CN1163635A - Macrophage derived chemokine and chemokine analogs - Google Patents

Abstract

The present invention provides purified and isolated polynucleotide sequences encoding a novel human macrophage-derived C-C chemokine designated MDC, and polypeptide analogs thereof. Also provided are materials and methods for the recombinant production of thechemokine, and purified and isolated chemokine protein, and polypeptide analogs thereof.

Description

Macrophage derived chemokine and chemokine analogue

The application is a U.S. Patent application, serial number 08/558,658, and the part in November 6 nineteen ninety-five submission date continues, and this application is the U.S. Patent application of submitting to June 7 nineteen ninety-five, and the part of serial number 08/479,620 continues.

Invention field

Generally, the present invention is relevant chemokine, more specifically be purifying and the polynucleotide that separate a kind of new people C-C chemokine of coding, purifying and the chemokine protein that separates by this polynucleotide encoding, and the material and the method for the production of the recombinant chou of new chemokine protein.

Background

Chemokine is also referred to as " intercellular excreted factor " and " SIS cytokine ", forms the little secretory protein of gang (for example, a 70-100 amino acid and 8-10 kilodalton), its attraction and activated leukocyte cell, thereby the activation of skeptophylaxis system and adjusting.The name of " chemokine " is from chemotactic cytokine, because of these albumen can stimulate leukocytic chemotaxis.Really, chemokine may constitute the main attraction molecule that inflammatory cell enters pathological tissue.Generally with reference to Ba Geoulini etc., immunology progress, 55:97-179 (1994).Though white corpuscle has abundant chemokine, different chemokines is also expressed in a large amount of tissues.The same magazine, table 2.

The chemokine of Que Rening in the past generally has the amino acid identity of 20-70% between mutually, contains the cysteine residues of 4 high conservatives.According to the relative position of the first two halfcystine, chemokine can be further divided into two subtribes." C-X-C " or " α " subtribe, the gene of coding are positioned on No. 4 karyomit(e) of people, and the first two halfcystine is separated by an amino acid." C-C " or " β " subtribe, the gene of coding are positioned on No. 17 karyomit(e)s of people, and the first two halfcystine is adjacent.The X-radiocrystallography studies show that to different chemokines that with NMR in each family, first and the 3rd halfcystine forms article one disulfide linkage, and second and the 4th halfcystine forms the second disulfide linkage, influences proteic native conformation consumingly.Only in the mankind, having described each chemokine subtribe has nearly ten different sequences.The chemokine of two class subtribes has 20-25 amino acid whose characteristic leader sequence.

The C-X-C chemokine comprises IL-8, GRO α/β/γ, platelet basic protein, platelet factor 4 (PF4), IP-10 and other kind; When relatively more wantonly two aminoacid sequences (remove between GRO α/β/γ member, have mutually the identity of 84-88% outer), enjoy the identity of about 25%-60%.Most of C-X-C chemokines (not comprising IP-10 and platelet factor 4) have common E-L-R three peptide motifs (motif) in the first two cysteine residues upstream; They are strong stimulation factors of neutrophilic granulocyte, cause metamorphosis fast, chemotaxis, respiratory burst and threshingization.These effects are by seven membrane-spanning domains, and the g protein coupled receptor of Visual purple sample mediates; To the special acceptor of IL-8 by Hellems etc., science, 253:1278-80 (1991) clone comes out, and identification IL-8, the similar acceptor of GRD and NAP2 (77% identity) is by Mo Fei and Di Feini, science, 253:1280-83 (1991) clone.Remove a certain C-X-C chemokine step by step, comprise IL-8, the N terminal amino acid sequence, relevant with active remarkable increase.

The C-C chemokine comprises macrophage inflammatory protein MIP-1 α and MIP-1 β, monocyte chemical attractants albumen 1,2 and 3 (MCP-1/2/3), and RANTES, I-309 and other kind are enjoyed 25%-70% amino acid identity between mutually.Confirmed C-C chemokine energy activated monocyte causes calcium current and chemotaxis before all.More optionally effect is to lymphocyte, as the T lymphocyte RANTES is had optimum response.Up to now, the Chemokine Receptors of seven membrane-spanning domain G of five tools albumen coupling is cloned, C-C Chemokine Receptors-1 (CCR1) (the interior top grade that comprises identification MIP-1 α and RANTES, cell, 72:415-425 (1993)), the CCR2 acceptor of identification MCP-1 (looking into sieve etc., institute of NAS newspaper, 91:2752-56 (1994)); The CCR3 (Kang Padier, journal of biological chemistry, 270:16491 (1995)) of identification eotaxin; The CCR4 (Pu'an etc., journal of biological chemistry, 270:19495 (1995)) of identification MIP-1 α, RANTES and MCP-1; CCR5 (Samson etc., biological chemistry, 35:3362 (1996)) with identification MIP-1 α, MIP-1 β and RANTES.

