucleotide can be that the part of carrier and/or such polynucleotide or polypeptide can be the parts of composition, and it remains isolating, and this is because this carrier or composition are not the parts of its natural surroundings.

The present invention also relates to comprise the carrier of polynucleotide of the present invention and the host cell that produces through genetically engineered with carrier of the present invention, and the method that produces polypeptide of the present invention through recombinant technology.

Host cell produces (transduction, conversion or transfection) with carrier of the present invention through genetically engineered, and said carrier can be cloning vector or expression vector.This carrier for example can be, forms such as plasmid, virion, phage.Said engineering host cell can be cultivated in the conventional nutritional medium of improvement, and described substratum is improved to such an extent that be suitable for activating promotor, selects transformant or amplification Ck β-8, MIP-4 and Ck β-1 gene.Culture condition, for example pH value and temperature etc. are to be used to select those of expression host cell in the past, are conspicuous to those of ordinary skill.

Polynucleotide of the present invention can be used for producing polypeptide through recombinant technology.Like this, for example, polynucleotide can be included in various expression vectors any, particularly the carrier of express polypeptide or plasmid.Such carrier comprises karyomit(e), non-chromosome and synthetic dna sequence dna, for example simian virus 40 derivative; Bacterial plasmid; Phage DNA; Yeast plasmid; From plasmid and phage DNA combination deutero-carrier; Viral DNA (as cowpox, adenovirus, fowl avipoxvirus, and pseudorabies virus).Yet any other plasmid or carrier also can use, as long as its reproducible and survival in the host.

Can suitable dna sequence dna be inserted in the carrier with several different methods.In general, with methods known in the art dna sequence dna is inserted into suitable restriction endonuclease site.Such method and other method are considered in those skilled in the art's ken.

Said dna sequence dna in expression vector can effectively be connected on the suitable expression control sequenc (promotor), and is synthetic to instruct mRNA.The representative example of such promotor can should be mentioned that: LTR or simian virus 40 promotor, intestinal bacteria, lac or trp, phage P _LPromotor and known other promotor that controlling gene is expressed in protokaryon or eukaryotic cell or their virus.Said expression vector also comprises the ribosome bind site that is used for translation initiation and Transcription Termination.This carrier also can comprise the suitable sequence of expressing for amplification.

In addition, expression vector preferably comprises a gene, to be provided for selecting the phenotypic characteristic of transformed host cells, and Tetrahydrofolate dehydrogenase or the neomycin resistance cultivated of eukaryotic cell for example, or tsiklomitsin and the amicillin resistance in the intestinal bacteria for example.

Comprise above-described suitable dna sequence dna and suitable promotor or the carrier of control sequence and can be used to transform suitable host, so that it can marking protein.

As the representative example of suitable host, can should be mentioned that here: bacterial cell, as intestinal bacteria, streptomycete, Salmonella typhimurium; The fungal cell is as yeast; Insect cell such as Drosophila (Drosophila) S2 and Sf9; Adenovirus; Zooblast is as CHO, COS or Bowes melanoma; Vegetable cell etc.By the elaboration of this paper, suitable host's selection is considered within those skilled in the art's ken.

More particularly, the present invention also comprises recombinant precursor, and this construct comprises above broadly described one or more sequences.This construct comprises carrier, and the carrier as plasmid or virus has wherein inserted sequence of the present invention forward or backwards.Under the even more ideal situation of this embodiment, construct also comprises the adjusting sequence that can effectively be connected on the described sequence, comprise, for example, promotor.A large amount of carrier and promotors that are fit to are well known by persons skilled in the art, and are obtainable by commercial sources.Provide following carrier by way of example.Bacterium: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).Eucaryon: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Parmacia).Yet any other plasmid or carrier are as long as their reproducible and survivals in the host can be used.

Can have the carrier of selectivity border note from any required gene Selection promoter region with CAT (CAT) carrier or other.Two kinds of suitable carriers are PKK232-8 and PCM7.The bacterium promotor of mentioning especially comprises lacI, lacZ, T3, T7, gpt, λ P _R, P _L, and trp.Promoter in eukaryote comprises that CMV is early stage immediately, HSV thymidine kinase, early stage and late period SV40, derive from retroviral LTRs and mouse metallothionein(MT)-I.Within the level that is chosen in those of ordinary skills to appropriate carriers and promotor.

In another embodiment, the present invention relates to comprise the host cell of the above construct.Said host cell can be a higher eucaryotic cells, as mammalian cell, or eukaryotic cell such as low, as yeast cell, perhaps host cell can be a prokaryotic cell prokaryocyte, as bacterial cell.Can be by the transfection of calcium phosphate transfection, DEAE-dextran mediation, or electroporation is incorporated into construct (Davis, L., Dibner, M., Battey, I., the basic skills in the molecular biology (1986)) in the host cell effectively.

Construct in the host cell can be used for producing in a usual manner the gene product by the recombination sequence coding.In addition, polypeptide of the present invention can produce by conventional peptide synthesizer is synthetic.

Mature protein can be at mammalian cell, yeast, and bacterium, or express in other cell under the control of suitable promotor.Employing derives from the RNA of DNA construct of the present invention, and cell free translation system also can be used for producing this protein.Sambrook etc., molecular cloning: laboratory manual, second edition, the cold spring port, N.Y., the suitable clone and the expression vector that use with protokaryon and eucaryon host have been described in (1989) (this paper is reference in the lump).

Be inserted into enhancer sequence in the carrier and strengthened encode the transcribing of DNA of polypeptide of the present invention of higher eucaryote.Enhanser is the cis-acting elements of DNA, and usually about 10 to 300bp, and acting on increases it and transcribe on the promotor.Example comprises the simian virus 40 enhanser of replication orgin rear side 100 to 270bp, cytomegalovirus early promoter enhanser, polyoma enhanser on the replication orgin rear side and adenovirus enhanser.

Usually, recombinant expression vector comprises replication orgin and allows the selected marker (for example, the ampicillin resistance gene of intestinal bacteria and yeast saccharomyces cerevisiae TRP1 gene) of host cell conversion and the promotor that instructs the downstream configurations sequence to transcribe that comes from cance high-expression gene.Such promotor can be come own coding glycolytic ferment (for example glycerol 3-phosphate acid kinase (PGK)), α-factor, the operon of acid phosphatase or heat shock protein etc.The allos structure sequence is in suitable stage and translation initiation and terminator sequence assembling, and preferably, the leader sequence that advances periplasmic space or extracellular substratum with the protein secreting that can instruct translation assembles.Heterologous sequence can be also encoding fusion protein not, comprise the terminal peptide of differentiating of the N-that gives required feature, required feature for example, the recombinant products stabilization of expression or simple purifying.

By with the reading operated with functional promotor mutually the suitable translation initiation in (operable reading phase) and the termination signal structural DNA sequence construct that inserts the coding desired protein together be used for the useful expression vector of bacterium.Said carrier comprises one or more Phenotypic Selection marks and replication orgin, with the maintenance that guarantees carrier with amplification is provided in the host when appropriate.The prokaryotic hosts that be fit to transform comprises intestinal bacteria, subtilis, Salmonella typhimurium, and Rhodopseudomonas, streptomyces, each of Staphylococcus kind, though other also can select use.

As one representational but be not restrictive example, the useful expression vector that is used for bacterium can comprise selected marker and the bacterium replication orgin that comes from commercially available plasmid (genetic elements that comprises well-known cloning vector pBR322 (ATCC37017)).Commercially available carrier like this comprises, for example, and pKK223-3 (Pharmacia Fine chemical company, Uppsala, Sweden) and GEM1 (PromegaBiotec, Madison, WI, the U.S.).These pBR322 " skeleton " part and suitable promotor and structure sequence combination to be expressed.Transform and appropriate host strain growth extremely after the suitable cell density at the appropriate host bacterial strain, induce the promotor of selection with suitable method (for example temperature inversion or chemical induction), other for some time of cell cultures.

Typically,, keep formed crude extract to be used for further purifying through physics or chemical process smudge cells through centrifugal cell harvesting.

Can be used for the microorganism cells of marking protein through the method fragmentation of any routine, described method comprises to be freezed-thaw cycles, sonication, and Mechanical Crushing or use lysis agent, these methods those skilled in the art will know that.

Various mammalian cell culture systems also can be used for the express recombinant body protein.The example of mammalian expression system comprises by the monkey kidney inoblast COS-7 clone of Gluzman (cell, 23:175 (1981)) description and other can express the clone of compatible carrier, for example, and C127,3T3, CHO, HeLa and bhk cell system.Mammiferous expression vector comprises replication orgin, and suitable promotor and enhanser also can comprise any essential ribosome bind site, polyadenylation site, donor splicing site and acceptor site, transcription termination sequence and 5 ' flank non-transcribed sequence.The dna sequence dna and the polyadenylation site that derive from the SV40 montage can be used to provide required non-transcribed genetic elements.Ck β-8, MIP-4 and Ck β-1 can reclaim and purifying from the reconstitution cell culture with several different methods, and described method comprises ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatograph, affinity chromatograph, hydroxylapatite chromatography and phytohemagglutinin chromatogram.In finishing the configuration of mature protein, can use the protein refolding step when needing.At last, can use high performance liquid chromatography (HPLC) as last purification step.

Polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis process, or produce from protokaryon or eucaryon host (for example bacterium, yeast, higher plant, the insect of cultivation and mammalian cell) through recombinant technology.According to the host who uses in the reorganization production method, polypeptide of the present invention can be glycosylated by Mammals or other eukaryotic carbohydrate, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise the initial methionine amino-acid residue.

Polypeptide of the present invention can be used for various immunomodulatorys and inflammation function, also can be used for various diseases.Ck β-8, MIP-4 and Ck β-1 belong to chemokine family, so they are chemoattractants of white corpuscle (as monocyte, neutrophil(e) cell, T lymphocyte, eosinocyte, basophilic leukocyte etc.).

The Northern engram analysis shows that Ck β-8, MIP-4 and Ck β-1 preferably express in the tissue of hemocytopoietic organ.

Ck β-8 plays an important role in adjusting immune response and aspect of inflammation.Figure 22 shows that lipopolysaccharide-induced human monocyte expresses Ck β-8.Therefore, endotoxic in response existence, monocytes Ck β-8 is so the use of Ck β-8 can be used for regulating host's immune response.