Under different pathological states, the existing sufficient document record of the effect of many chemokines, particularly IL-8.Generally can be referring to Ba Geoulini etc., the same, table 7.Relevant with the excessive generation of IL-8 as psoriasis, different researchs are observed: the patient of rheumatosis, osteoarthritis and gout, its inflamed joints synovia has high-caliber IL-8.

Although than the effect of IL-8 solve and lack, the effect of C-C chemokine is also on the books under pathological state.For example, in the synovia of rheumatoid arthritis patients, other arthritic's height of the concentration ratio of MCP-1.Stream is the critical event that idiopathic pulmonary fibrosis forms in the MCP-1 dependency of mononuclear phagocyte.The effect of C-C chemokine in the monocyte recruitement that enters the atherosclerosis district excites wide spread interest at present, in abundant arterial wall district of scavenger cell rather than general arterial tissue, detected MCP-1 and strengthened the property expression.The expression of MCP-1 in malignant cell shows the ability that suppresses to become in these cell paste knurl.(see U.S. Patent number 5,179,078, be hereby incorporated by).Thereby be necessary additional C-C chemokine is added their confirmation, its feature is described, further specify the important family of this molecule in pathological state effect and use the product be derived from chemokine, these pathological states are improved treatment.

The chemokine that has shown the C-C subtribe can be used for medical imaging, and as to infecting, imaging is carried out in inflammation site and other site with C-C Chemokine Receptors molecule.As see elder brother Ke Er etc., U.S. Patent number 5,413,778 is hereby incorporated by.The step that this kind method relates to is, with artificial recognition technology (as seeing U.S. Patent number 4,965,392 and 5,037,630, be hereby incorporated by), on the C-C chemokine, allow marking agent (as radio isotope) chemical attachment the experimenter take the chemokine of mark with pharmaceutically acceptable carrier, the chemokine of mark is accumulated, to the chemokine target position imaging in vivo of mark at target position.This area needs new C-C chemokine in addition, to increase effective storage of medical imaging instrument.

Also show: C-C chemokine RANTES, MIP-α and MIP-1 β are the main mediation person of human T-cell to human immunodeficiency virus's (HIV) retarding effect, and HIV causes causing people's acquired immune deficiency syndrome (AIDS) (AIDS).These chemokines show that suppressing the special strain of HIV infects and cultivate T clone, have dose-dependently [Cork is uncommon etc., science, 270:1811 (1995)].But, not all virus strain of being tried has identical susceptibility to this restraining effect; Thereby need additional C-C chemokine as the inhibitor of HIV strain.

More in general, because chemokine as chemotaxis and inflammation mediated person's importance, is necessary to confirm and separates new chemokine family member, to make things convenient for inflammation and immunoreactive adjusting.

For example, promote the material of inflammation can promote wound healing or acceleration rate as the recovery from the pneumonia state, inflammation is to eliminating infect important.The adjusting of inflammation is to the pathological state no less important of the obvious inflammation of tool.Crohn disease shows as the chronic inflammatory diseases of all layers of intestines, pain and diarrhoea, a kind of pathological state that comes to this.Mortality to Crohn disease pharmacological agent is quite high, even this disease patient accepts also often to recur after the surgical operation.Affirmation, separation and specificity analysis to new chemokine help the inflammation adjusting.

Similarly, the material of induce immune response can impel the mitigation or the disappearance of any pathological state.Because the vital role of white corpuscle (as neutrophil leucocyte and monocyte) in cell-mediated exempting from replied, and chemokine tailor-made really usefulness in leucocyte chemotaxis, be necessary to discern and separate new chemokine, to be convenient to the adjusting of immunne response.

In addition, the expression of chemokine, the definite relation between the condition of inflammation and the morbid state can and can play the antibody materials that specific immune reacts with chemokine chemokine, as the indication of diagnosis and prognosis; Be necessary to discern and separate new chemokine, with the convenient indication of doing diagnosis and prognosis.

Outside decapacitation attraction and the activated leukocyte cell, some chemokine such as IL-8, can influence the propagation of non-white corpuscle sexual cell.See Ta Siqieer, dermatitis research magazine, 99:294-298 (1992).Be necessary to discern and separate new chemokine, to promote the adjusting of these cell proliferations.

Be preceding being had reason of carrying, need the method for the newfound chemokine of recombinant production, this method promotes to relate to the clinical application of chemokine and CFI.

Summary of the invention

The invention provides new purifying and the method for separating polynucleotide and polypeptide, to satisfy the demand of above generalized one or more kinds.

For example, purifying and isolating polynucleotide (that is, DNA and RNA, justice and antisense strand) that the present invention proposes, its encode human chemokine of a kind of new C-C subtribe is at this called after " from the chemokine of scavenger cell " or " MDC ".The preferred dna sequence dna of the present invention comprises genome and cDNA sequence, and the dna sequence dna of chemosynthesis.

The cDNA nucleotide sequence, called after MDC cDNA, this chemokine of encoding is listed among the SEQID NO:1, and this sequence comprises 5 ' and 3 ' non-coding sequence.The preferred DNA of the present invention comprises the 20-298 position Nucleotide of SEQ ID NO:1, and this Nucleotide is formed the encoding sequence of MDC.

MDC albumen comprises 24 amino acid whose signal sequences of supposition at aminoterminal.The preferred DNA of the present invention forms the Nucleotide of SEQ ID NO:192-298, and these Nucleotide comprise the proteic encoding sequence of maturation (secretion) MDC of supposition, do not have signal sequence.

The aminoacid sequence of chemokine MDC is listed among the SEQ ID NO:2.Except that above-mentioned polynucleotide, the preferred polynucleotide of the present invention comprise encoding lists in aminoacid sequence among the SEQ ID NO:2, it with former joints describe only because the polynucleotide of well-known genetic code degeneracy are different.

Similarly, because 24 amino acid of SEQ ID NO:2 (24 to-1) are formed the signal peptide of generally acknowledging that the energy cracking produces sophisticated MDC chemokine, preferred polynucleotide comprise the amino acid whose part of 1-69 of coding SEQ ID NO:2.Like this, preferred polynucleotide are purified polynucleotides, and its encoded polypeptides contains the aminoacid sequence that comprises SEQ ID NO:21-69 amino acids.

Can be used as hybridization probe when using polynucleotide of the present invention, identification and separates the genomic dna of the people MDC that encodes, this gene may contain distinctive three exon/two intron structure of C-C chemokine gene (Bei Bageoulini etc., the same); Identification and the dna sequence dna that separates with MDC homologous coding non-human protein; Discern people similar and inhuman chemokine to MDC, and the condition of the cell of recognition expression MDC and this protein expression.

Hybridization probe of the present invention also has diagnostic uses, as screening the inflammation such as people's tissue of colon.More specifically, the hybridization research of use MDC multi-nucleotide hybrid probe can be distinguished colon's (MDC hybridization detection epithelium of segmental colitis patient, lamina propria, Pei Ye (payer ' s) spot and unstriated muscle) and normal people colon (can not hybridize under the above-mentioned background).

In general, have an appointment at least sequential portion of 14 preferably about 18 Nucleotide of MDC cDNA of the present invention is to being useful as hybridization probe of the present invention.Like this, in one embodiment, the DNA that the present invention includes contains the sequential portion of SEQ ID NO:1 or corresponding non-coding complementary nucleotide sequence, and this part contains 18 Nucleotide at least, and this DNA can be hybridized with the coding or the noncoding strand of people MDC gene under rigorous condition.When hybridization probe of the present invention is made diagnostic uses, more preferably show as hybridization specificity to the MDC gene order.Like this, in preferred embodiments, hybridization probe DNAs of the present invention under rigorous condition not can with other the human chemokine gene recombination (as, MCP-1 gene, MCP-2 gene, MCP-3 gene, RANTES gene, MIP-1 α gene, MIP-1 β gene and I-309 gene etc.).