As shown in figure 10, the activity of chemoattractant is tangible in THP-1 cell (A) and PBMCs (B).Ck β-8 induces tangible calcium motion (Figure 11) in the THP-1 cell, this shows that Ck β-8 pair monocyte has biological action.And Ck β-8 produces the chemotaxis and the calcium motion response of dependent dose in the human monocyte.

Therefore, use Ck β-8, MIP-4 and Ck β-1 help promoting wound healing by the infiltration of control target immunocyte to the wound district.In a similar fashion, polypeptide of the present invention can improve the host and by them chronic infection (for example, mycobacterial infections) defendd in microbicidal leukocytic attraction and activation.

Be injected in the tumour because evidence suggests the cell of will express chemokine, can cause the degeneration (for example, in the treatment of Karposi sarcoma) of tumour, so polypeptide of the present invention can also be used for tumor treatment.Figure 12 and 13 analysis revealed Ck β-8 induce TPH-1 emiocytosis TNF-α, and TNF-α is the reagent that becomes known for regressing tumors.The Ck β-8 of 250ng/ml induces generation and the secretion of 1200 slight grams per milliliter TNF-α.Ck β-8 also induces the human monocyte to secrete other tumour and cancer inhibitor significantly, as IL-6, IL-1 and G-CSF.In addition, Ck β-8, MIP-4 and Ck β-1 defend the intrusion and the activation of (tumoricidal) cell (as cytotoxic T cell and scavenger cell) by their chemotactic activity stimulation of host, and also can be used for treating the solid cancer in this way.

Said polypeptide also can be used for suppressing hemapoietic propagation and differentiation, therefore can be used for protecting bone marrow stem cell, prevents and treats it suffers chemotherapy agents at chemotherapeutic period injury.Figure 14 and 15 shows that Ck β-8 and Ck β-1 suppress the colony formation of low multiplication potentiality colony forming cell, and shows Ck β-the 8th, the strong and specific inhibition of LPP-CFC colony growth.Figure 16 shows that Ck β-1 suppresses the colony formation that M-CSF stimulates specifically, and Ck β-8 then can not.Yet, show that also Ck β-8 and Ck β-1 suppress the growth or the differentiation of medullary cell significantly.This antiproliferative effect allows to be exposed in the chemotherapy agents more widely, so chemotherapy is more effective.

Can use Ck β-1 and the restraining effect of Ck β-8 polypeptide in the subgroup (for example granulocyte, monocytes/macrophages) of typing ancester cell in the treatment to suppress leukemia cell's propagation.

In Figure 17,18 and 19, remove used cytophyletic committed cell, the cell colony that obtains is contacted with Ck β-8 with Ck β-1.Ck β-1 causes the reduction near 50% in Mac-1 positive cell colony, this result and the Ck β-1 that Figure 16 shows induce the inhibiting result of the colony forming cell of response M-CSF consistent.As shown in figure 19, Ck β-8 suppresses the ability of the formation colony of typing ancester cell response IL-3, GM-CSF and M-CSF.And, as shown in figure 20, shown that Ck β-8 suppresses the dose response that colony forms.This restraining effect may be because the special of differentiation signal of these factor mediations blockaded, or because cytotoxic effect in the ancester cell.

Another effect of said polypeptide is, for example in autoimmune disease and lymphocytic leukemia, and the propagation of suppressor T cell by the biosynthesizing that suppresses IL-2.

Produce MIP-1 α because found the langerhans' cells in the skin, so can use Ck β-8, MIP-4 and Ck β-1 to suppress the propagation (keratinocyte hyper-proliferative) of psoriasic epidermal keratinocytes.

Can use Ck β-8, MIP-4 and Ck β-1 by raising the excision fragment and promoting the inflammatory cell of reticular tissue and by controlling the appearance that the beta mediated fibrosis of excessive TGF prevents scar.In addition, because Ck is β-8, MIP-4 and Ck β-1 increase the perviousness of blood vessel, so can use these polypeptide treatment apoplexy, thrombocythemia, pulmonary infarction and bone marrow proliferation disorder.

Ck β-8 can be used for the treatment of leukemia and undesired proliferating cells (for example tumour cell) by inducing apoptosis.As shown in figure 21, Ck β-8 inducing apoptosis in hematopoiesis ancester cell colony.

The polynucleotide of polypeptide of the present invention and these polypeptide of coding can be used as the in vitro study reagent relevant with scientific research, DNA is synthetic and the generation of dna vector, and can be used for developing therapeutics and diagnostics, and purpose is the treatment human diseases.For example, Ck β-1 and Ck β-8 can be used to expand them by the differentiation that temporarily prevents immature hematopoiesis ancester cell (as granulocyte, scavenger cell and monocyte).These medullary cells can be in vitro culture.

Can use Ck β-8, the MIP-4 of total length and Ck β-1 gene fragment hybridization probe as the cDNA library, so as the gene that separates this total length with separate other gene that gene therewith has height sequence similarity or similar biologic activity.Yet preferably this probe has at least 30 bases, for example can contain 50 or more base.Said probe also can be used to differentiate corresponding to cDNA clone, the genomic clone of total length transcript or comprise adjusting and promoter region, exon and intron interior complete genome clone.The embodiment of screening comprises the coding region that utilizes known dna sequence to separate this gene, with synthetic oligonucleotide probe.The oligonucleotide that has with the mark of gene order complementary sequence of the present invention can be used for screening human cDNA library, genomic dna or mRNA, to determine probe and which library member hybridization.

The present invention also relates to Ck β-8, MIP-4 and Ck β-1 gene be as the purposes of part diagnostic reagent, detect with said nucleotide sequence sudden change have diseases associated and to the susceptibility of this disease.The expression deficiency of these diseases and chemokine polypeptides is relevant.

Can on dna level, detect with various technology and have Ck β-8, the individuality of MIP-4 and Ck β-1 transgenation.Can obtain diagnostic nucleic acid from patient's cell (as blood, urinate, saliva is organized examination of living tissue and necrotomy material).Genomic dna can be directly used in detection, perhaps uses PCR enzymatic amplification (Saiki etc., nature, 324:163-166 (1986)) before analysis.RNA or cDNA also can be used for identical purpose.As an example, can use and coding Ck β-8, Ck β-8 is differentiated and analyzed to the nucleic acid complementary PCR primer of MIP-4 and Ck β-1, MIP-4 and Ck β-1 sudden change.For example, can be by comparing the variation detection disappearance of amplified production size with normal genotype or inserting.Point mutation can be through the DNA and the radiolabeled Ck β-8 of amplification, MIP-4 and Ck β-1 RNA or radiolabeled Ck β-8, and MIP-4 and the hybridization of Ck β-1 antisense dna sequence are differentiated.Through ribonuclease A digestion, perhaps distinguish perfect paired sequence and mispairing duplex from the difference of melting temperature.

Can finish by detecting when being with or without denaturing agent on the gel change of dna fragmentation electrophoretic mobility based on the genetic test of dna sequence dna difference.Little sequence deletion and insertion can be demonstrated by the high resolving power gel electrophoresis.Not homotactic dna fragmentation can be distinguished on sex change methane amide gradient gel, the migration of wherein different dna fragmentations according to its specific fusing point or part melt temperature and be stuck in gel different positions (referring to, for example, Myers etc., science, 230:1242 (1985)).

Also can protect assay method to disclose sequence variation on the specific position, described assay method such as ribonuclease protecting and S1 protection or chemical cracking method (for example, Cotton etc., PNAS, the U.S., 85:4397-4401 (1985)) by nuclease.

Like this, can detect specific dna sequence by following method, described method is for example hybridized; ribonuclease protecting, chemical cracking, directly dna sequencing; or the Southern blotting of use restriction enzyme (for example, limited fragment length polymorphism (RFLP)) and genomic dna.

Except that conventional gel-electrophoresis and dna sequencing method, also available original position analyzing and testing sudden change.

Because (for example can detect certain disease with respect to the said proteinic overexpression of normal control tissue sample, tumour) existence or to the susceptibility of certain disease, therefore, the present invention also relates to be used for detect the diagnostic analysis method of the level of various tissue Ck β-8, MIP-4 and the proteic variation of Ck β-1.Being used for detecting from host's sample Ck β-8, the MIP-4 and the analytical procedure of Ck β-1 protein level is known to those skilled in the art, and said method comprises: radioimmunoassay, competition are measured and " interlayer " mensuration in conjunction with mensuration, Western engram analysis, ELISA.ELISA measures the antigenic specific antibody that (Coligan etc., immunology latest draft, 1 (2), the 6th chapter, (1991)) comprise preparation Ck β-8, MIP-4 and Ck β-1 albumen at first, preferably monoclonal antibody.In addition, the receptor antibody of preparation monoclonal antibody.With detectable reagent and receptor antibody combination, said reagent such as radioactivity, fluorescence or be horseradish peroxidase in this embodiment.By obtaining sample among the host, and with its with sample in the solid support (as the polystyrene ware) of protein bound go up and cultivate.By cultivating together, will cover any protein binding site freely in the ware with nonspecific proteins matter (as BSA).Next, in monoclonal antibody with during being attached to Ck β-8, MIP-4 and Ck β-1 protein binding on the polystyrene ware, monoclonal antibody is cultivated in ware.With damping fluid all unconjugated monoclonal antibodies are washed off.Now, the receptor antibody that will be connected with horseradish peroxidase is put into ware, and the result causes the monoclonal antibody combination on receptor antibody and any Ck of being attached to β-8, MIP-4 and Ck β-1 albumen.Then unconjugated monoclonal antibody is washed off.Add peroxidase substrate and typical curve then relatively in ware, the amount of the color that produces in preset time promptly is Ck β-8, MIP-4 and the proteic amount of Ck β-1 that exists in patient's sample of given volume.

Can use competition assay, wherein Ck β-8, MIP-4 and the special antibody of Ck β-1 are adsorbed on the solid support, Ck β-8, the MIP-4 of mark and Ck β-1 and the sample that is derived from the host be by solid support, and for example the amount of the mark by the detection of liquid scintillation chromatogram is relevant with proteic amount in the sample.

" interlayer " measured and is similar to ELISA mensuration.Ck β-8, MIP-4 and Ck β-1 be by solid support in " interlayer " measured, and with the antibodies that is adsorbed on the solid support.Then, second antibodies is on Ck β-8, MIP-4 and Ck β-1.Then mark and to special the 3rd antibody of second antibody by solid support and with second antibodies, carry out then quantitatively.