On the other hand, the invention provides purified polynucleotides, its under stringent condition can with the noncoding strand hybridization of the DNA of SEQID NO:1.Similarly, purified polynucleotides provided by the invention, if it were not for superfluous She's property (redundancy) of genetic code, under stringent condition can with the noncoding strand hybridization of the DNA of SEQID NO:1.Exemplary stringent hybridization condition is as follows: at 5 * SSC, and the 20mM sodium phosphate, pH6.8, in 50% methane amide, 42 ℃ of hybridization, 42 ℃ are cleaned down in 0.2 * SSC.Those skilled in the art understands, foundation is by the content experiment condition of the length of hybridization sequences and GC nucleotide base, be desirable, the formula of determining these variations is arranged [as seeing, holy nurse Brooker etc., molecular cloning: laboratory manual, second edition, the cold spring port, New York: cold spring harbor laboratory (1989) .].

On the other hand, the present invention includes the plasmid and the viral DNA carrier that have mixed DNA of the present invention, comprise any of above-mentioned or other local DNA that describes of this paper.Preferred carrier comprises expression vector, and in expression vector, the cDNA of the coding MDC that mixes operationally links to each other with endogenous or allogenic expression control sequenc.Above expression vector can further comprise the dna sequence dna of the coded polypeptide that functionally connects coding MDC dna sequence dna, and this carrier can be expressed and be produced the fusion rotein that contains important MDC polypeptide.

On the other hand, the present invention includes with DNA of the present invention or carrier stable transfection or conversion protokaryon or eukaryotic host cell.In preferred host cell, can express by DNA of the present invention or the coded sophisticated MDC polypeptide of carrier.DNA of the present invention, carrier and host cell are useful to a large amount of recombinant production as MDC polypeptide of the present invention.These methods are also among the present invention.For example, the present invention includes the method for a kind of MDC of production, host cell wherein of the present invention is grown in suitable nutritional medium, separates MDC albumen from cell or substratum.

On the one hand, the present invention includes purifying and the method for separating the MDC polypeptide again.Preferred peptide is the chemokine polypeptides of purifying, has the aminoacid sequence of the 1-69 amino acids that comprises SEQ ID NO:2.Polypeptide of the present invention can be from natural material purifying, but preferred, use DNAs of the present invention, carrier and/or host use recombination method production, or chemosynthesis.The polypeptide of purifying of the present invention according to situations such as the method for selected host cell, recombinant production, separation method, treating processes, storage buffer, can be glycosylation or nonglycosylated, and is water-soluble or insoluble, oxidation, reductive etc.

In addition, one aspect of the present invention comprises the MDC polypeptide analog, wherein is to add from one of MDC polypeptide of the present invention or many outer amino-acid residues disappearance or replace and form the distinctive one or more biologic activity of analogue reservation C-C chemokine.The chemosynthesis that helps above polypeptide analog for a short time of MDC, with the method for many determinations of activity described herein, the analogue of the biological activity of screening tool MDC (as: induce the chemotactic ability of scavenger cell, suppress the ability of monocyte chemotaxis).Alternative method is that these polypeptide analogs can produce with the method reorganization of knowing, as the DNA rite-directed mutagenesis of MDC that the present invention is encoded.

Relevant aspect, the present invention includes the polypeptide analog that is added, lacks or replace from the one or more amino-acid residues of MDC polypeptide of the present invention, this analogue lacks the biologic activity of C-C chemokine or MDC, but energy competitiveness or noncompetitive ground suppress the combination of MDC polypeptide and C-C Chemokine Receptors.Such polypeptide is useful, as is used to regulate the biologic activity of host's endogenous MDC, also can be used for above-mentioned medical image method.

The special analogue of some of MDC can design structure, intermolecular binding characteristic and the biologic activity of making to regulate MDC.For example, can design the 26S Proteasome Structure and Function of analogue (brachymemma) the change MDC of disappearance N-terminal (N-end) and C-terminal (C-end) especially.