The invention provides the method for the acceptor that is used to differentiate chemokine polypeptides.Can come the gene of the said acceptor of identifier number with many methods well known by persons skilled in the art, as part elutriation and FACS classification (immunology latest draft such as Coligan, the 1 (2), the 5th chapter, (1991)).What preferably use is cloning by expression, and polyadenylic acid RNA wherein is divided into different storehouses to the cDNA library of being set up by said RNA, other cell that is used for rotaring redyeing COS cell or said polypeptide is not responded by the cell preparation to said polypeptide response.The transfectional cell that is grown on the slide glass is exposed in the polypeptide of said mark.Said polypeptide can be with the various means marks that comprise the recognition site iodate of locus specificity protein kinase or its inclusion body.Fixing and cultivate after, slide glass is carried out radioautographic analysis.Differentiate positive storehouse, utilize repetition word bank collection (iterative sub-pooling) and preparation of screening (rescreen) method and transfection word bank more simultaneously, final generation coding is inferred the mono-clonal of acceptor.

As another kind of acceptor discrimination method, the polypeptide of mark can be a photoaffinity, can be connected with the cytolemma or the extract formulation of energy expressed receptor molecule.With PAGE differentiate cross-coupled material and its exposure on X-ray film.Can cut off the labeled complex of the acceptor that comprises said polypeptide, resolve into protein fragments and carry out albumen micrometering preface.The aminoacid sequence that obtains from the micrometering preface can be used for designing a cover degenerate oligonucleotide probe and be used for the cDNA library of the acceptor gene that identifier number infers with screening.

The present invention also relates to SCREENED COMPOUND with the stimulant of discriminating chemokine polypeptides of the present invention and the method for antagonist.Stimulant is the compound that contains the similar biological function of said polypeptide, and antagonist is then blocked these functions.Can be by measuring chemotaxis at the top placement cell of band foraminous strainer (said hole has enough diameters (about 5 μ m), allow cell by), said cell is subjected to the chemotactic of any polypeptide of the present invention.Potential stimulant solution is placed on the bottom of chamber, is suitable control medium in the top compartment, like this, migrates in time or measures anti-depressant concentration gradient by the cell of pore membrane through counting.

When measuring antagonist, chemokine polypeptides of the present invention is placed in the following chamber, add the potential antagonist and whether stoped with the chemotaxis of determining said cell.

In addition, under the situation that this compound exists, the mammalian cell or the film preparation of expressing said polypeptide receptor are cultivated with the chemokine polypeptides (as radioactivity) of mark.Then, can measure this compound and stop this interactional ability.When measuring stimulant by this way, do not add chemokine, measure the interactional ability of stimulant itself and said acceptor.

Potential Ck β-8, the example of MIP-4 and Ck β-1 antagonist comprises antibody, or is in some cases and this peptide species bonded oligonucleotide.Another example of potential antagonist is the negative dominant mutant of said polypeptide.Negative dominant mutant is the polypeptide with the receptors bind of said wild type peptide, but does not keep biologic activity.

Adopting the antisense constructs of antisense technology preparation also is the potential antagonist.The expression that antisense technology can come controlling gene by triple helical formation or antisense DNA or RNA, these two kinds of methods are all based on the combination of polynucleotide and DNA or RNA.For example, the encode 5 ' encoding part of polynucleotide sequence of mature polypeptide of the present invention can be used for the antisense rna oligonucleotide of about 10 to 40 base pairs of design length.Design a kind of with transcribe related gene regions complementary DNA oligonucleotide (triple helical-referring to Lee etc., nucleic acids research, 6:3073 (1979); Cooney etc., science, 241:456, (1988); With Dervan etc., science, 251:1360 (1991)), and then stop transcribing and producing of chemokine polypeptides.Hybridize on the mRNA in the antisense rna oligonucleotide body, and the translation becoming of blocking-up mRNA molecule said polypeptide (antisense-Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotide is as the antisense inhibitor (CRC press, Boca Raton, FL (1988)) of genetic expression.Oligonucleotide described above can be sent in the cell, so that can expression in vivo sense-rna or DNA, and the generation that comes the chemokine inhibiting polypeptide.

The antagonist of another kind of potential chemokine is the peptide derivant of said polypeptide, this peptide is modified into the analogue of said polypeptide by natural or synthetic mode, it has lost biologic activity, but still can discern and in conjunction with the acceptor of said polypeptide, therefore suppress acceptor effectively.The example of polypeptide derivative includes but not limited to little peptide or class peptide molecule.

Can use these antagonist for treating MIP inductive or promoted disorder, for example autoimmunization, chronic inflammatory diseases and communicable disease.The example of autoimmune disease comprises multiple sclerosis and insulin-dependent diabetes.

These antagonists also can be used for by stop monokaryon phagocytal raise and activate treat transmissible disease, said transmissible disease comprises silicosis, interior sample knurl disease, spontaneous pulmonary fibrosis.By stoping eosinophilic generation and migration, these compounds also can be used for treating the spontaneous eosin syndromes of too much having a liking for.These antagonists also can be by stoping scavenger cell migration and produce chemokine polypeptides of the present invention and treat endotoxin shock.

By stoping monocytic infiltration in the arterial wall, these antagonists also can be used for treating atherosclerosis.

By chemokine inhibiting inductive mastocyte and have a liking for the threshing of alkali and the release of histamine, these antagonists also can be used for treating the anaphylaxis and the immunologic derangement (comprising late phase allergic responses, chronic urticaria atopic dermatitis) of histamine-mediated.Also can treat the anaphylaxis of IgE mediation, as allergic asthma, rhinitis and eczema.

By stoping the wound district to monocytic attraction, these antagonists also can be used for treating chronic and acute inflammation.Because chronic with acute struvite pulmonary disorder and lung monokaryon be phagocytal compile relevant, so these antagonists can be used for regulating scavenger cell colony of normal lung.

To monocytic attraction, antagonist also can be used for treating rheumatic arthritis by the synovia in the prevention patient joint.Monocytic inflow and activate degenerate and inflammatory arthropathy pathogenic in play an important role.

These antagonists also can be used for disturbing the deleterious cascade reaction of important IL-1 of coming from and TNF.By this way, antagonist can be used for stoping inflammation.Antagonist also can be used for the heating that the chemokine inhibiting inductive relies on prostaglandin(PG).

These antagonists also can be used for treating the incapability of marrow, as aplastic anemia and myelodysplasia syndromes.

By stoping eosinophilic accumulation in the lung, these antagonists also can be used for treating asthma and allergy.These antagonists also can be used for treating subepithelial basilar membrane fibrosis, and it is the notable feature of asthma lung.

Antagonist can be used to be combined into composition with pharmaceutically acceptable carrier, and is for example, described below.

Said chemokine polypeptides, stimulant and antagonist can be used in combination with suitable pharmaceutical carrier.Such composition comprises said polypeptide and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.Such carrier includes but not limited to salt solution, buffer saline, dextrose, water, glycerine, ethanol and their composition.The mode that its prescription should be suitable for using.

The present invention also provides pharmaceutical pack or test kit, and they comprise one or more containers that are filled with one or more compositions of pharmaceutical composition of the present invention.What be associated with this container can be the bulletin of government organs' defined form of management medicine and biological products manufacturing, use or sale, and this bulletin has reflected manufacturing, use or sold the human allowance that makes the government organs of articles for use.In addition, said polypeptide, stimulant and antagonist can be treated compound with other and be used in combination.

Described pharmaceutical composition can be used in mode easily, for example part, intravenously, intraperitoneal, intramuscular, tumour are interior, subcutaneous for described mode, in the nose or the intradermal approach use.Said pharmaceutical composition is used with the significant quantity that treats and/or prevents specified disease.In general, they are used with the amount of about at least 10 micrograms/kg body weight, and in most of the cases, they are used with the amount that is no more than about 8 mg/kg body weight every day.Under most applications, consider factors such as route of administration and illness, dosage from every day about 10 microgram/kilograms to 1 mg/kg body weight.

The stimulant of chemokine polypeptides and polypeptide form or antagonist can use by the such polypeptide of expression in vivo according to the present invention, and this often is known as " gene therapy ".

Like this, for example, can carry out the genetically engineered operation with the polynucleotide (DNA or RNA) of external coded polypeptide to patient's cell, the patient who treats with this polypeptide to desire provides said engineering cell then.Such method is well known in the art.For example, can use the retroviral particle pair cell of the RNA that comprises the polypeptide of the present invention of encoding to carry out the genetically engineered operation through method well known in the art.

Similarly, can carry out the genetically engineered operation by pair cell in the methods known in the art body for example, so that the expression in vivo polypeptide.As known in the art, generation can be comprised that the production cell of retroviral particle of the RNA of the polypeptide of the present invention of encoding is administered to the patient, so that pair cell carries out genetically engineered and handles and the expression in vivo polypeptide in the body.By elaboration of the present invention, it will be apparent to those skilled in the art that by this mode these and other method through using polypeptide of the present invention.For example, the expression vector of through engineering approaches cell can not be a retroviral vector, as, with suitable transport carrier and combine after, adenovirus can be used for through engineering approaches cell in the body.

Retroviral plasmid vector can be from retrovirus, and these viruses include but not limited to: moloneys mouse sarcoma virus, moloneys mouse leukosis virus, spleen necrosis virus, Rous sarcoma virus and Harvey sarcoma virus.

In a preferred embodiment, the both sides of retrovirus expression vector (pMV-7) are the sarcoma viral length of moloneys mouse terminal repetition (LTRs), and comprise the selectable drug resistance gene neo under the adjusting of the simple venereal disease poison of bleb (HSV) thymidine kinase (tk) promotor.Unique EcoRI and HindIII site help the introducing (Kirschmeier, P.T. etc., DNA, 7:219-25 (1988)) of encoding sequence.

Said carrier comprises one or more suitable promotors, and these promotors include but not limited to: retrovirus LTR; The simian virus 40 promotor; With (biotechnologys such as Miller, vol.7, No.9,980-990 (1989)) human cell who describes loose virus (CMV) promotor or other promotor are for example as the cell promotor of eukaryotic cell promotor (promotor include but not limited to histone, pol III and beta-actin promotor).By the elaboration of this paper, the selection of suitable promotor is apparent to one skilled in the art.

The nucleotide sequence of code book invention polypeptide is under suitable promotor control, and these promotors include but not limited to: viral thymidine kinase promoter, as the simple property of bleb thymidine kinase promoter; Retrovirus LTRs; The beta-actin promotor; Natural promoter with the gene of controlling the said polypeptide of coding.

Use retroviral plasmid vector transduction package cell line to form producer's clone.The example of packing cell that can transfection includes but not limited to: PE501, PA317 and GP+am12.Said carrier can be by any known method in this area these packing cells of transduceing.These methods include but not limited to: electroporation, use liposome and CaPO ₄Precipitation.

Producer's clone produces the infectious retroviral carrier granule of the nucleotide sequence that comprises the said polypeptide of encoding.Can use the external or interior transduction of the body eukaryotic cell of these retroviral vector particles.The eukaryotic cell of transduction will be expressed the nucleotide sequence of the said polypeptide of coding.The eukaryotic cell that can be transduceed includes but not limited to inoblast and endotheliocyte.

Sequence of the present invention is differentiated also valuable to karyomit(e).The specific position of the single human chromosome of said sequence-specific ground guiding, and hybridization with it.In addition, the current specific site that also needs on the differential staining body.Few chromosome marking reagent based on actual sequence data (repetition polymorphism) can be used to the marker chromosomes position now.In those sequences of related and disease related gene, be the important the first step according to chromosomal DNA mapping of the present invention.

In brief, can map on the karyomit(e) to sequence by prepare PCR primer (preferably 15-25bp) from cDNA.Use the Computer Analysis of cDNA to select primer rapidly, this primer is not crossed over the exon in the more than one genomic dna, makes amplification method complicated like this.Then, these primers are used for the somatic cell hybrid that the PCR screening comprises independent human chromosome.Only comprise those hybrids generation amplified fragments corresponding to the people's gene of primer.

The PCR mapping of somatic cell hybrid is to specify specific DNA to specific chromosomal fast method.Utilize the present invention and identical Oligonucleolide primers, can obtain inferior location in a similar fashion by one group of specific chromosomal fragment or one group of big genomic clone.Can use other to be used for mapping strategy to chromosome mapping in a similar fashion, described strategy comprises in situ hybridization, with the airflow classification karyomit(e) prescreen of mark with through hybridizing preselected structure chromosome specific-cDNA library.

In a step, the cDNA clone's of Metaphase Chromosome diffusion fluorescence in situ hybridization (FISH) can be used to provide accurate chromosome position.This technology can be used with the cDNA that is as short as 500 or 600 bases.The summary of this technology is referring to Verma etc., human chromosome: basic fundamental handbook, Pergamon press, New York (1988).

In case sequence is mapped to accurate chromosome position, the physical location of the sequence on the karyomit(e) just can with the genetic map data association.Such data can be at for example V.McKusick, finds in the Mendelian inheritance in the people (can connect on the Johns Hopkins Welch of the university medical science library and use).Then, the mutual relationship between the disease of the gene and the identical chromosomal region of having mapped is differentiated through linkage analysis (the common heredity of physics contiguous gene).

Then, be necessary determine to infect and the difference in cDNA or the genome sequence between the infected individuals not.If observe sudden change in some or all infected individuals, but do not observe in any normal individual, then described sudden change is likely the cause of disease of disease.

Adopt the physical mapping and the genetic mapping technology of existing resolving power, accurately navigate to and the chromosomal region of disease-related on cDNA be one (this supposes 1 megabasse mapping resolving power, gene of every 20kb) of the potential pathogenic gene between 50 and 500.

Said polypeptide, its fragment or other derivative or its analogue, or the cell of expressing them can produce antibody thus as immunogen.These antibody for example can be, polyclone or monoclonal antibody.That the present invention also comprises is chimeric, the antibody and the Fab fragment of strand and peopleization, or the product of Fab expression library.The whole bag of tricks as known in the art can be used to produce such antibody and fragment.

Can be by using the next anti-antibody of this peptide species acquisition generation corresponding to polypeptide of sequence of the present invention to the said polypeptide of animal direct injection or to animal (preferably non-human animal).The antibodies polypeptide itself that obtains like this then.In such a way, even a fragments sequence of coded polypeptide also can be used to produce in conjunction with all natural polypeptide antibody.With this antibody isolated polypeptide from the tissue of express polypeptide.

For MONOCLONAL ANTIBODIES SPECIFIC FOR, can use any technology that the antibody that is produced by the continuous cell line culture is provided.Example comprises hybridoma technology (Kohler and Milstein, 1975, nature, 256:495-497), trioma technology, people B-quadroma technology (Kozbor etc., 1983, current immunology is 4:72) with the EBV-hybridoma technology (Cole that produces human monoclonal antibodies, Deng, 1985, monoclonal antibody and cancer therapy, Alan R.Liss, company, pp.77-96).

The technology (United States Patent (USP) 4,946,778) that relevant generation single-chain antibody is described can adopt the single-chain antibody that produces anti-immunogenic polypeptide product of the present invention.Also can utilize transgenic mice to express the antibody of the peopleization of anti-immunogenic polypeptide product of the present invention.

Further describe the present invention with reference to following examples.Yet should know and the invention is not restricted to these embodiment.Unless specialize, all umbers or quantity all refer to weight.

In order to help understanding the following example, method and/or term that some often occur are described below.

" plasmid " is by the small letter p of front and/or follow-up capitalization and/or figure denote.The initial plasmid of this paper be commercially available, the unconfined public obtainable or can be by published method from obtainable plasmid construction.In addition, the plasmid that is equal to of described plasmid is well known in the art, and is conspicuous to those of ordinary skill.

DNA's " digestion " refer to that with restriction enzyme catalyze cleavage DNA, this restriction enzyme only acts on the certain sequence of DNA.Various restriction enzyme used herein is commercially available, uses their reaction conditions, and cofactor and other need be the knowledge that should be appreciated that as those of ordinary skill.For the purpose of analyzing, usually be the enzyme that in about 20 microlitre damping fluids, uses 1 microgram plasmid or dna fragmentation and about 2 units.Carrying out the purpose of plasmid construction for the DNA isolation fragment, usually is with enzymic digestion 5 to 50 micrograms of DNA of 20 to 250 units in comparatively large vol.The damping fluid that the specific limited enzyme is adopted and the amount of substrate are stipulated by the producer.Usually use 37 ℃ of about incubation times of 1 hour, but can change according to supplier's explanation.Digestion after, reactant directly on polyacrylamide gel electrophoresis to separate required fragment.

With Goeddel etc., nucleic acids research, 8% polyacrylamide gel that 8:4057 (1980) describes carries out the size separation of cutting fragment.

" oligonucleotide " refers to strand poly deoxynucleosides or two complementary poly deoxynucleosides chains, and it can be chemosynthesis.The synthetic oligonucleotide does not have 5 ' phosphate like this, and when not adding the phosphoric acid salt that has ATP simultaneously in the presence of kinases, it does not connect another oligonucleotide.The synthetic oligonucleotide will connect does not have dephosphorylized fragment.

" connection " refers between two double stranded nucleic acid fragments to form the process of phosphodiester bond, and (Maniatis, T. is etc., Id., p.146).Unless alternate manner is provided, can adopts known damping fluid to be connected with dna fragmentation to be connected 10 T4DNA of the unit ligase enzymes (" ligase enzyme ") of condition with the about equimolar amount of per 0.5 microgram.

Unless other explanation is arranged, press Graham, F. and van der Eb, A., virusology, the description of 52:456-457 (1973) transforms.

Embodiment 1

The bacterial expression of Ck β-8 and purifying

Use corresponding to 5 of finished Ck β-8 protein (subtraction signal peptide sequence) ' and the PCR Oligonucleolide primers of 3 ' end sequence and dna sequence dna of the initial amplification coding Ck of Ck β-8 gene 3 ' carrier sequence β-8, ATCC#75676.Additional nucleotide corresponding to Bam HI and XbaI is joined 5 respectively ' and 3 ' sequence in.5 ' Oligonucleolide primers with 5 ' TCAGGATCCGTCACAAAAGATGCAGA 3 ' (SEQ ID NO.7) sequence comprises a BamHI restriction endonuclease sites, is thereafter 18 Nucleotide from Ck β-8 encoding sequence of supposing finished proteinic end amino acid codon.Sequence 5 ' the CGCTCTAGAGTAAAACGACGGCCAGT3 ' (SEQ ID NO.8) of 3 ' end comprises and XbaI site complementary sequence.Said restriction endonuclease sites is equivalent to bacterial expression vector pQE-9 (Qiagen company, Chat sworth, restriction endonuclease sites CA).PQE-9 coding antibiotics resistance (Amp ^r), bacterium replication orgin (ori), IPTG-regulate promotor operon (P/0), ribosome bind site (RBS), 6-histidine mark and restriction endonuclease sites.With BamHI and Xba I digestion pQE-9.The sequence of amplification is connected on the pQE-9, and is inserted in the framework of the sequence that has encoding histidine mark and RBS.With connecting mixture transformed into escherichia coli bacterial strain M15/rep4 (can obtain) from Qiagen.M15/rep4 comprises the plasmid pREP4 of multiple copied, and this plasmid expression lacI suppresses son and gives kalamycin resistance (Kan ^r).Differentiate that by its energy for growth on the LB flat board this transformant selects the bacterium colony of anti-penbritin/kantlex simultaneously.Isolated plasmid dna, and confirm by restriction analysis.(O/N) cultivation of spending the night in the liquid culture of the LB substratum that replenishes Amp (100 ug/ milliliter) and Kan (25ug/ milliliter) comprises the clone of required construct.Said O/N culture is used for inoculating a large amount of cultures with 1: 100 to 1: 250 ratio.Make cell grow to optical density(OD) 600 (0.D. between 0.4 and 0.6 ⁶⁰⁰).Add IPTG (sec.-propyl-B-D-sulfur sugar pyranoside) to final concentration 1mM.IPTG suppresses son by deactivation lacI, removes to cause the P/O of the genetic expression that increases to induce.Cell was cultivated 3 to 4 hours in addition.Then by centrifugal cell harvesting.The cell precipitation that dissolving obtains in chaotropic agent (6 moles Guanidinium hydrochlorides).After the clarification, under the condition that the protein that allows to contain the 6-histidine mark is combined closely from then in the solution through nickel chelate column chromatography purification dissolved Ck β-8 (Hochuli, E. etc., chromatography magazine 411:177-184 (1984)).From then in the post with 6 moles Guanidinium hydrochloride (pH5.0) wash-out Ck β-8 (95% purity), for sex change elutriant is transferred to 3 moles Guanidinium hydrochloride, 100mM sodium phosphate, the gsh (reduced form) of 10 mmoles and the gsh (oxidized form) of 2 mmoles.Incubation is after 12 hours in this solution, with the sodium phosphate of the 10 mmoles said protein of dialysing.