In addition, can design following monamino acid especially and change body (independent or combination): (1) non-basic aminoacids is replaced 24 and 27 alkaline arginine and/or the Methionin of SEQ ID NO:2 respectively; (2) charged or polare Aminosaeren (as Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine or halfcystine) is replaced the tyrosine of SEQID NO:230 position, replace 59 the tryptophane of SEQ D NO:2, and/or the Xie Ansuan of SEQ ID NO:260 position; (3) with L-glutamic acid alkaline or uncharged p1 amino acid (as Methionin, arginine, Histidine, glycine, L-Ala) replacement SEQ ID NO:250 position.Special analogue with these amino acid changes is included in (SEQID NO:25) in the following general formula:

Met?Ala?Arg?Leu?Gln?Thr?Ala?Leu?Leu?Val?Val?Leu?Val?Leu?Leu?Ala

-24?????????????-20?????????????????-15?????????????????-10

Val?Ala?Leu?Gln?Ala?Thr?Glu?Ala?Gly?Pro?Tyr?Gly?Ala?Asn?Met?Glu

-5???????????????????1???????????????5

Asp?Ser?Val?Cys?Cys?Arg?Asp?Tyr?Val?Arg?Tyr?Arg?Leu?Pro?Leu?Xaa

10??????????????????15??????????????????20

Val?Val?Xaa?His?Phe?Xaa?Trp?Thr?Ser?Asp?Ser?Cys?Pro?Arg?Pro?Gly

25??????????????????30??????????????????35??????????????????40

Val?Val?Leu?Leu?Thr?Phe?Arg?Asp?Lys?Xaa?Ile?Cys?Ala?Asp?Pro?Arg

45??????????????????50??????????????????55

Val?Pro?Xaa?Xaa?Lys?Met??Ile?Leu?Asn?Lys?Leu?Ser?Gln

60 65 wherein 24 amino acid can be selected from arginine, glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), Serine, Threonine, phenylalanine, tyrosine, tryptophane, aspartic acid, L-glutamic acid, l-asparagine, glutamine, halfcystine and methionine(Met); Wherein 27 amino acid can be selected from Methionin, glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), Serine, Threonine, phenylalanine, tyrosine, tryptophane, aspartic acid, L-glutamic acid, l-asparagine, glutamine, halfcystine and methionine(Met) by oneself; Wherein 30 amino acid can be selected from tyrosine, Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine and halfcystine by oneself; Wherein 50 amino acid can be selected from L-glutamic acid, Methionin, arginine, Histidine, glycine and L-Ala by oneself; Wherein 59 amino acids can be selected from tryptophane, Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine and halfcystine by oneself; Wherein 60 amino acids can be selected from Xie Ansuan, Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine and halfcystine by oneself.These MDC polypeptide analogs can be specifically designed to regulate the binding characteristic of MDC to Chemokine Receptors and/or other molecule (as heparin, glycosaminoglycan, red corpuscle Chemokine Receptors), these characteristics MDC be think when passing its acceptor important.In an embodiment preferred, MDC polypeptide analog of the present invention comprises the amino acid of the 1-69 of SEQ ID NO:25.

The following other analogue of synthetic also becomes aspect of the present invention: the polypeptide that (a) comprises the aminoacid sequence of the 1-70 position that is accredited as SEQ ID NO:30; (b) comprise the polypeptide of the aminoacid sequence of the 9-69 position that is accredited as SEQ ID NO:2; (c) comprise the polypeptide of the aminoacid sequence of the 1-69 position that is accredited as SEQ ID NO:31; (d) comprise the polypeptide of the aminoacid sequence of the 1-69 position that is accredited as SEQID NO:32.

Related aspect is the method that the invention provides purifying and the polynucleotide that separate the above MDC polypeptide analog of encoding, and these polynucleotide are to producing the MDC polypeptide analog as recombination method; Above polynucleotide are connected with virus vector with plasmid, stably transform protokaryon and eukaryotic host cell is useful with this DNA or carrier.

The present invention includes on the other hand can and MDC polypeptide of the present invention and polypeptide analog play immunoreactive antibody materials (for example, mono-clonal and polyclonal antibody, single-chain antibody, chimeric or humanized antibody etc.).Above antibody is to such as purifying polypeptide of the present invention, as with the endogenic MDC of elisa technique quantitative assay host that knows with to regulate MDC be useful to the combination of its acceptor.The present invention and then comprise the hybridoma cell line of production antibody materials of the present invention.

The MDC polypeptide of the present invention reorganization can be used for association reaction with polypeptide analog as antibody, with the cell of recognition expression MDC acceptor with the clonal expression technology of standard, separates the polynucleotide of the acceptor of encoding.These MDC polypeptide, MDC polypeptide analog and MDC receptor polypeptides are to the active adjusting of MDC chemokine, and (as small molecules) the MDC agonist of polypeptide and chemical and the discriminating of antagonist are useful.