Embodiment 2

The bacterial expression of MIP-4 and purifying

Use corresponding to 5 of finished MIP-4 protein (subtraction signal peptide sequence) ' and dna sequence dna of the initial amplification coding MIP-4 of PCR Oligonucleolide primers of 3 ' sequence, ATCC#75675.Additional nucleotide corresponding to Bam HI and XbaI is joined 5 respectively ' and 3 ' end sequence in.5 ' Oligonucleolide primers with 5 ' TCAGGATCCTGTGCACAAGTTGGTACC 3 ' (SEQ ID NO.9) sequence comprises a BamHI restriction endonuclease sites, is thereafter 18 Nucleotide from the MIP-4 encoding sequence of supposing finished proteinic end amino acid codon; Sequence 5 ' the CGCTCTAGAGTAAAACGACGGCCAGT 3 ' (SEQ ID NO.10) of 3 ' end comprises and Xba I site complementary sequence.Said restriction endonuclease sites is equivalent to bacterial expression vector pQE-9 (Qiagen company, Chat sworth, restriction endonuclease sites CA).PQE-9 coding antibiotics resistance (Amp ^r), bacterium replication orgin (ori), IPTG-regulate promotor operon (P/O), ribosome bind site (RBS), 6-histidine mark and restriction endonuclease sites.With BamHI and Xba I digestion pQE-9, the sequence that increases is connected on the pQE-9, and is inserted in the framework of the sequence that has encoding histidine mark and RBS.Then with connecting mixture transformed into escherichia coli bacterial strain (can obtain) from Qiagen.M15/rep4 comprises the plasmid pREP4 of multiple copied, and this plasmid expression lacI suppresses son and gives kalamycin resistance (Kan ^r).Differentiate that by its energy for growth on the LB flat board this transformant selects the bacterium colony of anti-penbritin/kantlex simultaneously.Isolated plasmid dna, and confirm by restriction analysis.(O/N) cultivation of spending the night in the liquid culture of the LB substratum that replenishes Amp (100ug/ milliliter) and Kan (25ug/ milliliter) comprises the clone of required construct.Said O/N culture is used for inoculating a large amount of cultures with 1: 100 to 1: 250 ratio.Make cell grow to optical density(OD) 600 (O.D. between 0.4 and 0.6 ⁶⁰⁰).Add IPTG (sec.-propyl-B-D-sulfur sugar pyranoside) to final concentration 1mM.IPTG suppresses son by deactivation lacI, removes to cause the P/O of the genetic expression that increases to induce.Cell was cultivated 3 to 4 hours in addition.Then by centrifugal cell harvesting.The cell precipitation that dissolving obtains in chaotropic agent (6 moles Guanidinium hydrochlorides).After the clarification, under the condition that the protein that allows to contain the 6-histidine mark is combined closely from then in the solution through nickel chelate column chromatography purification dissolved MIP-4 (Hochuli, E. etc., chromatography magazine 411:177-184 (1984)).From then in the post with 6 moles Guanidinium hydrochloride (pH5.0) wash-out MIP-4 (95% purity), for sex change elutriant is transferred to 3 moles Guanidinium hydrochloride, 100mM sodium phosphate, the gsh (reduced form) of 10 mmoles and the gsh (oxidized form) of 2 mmoles.Incubation is after 12 hours in this solution, with the sodium phosphate of the 10 mmoles said protein of dialysing.

Embodiment 3

The bacterial expression of Ck β-1 and purifying

Use corresponding to 5 of finished Ck β-1 protein (subtraction signal peptide sequence) ' and dna sequence dna of the initial amplification coding Ck of the PCR Oligonucleolide primers β-1 of 3 ' end sequence, ATCC#75572, and join 5 respectively corresponding to the additional nucleotide of Bam HI and XbaI ' and 3 ' sequence in.5 ' Oligonucleolide primers with 5 ' GCCCGCGGATCCTCCTCACGGGGACCTTAC3 ' (SEQ ID NO.11) sequence comprises a BamHI restriction endonuclease sites, is thereafter 15 Nucleotide from Ck β-1 encoding sequence of supposing finished proteinic end amino acid codon; Sequence 5 ' the GCCTGCTCTAGATCAAAGCAGGGAAGCTCCAG 3 ' (SEQ ID NO.1 2) of 3 ' end comprises and Xba I site complementary sequence, last 20 Nucleotide of translation stop codon and Ck β-1 encoding sequence.Said restriction endonuclease sites is equivalent to bacterial expression vector pQE-9 (Qiagen company, Chatsworth, restriction endonuclease sites CA).PQE-9 coding antibiotics resistance (Amp ^r), bacterium replication orgin (ori), IPTG-regulate promotor operon (P/O), ribosome bind site (RBS), 6-histidine mark and restriction endonuclease sites.With BamHI and XbaI digestion pQE-9, the sequence that increases is connected on the pQE-9, and is inserted in the framework of the sequence that has encoding histidine mark and RBS then.Then with connecting mixture transformed into escherichia coli bacterial strain (can from M15/rep 4 trade marks of Qiagen, obtain).M15/rep4 comprises the plasmid pREP4 of multiple copied, and this plasmid expression lacI suppresses son and gives kalamycin resistance (Kan ^r).Differentiate that by its energy for growth on the LB flat board transformant selects the bacterium colony of anti-penbritin/kantlex simultaneously.Isolated plasmid dna, and confirm by restriction analysis.(O/N) cultivation of spending the night in the liquid culture of the LB substratum that replenishes Amp (100ug/ milliliter) and Kan (25ug/ milliliter) comprises the clone of required construct.Said O/N culture is used for inoculating a large amount of cultures with 1: 100 to 1: 250 ratio.Make cell grow to optical density(OD) 600 (O.D. between 0.4 and 0.6 ⁶⁰⁰).Add IPTG (sec.-propyl-B-D-sulfur sugar pyranoside) to final concentration 1mM.IPTG suppresses son by deactivation lacI, removes to cause the P/O of the genetic expression that increases to induce.Cell was cultivated 3 to 4 hours in addition.Then by centrifugal cell harvesting.The cell precipitation that dissolving obtains in chaotropic agent (6 moles Guanidinium hydrochlorides).After the clarification, under the condition that the protein that allows to contain the 6-histidine mark is combined closely from then in the solution through nickel chelate column chromatography purification dissolved Ck β-1 (Hochuli, E. etc., chromatography magazine 411:177-184 (1984)).From then in the post with 6 moles Guanidinium hydrochloride (pH5.0) wash-out Ck β-1 (95% purity), for sex change elutriant is transferred to 3 moles Guanidinium hydrochloride, 100mM sodium phosphate, the gsh (reduced form) of 10 mmoles and the gsh (oxidized form) of 2 mmoles.Incubation is after 12 hours in this solution, with the sodium phosphate of the 10 mmoles said protein of dialysing.

Embodiment 4

The expression of recombinant C k β-8 in the COS cell

The expression of plasmid CMV-Ck β-8 HA derives from carrier pcDNAI/Amp (Invitrogen), it comprises: the 1) replication orgin of simian virus 40,2) ampicillin resistance gene, 3) colibacillary replication orgin, 4) CMV promotor is thereafter polylinker district, simian virus 40 intron and polyadenylation site.With the dna fragmentation of coding total length Ck β-8 precursor be fused to HA marker clone in its 3 ' end frame to the polylinker district of said carrier, therefore, being expressed under the control of CMV promotor of recombinant protein.The HA mark is equivalent to from the proteinic epi-position of influenza hemagglutinin, and this point was described (I.Wilson, etc., cell 37:767 (1984)) in the past.The HA mark is inculcated to target cell is proteic, makes the recombinant protein that has antibody (its identification HA epi-position) be easy to measure.

The plasmid construction strategy is described below:

Use two primers to make up the dna sequence dna (ATCC#75676) of coding Ck β-8 through PCR, said primer: 5 ' primer, 5 ' GGAAAGCTTATGAAGGTCTCCGTGGCT3 ' (SEQ ID NO:13) comprises the HindIII site, is 18 Nucleotide of Ck β-8 encoding sequence that begun by initiator codon afterwards; 3 ' sequence, 5 ' CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAATTCTTCCTGGTCTTGA TCC3 ' (SEQ ID NO:14) comprises last 20 Nucleotide (not comprising terminator codon) of complementary sequence, translation stop codon, HA mark and Ck β-8 encoding sequence in Xba I site.Therefore, the PCR product comprises the HindIII site, and Ck β-8 encoding sequence is the HA mark that merges in framework afterwards, is translation stop codon and Xba I site behind the HA mark.With HindIII and XbaI digestion with restriction enzyme and dna fragmentation that is connected pcr amplification and carrier pcDNAI/Amp.To connect mixture and be transformed among the coli strain SURE that (Stratagene cloning system, La Jolla CA), are seeded in the culture that transforms on the ampicillin medium flat board, and screening resistance bacterium colony.Isolated plasmid dna from transformant, and detect whether there is correct fragment with restriction analysis.For expression recombinant Ck β-8, by DEAE-DEXTRAN method expression vector rotaring redyeing COS cell (J.Sambrook, E.Fritsch, T.Maniatis, molecular cloning: laboratory manual, press of cold spring harbor laboratory, (1989)).Detect Ck β-8-HA protein expression (E.Harlow, D.Lane, antibody: laboratory manual, press of cold spring harbor laboratory, (1988)) by radio-labeled and immuno-precipitation.With the two day usefulness of cell after transfection ³⁵S-halfcystine mark 8 hours.Collect substratum then and with washing agent lysing cell (RIPA damping fluid (0.1%SDS, 1%NP-40,0.5%DOC, 50mM Tris, pH 7.5 for 150mM NaCl, 1%NP-40) (Wilson, I. etc., Id.37:767 (1984)).Monoclonal antibody specific sedimentation cell lysate and substratum with HA.On 15% SDS-PAGE gel, analyze sedimentary protein.

Embodiment 5

The expression of reorganization MIP-4 in the COS cell

The expression of plasmid CMV-MIP-4 HA derives from carrier pcDNAI/Amp (Invitrogen), it comprises: the 1) replication orgin of simian virus 40,2) ampicillin resistance gene, 3) colibacillary replication orgin, 4) CMV promotor is thereafter polylinker district, simian virus 40 intron and polyadenylation site.With the dna fragmentation of coding total length MIP-4 precursor with in frame, be integrated into the polylinker district of its 3 ' terminal HA marker clone to said carrier, therefore, being expressed under the control of CMV promotor of recombinant protein.The HA mark is equivalent to from the proteinic epi-position of influenza hemagglutinin, and this point was described (I.Wilson, etc., cell 37:767 (1984)) in the past.The HA mark is inculcated to target cell is proteic, makes the recombinant protein that has antibody (its identification HA epi-position) be easy to measure.

The plasmid construction strategy is described below:

Use two primers to make up the dna sequence dna (ATCC#75675) of coding MIP-4 through PCR, said primer: 5 ' primer, 5 ' GGAAAGCTTATGAAGGGCCTTGCAGCTGCC3 ' (SEQ ID NO:15) comprises the HindIII site, is 20 Nucleotide of the MIP-4 encoding sequence that begun by initiator codon afterwards; 3 ' sequence, 5 ' CGCTCTAGATCAABCGTAGTCTGGGACGTCGTATGGGTAGGCATTCAGCTTCAGGT C3 ' (SEQ ID NO:16) comprises last 19 Nucleotide (not comprising terminator codon) of complementary sequence, translation stop codon, HA mark and the MIP-4 encoding sequence in Xba I site.Therefore, the PCR product comprises the HindIII site, and the MIP-4 encoding sequence is the HA mark that merges in framework afterwards, is translation stop codon and XbaI site behind the HA mark.With HindIII and XbaI digestion with restriction enzyme and dna fragmentation that is connected pcr amplification and carrier pcDNAI/Amp.To connect mixture and be transformed among the coli strain SURE that (Stratagene cloning system, La Jolla CA), are seeded in the culture that transforms on the ampicillin medium flat board, and screening resistance bacterium colony.Isolated plasmid dna from transformant, and detect whether there is correct fragment with restriction analysis.For expression recombinant MIP-4, by DEAE-DEXTRAN method expression vector rotaring redyeing COS cell (J.Sambrook, E.Fritsch, T.Maniatis, molecular cloning: laboratory manual, press of cold spring harbor laboratory, (1989)).Detect MIP-4-HA protein expression (E.Harlow, D.Lane, antibody: laboratory manual, press of cold spring harbor laboratory, (1988)) by radio-labeled and immuno-precipitation.With the two day usefulness of cell after transfection ³⁵S-halfcystine mark 8 hours.Collect substratum then and with washing agent lysing cell (RIPA damping fluid (0.1%SDS, 1%NP-40,0.5%DOC, 50mM Tris, pH 7.5 for 150mM NaCl, 1%NP-40) (Wilson, I. etc., Id.37:767 (1984)).Monoclonal antibody specific sedimentation cell lysate and substratum with HA.On 15% SDS-PAGE gel, analyze sedimentary protein.

Embodiment 6

The expression of recombinant C k β-1 in the COS cell

The expression of plasmid CMV-Ck β-1 HA derives from carrier pcDNAI/Amp (Invitrogen), it comprises: the 1) replication orgin of simian virus 40,2) ampicillin resistance gene, 3) colibacillary replication orgin, 4) CMV promotor is thereafter polylinker district, simian virus 40 intron and polyadenylation site.With the dna fragmentation of coding total length Ck β-1 precursor be fused to HA marker clone in its 3 ' end frame to the polylinker district of said carrier, therefore, being expressed under the control of CMV promotor of recombinant protein.The HA mark is equivalent to from the proteinic epi-position of influenza hemagglutinin, and this point was described (I.Wilson, etc., cell 37:767 (1984)) in the past.The HA mark is inculcated to target cell is proteic, makes the recombinant protein that has antibody (its identification HA epi-position) be easy to measure.

The plasmid construction strategy is described below:

Use two primers to make up the dna sequence dna (ATCC#75572) of coding Ck β-1 through PCR, said primer: 5 ' primer, 5 ' GGAAAGCTTATGAAGATTCCGTGGCTGC3 ' (SEQ ID NO:17) comprises the HindIII site, is 20 Nucleotide of Ck β-1 encoding sequence that begun by initiator codon afterwards; 3 ' sequence, 5 ' CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGTTCTCCTTCATGTCCT TG3 ' (SEQ ID NO:18) comprises last 19 Nucleotide (not comprising terminator codon) of complementary sequence, translation stop codon, HA mark and Ck β-1 encoding sequence in Xba I site.Therefore, the PCR product comprises the HindIII site, and Ck β-8 encoding sequence is the HA mark that merges in framework afterwards, is translation stop codon and XbaI site behind the HA mark.With HindIII and XbaI digestion with restriction enzyme and dna fragmentation that is connected pcr amplification and carrier pcDNAI/Amp.To connect mixture and be transformed among the coli strain SURE that (Stratagene cloning system, La Jolla CA), are seeded in the culture that transforms on the ampicillin medium flat board, and screening resistance bacterium colony.Isolated plasmid dna from transformant, and detect whether there is correct fragment with restriction analysis.For expression recombinant Ck β-1, by DEAE-DEXTRAN method expression vector rotaring redyeing COS cell (J.Sambrook, E.Fritsch, T.Maniatis, molecular cloning: laboratory manual, press of cold spring harbor laboratory, (1989)).Detect Ck β-8-HA protein expression (E.Harlow, D.Lane, antibody: laboratory manual, press of cold spring harbor laboratory, (1988)) by radio-labeled and immuno-precipitation.With the two day usefulness of cell after transfection ³⁵S-halfcystine mark 8 hours.Collect substratum then and with washing agent lysing cell (RIPA damping fluid (0.1%SDS, 1%NP-40,0.5%DOC, 50mM Tris, pH 7.5 for 150 mM NaCl, 1%NP-40) (Wilson, I. etc., Id.37:767 (1984)).Monoclonal antibody specific sedimentation cell lysate and substratum with HA.On 15% SDS-PAGE gel, analyze sedimentary protein.

Embodiment 7

The expression pattern of Ck β-8 in human tissue

Carry out the Northern engram analysis to detect the expression level of Ck β-8 in human tissue.Use RNAzol ^TMThe B system separate total cell RNA sample (the Biotecx laboratory, company, Houston, TX77033).Will be from each specific human tissue total RNA of isolating about 10 μ g separate and on the trace NF (Sambrook, Fritsch, Maniatis, molecular cloning, press of cold spring harbor laboratory, (1989)) at 1% sepharose.Carry out labeled reactant according to the Stratagene Prime-It test kit that has the 50ng dna fragmentation.DNA with Select-G-50 column purification mark.(5 Prime-3 Prime, the Boulder of company, CO).Use radiolabeled total length Ck β-8 gene with 1,000 then, 000 cpm/ml is at 0.5 M NaPO ₄, the said filter of hybridization spends the night under pH 7.4 and 7%SDS and 65 ℃.With O.5 * SSC and 0.1%SDS washed twice at room temperature, 60 ℃ of following washed twice, then under-70 ℃, this filter is crossed night dew puts and strengthening on the screen.

Embodiment 8

The expression pattern of MIP-4 in the human cell

Carry out the Northern engram analysis to detect the expression level of MIP-4 in the human cell.Use RNAzol ^TMThe B system separates total cell RNA sample (Biotecx laboratory, company, Houstonl TX77033).Will be from each specific human tissue total RNA of isolating about 10 μ g separate and trace (Sambrook, Fritsch, Maniatis, molecular cloning, press of cold spring harbor laboratory, (1989)) on NF at 1% sepharose.Carry out labeled reactant according to the Stratagene Prime-It test kit that has the 50ng dna fragmentation.DNA with Select-G-50 column purification mark.(5 Prime-3 Prime, the Boulder of company, CO).Use radiolabeled total length MIP-4 gene with 1,000 then, 000 cpm/ml is at 0.5 M NaPO ₄, the said filter of hybridization spends the night under pH 7.4 and 7%SDS and 65 ℃.With 0.5 * SSC and 0.1%SDS washed twice at room temperature,, then under-70 ℃, this filter is crossed night dew put and strengthening on the screen 60 ℃ of following washed twice.

Embodiment 9

The expression pattern of Ck β-1 in human tissue

Carry out the Northern engram analysis to detect the expression level of Ck β-8 in human tissue.Use RNAzol ^TMThe B system separates total cell RNA sample (Biotecx laboratory, company, Houstonl TX77033).Will be from each specific human tissue total RNA of isolating about 10 μ g separate and trace (Sambrook, Fritsch, Maniatis, molecular cloning, press of cold spring harbor laboratory, (1989)) on NF at 1% sepharose.Carry out labeled reactant according to the Stratagene Prime-It test kit that has the 50ng dna fragmentation.DNA with Select-G-50 column purification mark.(5 Prime-3 Prime, the Boulder of company, CO).Use radiolabeled total length Ck β-8 gene with 1,000 then, 000 cpm/ml is at 0.5 M NaPO ₄, the said filter of hybridization spends the night under pH 7.4 and 7%SDS and 65 ℃.With 0.5 * SSC and 0.1%SDS washed twice at room temperature,, then under-70 ℃, this filter is crossed night dew put and strengthening on the screen 60 ℃ of following washed twice.The messenger RNA(mRNA) that has abundant Ck β-1 in the spleen.

Embodiment 10

Use baculovirus expression system to express and purifying chemokine Ck β-8

Use recombinant baculovirus (designing this virus is in order to express Ck β-8 cDNA) to infect the SF9 cell.With 2 MOT cells infecteds, and under 28 ℃, cultivated 72-96 hour.From the culture that infects, remove cell debris by low-speed centrifugal.Add the protease inhibitor mixture in supernatant liquor, ultimate density is 20 μ g/ml Pefabloc SC, 1 μ g/ml leupeptin, 1 μ g/mlE-64 and 1mM ethylenediamine tetraacetic acid (EDTA).By only with the supernatant liquor application of sample of 20-30 μ l Ck β-8 level in the monitoring supernatant liquor to the 15%SDS-PAGE gel.Detect Ck β-8, the band of its visible 9Kd is equivalent to every liter several milligrams expression level.In addition by three step purifying method purifying Ck β-8: heparin is in conjunction with affinity chromatography.The supernatant liquor of baculovirus culture and the damping fluid that contains 100mMHEPES/MES/NaOAc (pH6) of 1/3 volume are mixed, and pass through the membrane filtration of 0.22 μ m.Then with this sample pipetting volume (HE1 poros 20, biological sensory perceptual system company) to the heparin column.In about 300 mM NaCl with the linear gradient elution Ck β-08 of 50 to the 500 mM NaCl solution of 50 mM HEPES/MES/NaOAc (pH 6); Cation-exchange chromatography.Will be from the heparin chromatography Ck β-8 usefulness of enrichment contain 5 times of the damping fluid dilutions of 50 mM HEPES/MES/NaOAc (pH 6).With the mixture application of sample that obtains in cationic exchange coloum (S/M poros 20, biological sensory perceptual system company).In 250 mM NaCl with the linear gradient elution Ck β-8 of 25 to the 300 mM NaCl solution of 50 mM HEPES/MES/NaOAc (pH 6); Exciusion chromatography.Behind cation-exchange chromatography, by (HW50, TOSO HAAS are further purified Ck β-8 on 1.4 * 45cm) to the screen analysis post with Ck β-8 application of sample.The Ck β-8 of fractional separation is equivalent to said proteic dipolymer form on the position that near molecular weight is 13.7Kd standard substance (Rnase A).

Behind three step purifying described above, judge by the Coomassie blue stain of SDS-PAGE gel whether the purity of the Ck β-8 that obtains surpasses 90% (Fig. 9).

Also checked the pollution of intracellular toxin/LPS of the Ck β-8 of purifying.Measure according to LAL, the content of LPS is less than 0.1ng/ml (BioWhittaker).

The effect that the colony of the new isolating medullary cell that the Ck β-1 of embodiment 11 baculovirus expressions and Ck β-8 couple M-CSF and SCF stimulate forms

Under 37 ℃, the low density colony of bone marrow cells in mice is cultivated in the tissue culture dish of handling 1 hour to remove monocyte, scavenger cell and other is adsorbed on the cell of frosting.Under factor existence or non-existent situation shown in Figure 16, will inoculate (10,000 cell/dishes) in containing the agar of growth medium then less than the cell colony of absorption.Under 37 ℃, these cultures are cultivated 10 days (88%N ₂, 5%CO ₂And 7%O ₂), and under inverted microscope, count the colony number.Represent the colony number with mean number, and obtain by three replications that carry out.

The increment of the medullary cell lin colony that embodiment 12Ck β-8 and Ck β-1 couple IL-3 and SCF stimulate and the effect of differentiation

Use negative system of selection to obtain to be rich in bone marrow cells in mice group in original hematopoiesis ancestors, wherein use the globule of one group of monoclonal antibody (anti-cd11b, CD4, CD8, CD45R and Gr-1 antigen) and magnetic to remove the cell of having finalized the design in this pedigree of major part.Under indication chemokine (50 ng/ml) existence or non-existent situation, with cell mass (lin cell) inoculation (5 * 10 that obtains ⁴Cell/ml) in the growth medium that has added IL-3 (50 ng/ml)+SCF (100 ng/ml).Under 37 ℃ at the insulation can (5%CO of humidity ₂, 7%O ₂, and 88%N ₂Environment) cultivate after 7 days in, harvested cell is also measured HPP-CFC and immature ancestors.In addition, the expression of some differentiation antigen by the FACScan analysis of cells.With mean number+/-SD represents the colony number, and obtains colony data (Figure 17) by the experiment of 6 dishes that every group of cells are carried out.

Embodiment 13

Ck β-8 suppresses response IL-3, the formation of the colony of M-CSF and GM-CSF

From femur and shin bone, wash bone marrow cells in mice, on the ficol density gradient, separate, and remove monocyte by plastics absorption.With the cell colony that obtains based on the interpolation of MEM IL-3 (5ng/ml), GM-CSF (5ng/ml), incubated overnight in the substratum of M-CSF (10ng/ml) and G-CSF (10ng/ml).At IL-3 (5ng/ml), GM-CSF (5ng/ml) or M-CSF (5ng/ml) and be with or without under the situation that Ck β-8 (50ng/ml) exists, these cells are seeded in the colony forming assay substratum based on agar with the density of 1,000 cell/dish.The percentage ratio of the colony number that the data that the expression colony forms form when being the specificity factor Individual existence.With two experiments of the described data representation of mean number as respectively repeating to coil, error bars is represented the standard deviation (Figure 19) of each experiment.

Embodiment 14

Expression through gene therapy

From the patient, obtain inoblast through Skin biopsy.Be placed on the tissue that obtains in the tissue culture medium (TCM) and be divided into fritter.Small tissue blocks is placed on the surface of wet tissue's culturing bottle, places about 10 for every bottle.With the bottle reversing, closely close, at room temperature spend the night.After at room temperature placing 24 hours, the bottle rotation is come, tissue block still anchors at the bottle bottom, adds fresh culture (as Ham ' s F12 substratum, wherein containing 10%FBS, penicillin and Streptomycin sulphate).Then, cultivate about 1 week down at 37 ℃.At this moment, add fresh culture, changed a subculture later on every several days.Cultivate again after 2 weeks, the inoblast of individual layer occurs.With the monolayer cell tryptic digestion, and divide equably by weight and install in the bigger bottle.

PMV-7 (Kirschmeier, P.T. etc., DNA, 7:219-25 (1988)) (its both sides are the terminal repetition districts of the length of Moloney murine sarcoma virus) with EcoRI and HindIII digestion, is handled with calf intestinal phosphatase enzyme afterwards.Fractional separation linear carrier on sepharose, and use the granulated glass sphere purifying.

Utilizing the encode cDNA of polypeptide of the present invention of PCR primer amplification, said primer is equivalent to 5 respectively ' and 3 ' end sequence.5 ' the primer that comprises EcoRI site and 3 ' primer also comprises the HindIII site.Under the situation that the T4 dna ligase exists, the Moloney murine sarcoma virus linear backbone of equivalent and EcoRI and HindIII fragment are added together.Under the condition that is suitable for these two kinds of fragments connections, preserve the reaction mixture that obtains.With connecting mixture transform bacteria HB101, then bacterium HB101 is seeded in the agar that comprises kantlex, to confirm whether to contain in the carrier interest genes of correct insertion.

Amphophilic pA317 or GP+am12 packing cell are cultivated in tissue culture, to its in the improved Eagles substratum of Dulbecco (DMEM) (containing 10% calf serum (CS), penicillin and Streptomycin sulphate) for being paved with density.The carrier that will contain said gene then joins in the substratum, and uses the carrier transduction packing cell.At this moment, packing cell produces the infectious virus particle (at this moment, packing cell is called producer's cell) that contains said gene.

Fresh culture is joined in producer's cell of transduction, subsequently, gather in the crops substratum being paved with on the 10cm flat board of producer's cell.The exhausted substratum that will contain infectious virus particle filters by millipore filter, to remove isolating producer's cell, infects inoblast with this substratum then.Never be paved with in fibroblastic flat board and remove substratum, and promptly replace with the substratum of producer's cell.This substratum is removed, replaced with fresh culture.If the titre of virus is very high, in fact all inoblasts then do not need to select all with infected.If the titre of virus is very low, be necessary to utilize to have the optionally retrovirus vector of mark (as neo or his).

Inoblast with through engineering approaches is expelled among the host then, perhaps separately or grow on cytodex 3 microcarrier beads and be paved with.At this moment, inoblast produces said protein.

According to many improvement of the present invention of description above and variation is possible, therefore, in the appended claims scope, can non-in addition specific description as mentioned implement the present invention.

Sequence table (1) general information: (i) applicant: LI etc. are denomination of invention (ii): human chemokine beta-8, chemokine beta-1 and macrophage inflammatory

Protein-4 is sequence number (iii): 18 (iv) addresses:

(A) addressee: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART and OLSTEIN

(B) street: 6 BECKER FARM ROAD

(C) city: ROSELAND

(D) state: NEW JERSEY

(E) country: the U.S.

(F) ZIP:07068 (v) computer-reader form:

(A) media type: 3.5 inches disks

(B) computer: IBM PS/2

(c) operating system: MS-DOS

The data of the current application of (D) software: WORD PERFECT 5.1 (Vi):

(A) application number:

(B) applying date:

(C) classification number: (Vii) in the data of first to file

(A) application number: 08/173,209

(B) applying date: on December 22nd, 1993 (Viii) is in the data of first to file

(A) application number: 08/208,339

(B) applying date: on March 8th, 1994 (ix) is in the data of first to file

(A) application number: PCT/US94/07256

(B) applying date: on June 28th, 1994 (ix) lawyer/proxy's information:

(A) name: FERRARO, GREGORY D.

(B) registration number: 36,134

(c) certificate/number of documents: 325800-289 (x) telecom information:

(A) phone: 201-994-1700

(B) fax: the information of 201-994-1744 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 363 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linear (ii) molecule type: cDNA (xi) sequence description: the information of SEQ ID NO:1ATGAAGGTCT CCGTGGCTGC CCTCTCCTGC CTCATGCTTG TTACTGCCCT TGGATCCCAG 60GCCCGGGTCA CAAAAGATGC AGAGACAGAG TTCATGATGT CAAAGCTTCC ATTGGAAAAT 120CCAGTACTTC TGGACAGATT CCATGCTACT AGTGCTGACT GCTGCATCTC CTACACCCCA 180CGAAGCATCC CGTGTTCACT CCTGGAGAGT TACTTTGAAA CGAACAGCGA GTGCTCCAAG 240CCGGGTGTCA TCTTCCTCAC CAAGAAGGGG CGACGTTTCT GTGCCAACCC CAGTGATAAG 300CAAGTTCAGG TTTGCATGAG AATGCTGAAG CTGGACACAC GGATCAAGAC CAGGAAGAAT 360TGA 363 (2) SEQ ID NO:2: (i) sequence signature:

(A) length: 120 amino acid

(B) type: amino acid

(C) chain:

(D) topological framework: linearity is molecule type (ii): protein (xi) sequence description: SEQ ID NO:2Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Lys Val Thr

-20 -15 -10Ala?Leu?Gly?Ser?Gln?Ala?Arg?Val?Thr?Lys?Asp?Ala?Glu?Thr?Glu

The information of-5 1 5Phe Met Met Ser Lys Leu Pro Leu Glu Asn Pro Val Leu Leu Asp10 15 20Arg Phe His Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr Pro25 30 35Arg Ser Ile Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn40 45 50Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly55 60 65Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys Gln Val Gln Val Cys70 75 80Met Arg Met Leu Lys Leu Asp Thr Arg Ile Lys Thr Arg Lys Asn85 90 95 (2) SEQ ID NO:3: (i) sequence signature:

(A) length: 282 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linear (ii) molecule type: cDNA (xi) sequence description: the information of SEQ ID NO:3ATGAAGATCT CCGTGGCTGC AATTCCCTTC TTCCTCCTCA TCACCATCGC CCTAGGGACC 60AAGACTGAAT CCTCCTCACG GGGACCTTAC CACCCCTCAG AGTGCTGCTT CACCTACACT 120ACCTACAAGA TCCCGCGTCA GCGGATTATG GATTACTATG AGACCAACAG CCAGTGCTCC 180AAGCCCGGAA TTGTCTTCAT CACCAAAAGG GGCCATTCCG TCTGTACCAA CCCCAGTGAC 240AAGTGGGTCC AGGACTATAT CAAGGACATG AAGGAGAACT GA 282 (2) SEQ ID NO:4: (i) sequence signature:

(A) length: 93 amino acid

(B) type: amino acid

(C) chain:

(D) topological framework: linearity is molecule type (ii): protein (xi) sequence description: SEQ ID NO:4Met Lys Ile Ser Val Ala Ala Ile Pro Phe Phe Leu Leu Ile Thr

-15 -10 -5Ile?Ala?Leu?Gly?Thr?Lys?Thr?Glu?Ser?Ser?Ser?Arg?Gly?Pro?Tyr

1 5 10His?Pro?Ser?Glu?Cys?Cys?Phe?Thr?Tyr?Thr?Thr?Tyr?Lys?Ile?Pro

15 20 25Arg?Gln?Arg?Ile?Met?Asp?Tyr?Tyr?Glu?Thr?Asn?Ser?Gln?Cys?Ser

30 35 40Lys?Pro?Gly?Ile?Val?Phe?Ile?Thr?Lys?Arg?Gly?His?Ser?Val?Cys

45 50 55Thr?Asn?Pro?Ser?Asp?Lys?Trp?Val?Gln?Asp?Tyr?Ile?Lys?Asp?Met

The information of 60 65 70Lys Glu Asn (3) SEQ ID NO:5: (i) sequence signature:

(A) length: 270 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linear (ii) molecule type: cDNA (xi) sequence description: the information of SEQ ID NO:5ATGAAGGGCC TTGCAGCTGC CCTCCTTGTC CTCGTCTGCA CCATGGCCCT CTGCTCCTGT 60GCACAAGTTG GTACCAACAA AGAGCTCTGC TGCCTCGTCT ATACCTCCTG GCAGATTCCA 120CAAAAGTTCA TAGTTGACTA TTCTGAAACC AGCCCCCAGT GCCCCAAGCC AGGTGTCATC 180CTCCTAACCA AGAGAGGCCG GCAGATCTGT GCTGACCCCA ATAAGAAGTG GGTCCAGAAA 240TACATCAGCG ACCTGAAGCT GAATGCCTGA 270 (2) SEQ ID NO:6: (i) sequence signature:

(A) length: 89 amino acid

(B) type: amino acid

(C) chain:

(D) topological framework: linearity, (ii) molecule type: protein, (xi) sequence description: SEQ ID NO:6Met Lys Gly Leu Ala Ala Ala Leu Leu Val Leu Val Cys Thr Met-20-15-10Ala Leu Cys Ser Cys Ala Gln Val Gly Thr Asn Lys Glu Leu Cys-5,15 10Cys Leu Val Tyr Thr Ser Trp Gln Ile Pro Gln Lys Phe Ile Val

15 20 25Asp?Tyr?Ser?Glu?Thr?Ser?Pro?Gln?Cys?Pro?Lys?Pro?Gly?Val?Ile

30 35 40Leu?Leu?Thr?Lys?Arg?Gly?Arg?Gln?Ile?Cys?Ala?Asp?Pro?Asn?Lys

45 50 55Lys?Trp?Val?Gln?Lys?Tyr?Ile?Ser?Asp?Leu?Lys?Leu?Asn?Ala

The information of 60 65 (2) SEQ ID NO:7: (i) sequence signature:

(A) length: 26 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:7TCAGGATCCG TCACAAAAGA TGCAGA 26 (2) SEQ ID NO:8: (i) sequence signature:

(A) length: 26 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:8CGCTCTAGAG TAAAACGACG GCCAGT 26 (2) SEQ ID NO:9: (i) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:9TCAGGATCCT GTGCACAAGT TGGTACC 27 (2) SEQ ID NO:10: (i) sequence signature:

(A) length: 26 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:10CGCTCTAGAG TAAAACGACG GCCAGT 26 (2) SEQ ID NO:11: (i) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:11GCCCGCGGAT CCTCCTCACG GGGACCTTAC 30 (2) SEQ ID NO:12: (i) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:12GCCTGCTCTA GATCAAAGCA GGGAAGCTCC AG 32 (2) SEQ ID NO:13: (i) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:13GGAAAGCTTA TGAAGGTCTC CGTGGCT 27 (2) SEQ ID NO:14: (i) sequence signature:

(A) length: 59 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:14CGCTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAA TTCTTCCTGG TCTTGATCC 59 (2) SEQ ID NO:15: (i) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:15GGAAAGCTTA TGAAGGGCCT TGCAGCTGCC 30 (2) SEQ ID NO:16: (i) sequence signature:

(A) length: 57 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:16CGCTCTAGAT CAABCGTAGT CTGGGACGTC GTATGGGTAG GCATTCAGCT TCAGGTC 57 (2) SEQ ID NO:17: (i) sequence signature:

(A) length: 28 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:17 GGAAAGCTTA TGAAGATTCC GTGGCTGC 28 (2) SEQ ID NO:18: (i) sequence signature:

(A) length: 58 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO:18CGCTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAG TTCTCCTTCA TGTCCTTG 58

Claims (22)

1. isolating polynucleotide, this polynucleotide comprise the member who is selected from the group of being made up of following material:

(a) a kind of coding comprises the polynucleotide of the amino acid-21 of SEQ ID NO.2 to amino acid 99 more than peptides;

(b) a kind of coding comprises the polynucleotide of the amino acid/11 of SEQ ID NO.2 to amino acid 99 more than peptides;

(c) a kind of can have the polynucleotide of at least 70% homogeny with (a) or multi-nucleotide hybrid (b) and with (a) or polynucleotide (b); With

(d) polynucleotide passage of a kind of (a) or polynucleotide (b).

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, this polynucleotide encoding comprises the polypeptide of the amino acid-21 of SEQ ID NO.2 to amino acid 99.

6. the polynucleotide of claim 2, this polynucleotide encoding comprises the polypeptide of the amino acid/11 of SEQ ID NO.2 to amino acid 99.

7. isolating polynucleotide, this polynucleotide comprise the member who is selected from the group of being made up of following material:

(a) a kind of polynucleotide of coded polypeptide, described polypeptide have the aminoacid sequence of being expressed by the DNA that is included in No. 75676 preservation things of ATCC;

(b) a kind of can have the polynucleotide of at least 70% homogeny with the multi-nucleotide hybrid of (a) and with the polynucleotide of (a);

(c) polynucleotide passage of a kind of (a) or polynucleotide (b).

8. the polynucleotide of claim 1, this polynucleotide comprise as in the sequence of Nucleotide 1 as shown in the SEQ ID NO.1 to Nucleotide 363.

9. carrier that comprises the DNA of claim 2.

10. host cell that obtains through the genetically engineered operation with the carrier of claim 9.

11. a method that produces polypeptide, this method comprises: the host cell expression of Accessory Right requirement 10 is by the polypeptide of said dna encoding.

12. the method for the cell that a generation can express polypeptide, this method comprises that the carrier pair cell with claim 9 carries out the genetically engineered operation.

13. polypeptide that is selected from the group of forming by the following member: (i) polypeptide of a kind of SEQ of having ID NO.2 aminoacid sequence of inferring and its fragment, analogue and derivative; (ii) a kind of by the cDNA encoded polypeptides of ATCC75676 preservation thing and fragment, analogue and the derivative of said polypeptide.

14. the polypeptide of claim 13, wherein said polypeptide has the aminoacid sequence that SEQ ID NO.2 infers.

15. the stimulant of peptide more than the claim 13.

16. the antagonist of peptide more than the anti-claim 13.

17. a method that is used for the treatment of the patient who needs Ck β-8, this method comprises: to the polypeptide of the claim 13 of said patient's administering therapeutic significant quantity.

18. the method for claim 17, the polypeptide of wherein said treatment significant quantity are by the DNA that the said polypeptide of coding is provided to the patient and express said polypeptide in vivo and use.

19. a method for the treatment of the patient that need more suppress Ck β-8 polypeptide, this method comprises: to the antagonist of the claim 16 of patient's administering therapeutic significant quantity.

20. the not enough diseases associated of the polypeptide expression of diagnosis and claim 13 or to the method for the susceptibility of this disease, this method comprises:

Determine the sudden change of the nucleotide sequence of the said polypeptide of coding.

21. a diagnostic method, this method comprises:

Whether analysis exists the polypeptide of claim 13 in deriving from host's sample.

22. an antagonist and an anti-depressant method of identifying the polypeptide of anti-claim 13, this method comprises:

Cell, compound to be screened are in the same place with said polypeptides in combination, and wherein said cell separates with said polypeptide by the porous filter; With

Determine the extent of migration of these cells, to determine whether said compound is effective antagonist or stimulant.

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