Another aspect of the present invention is the pharmaceutical application about MDC polypeptide of the present invention and polypeptide analog.For example, shown that MDC can regulate leucocyte chemotaxis.Especially, MDC can induce the scavenger cell chemotaxis and suppress monocyte chemotaxis.Thereby the present invention includes on the one hand the method for adjustings (as raising or reducing) mammals host's leucocyte chemotaxis, contain to the mammals host take the step of MDC polypeptide of the present invention and polypeptide analog, wherein MDC polypeptide or MDC polypeptide analog can be regulated the leukocytic chemotaxis of host.In a preferred method, white corpuscle is monocyte and/or scavenger cell.For example, take the MDC dosage (as in pharmaceutically acceptable carrier) that experiment is determined, inducing the scavenger cell chemotaxis or to suppress monocyte chemotaxis, and use the MDC polypeptide analog of inhibition to obtain reverse effect.

On the other hand, the invention provides the method that alleviates patient's inflammatory states, the feature of this state is (i) monocyte to described patient's inflammation site chemotactic or (ii) one of fibroblast proliferation at least, and this method comprises the step of the MDC treatment significant quantity that doses a patient with.In one embodiment, MDC treatment significant quantity is to suppress the amount of monocyte chemotaxis.In another embodiment, MDC treatment significant quantity is to suppress the amount of fibroblast proliferation.These treatment significant quantities are to determine with the dose response analysis experiment of recognized technology.

As additional content, the invention provides and contain MDC polypeptide of the present invention or the pharmaceutical compositions of polypeptide analog in pharmaceutically acceptable carrier.Similarly, the present invention has the composition application method of the relevant work according to the present invention as the morbid state treatment of inflammatory diseases state.In one embodiment, the feature of inflammatory diseases state is the inflammatory site chemotactic of monocyte in the patient of morbid state.In another embodiment, the feature of inflammatory diseases state is tool morbid state patient's a fibroblast proliferation.

Aforesaid content and a large amount of additional content are from following accompanying drawing with more obvious describing in detail.

The accompanying drawing summary

Fig. 1 is that the aminoacid sequence of people MDC (SEQ ID NO:2) and the aminoacid sequence of other people C-C chemokines of having described in the past compare, these chemokines are: MCP-3[Fan Daimu etc., The Journal of Experimental Medicine, 176:59 (1992)] (SEQ ID NO:18); MCP-1[Ma Tesuxuema etc., The Journal of Experimental Medicine, 169:1485 (1989)] (SEQ ID NO:19); MCP-2 (ripe attitude) [Fan Daimu etc., the same, normal etc., international immunology, 1:388 (1989)] (SEQID NO:20); RANTES[Shi Chaer etc., Journal of Immunology, 141:1018 (1988)] (SEQ IDNO:21); MIP-1 β [Blang etc., Journal of Immunology, 142:679 (1989)] (SEQ IDNO:22); MIP-1 α [interior high, molecular cytobiology, 10:3646 (1990)] (SEQ IDNO:23); With I-309[Miller etc., Journal of Immunology, 143:2907 (1989)] (SEQ IDNO:24).The site that the signal peptide that slash "/" expression is generally acknowledged is cut.Inserting dash makes sequence attractive in appearance neat.

Fig. 2 is described in the chemotaxis assay, increases the chemotactic influence figure (with flat fluorescent measure) of MDC concentration to person monocytic cell's migration.Solid circles is represented to reply from the monocytic of THP-1 clone.Open diamonds represents that to positive control zymosan activates replying of serum (2AS).

Fig. 3 describes to increase chemotactic influence figure (the use flat fluorescent) metering of MDC concentration to people's polymorphic nucleus (pmn) leucocyte migration.Solid circles represents that open diamonds is represented replying positive control IL-8 to the replying of MDC.

Fig. 4 describe to increase MDC concentration the chemotactic of scavenger cell and monocyte migration is influenced figure (measuring with flat fluorescent).Solid circles is represented scavenger cell the replying MDC from THP-1 clone.Open circles is represented monocyte the replying MDC from THP-1 clone.

Fig. 5 describes increase MDC concentration the chemotactic that the cavy peritoneal macrophages moves is influenced figure (measuring with flat fluorescent).Solid circles is represented scavenger cell replying MDC.Hollow triangle is represented replying zymosan activation serum (ZAS) of positive control.

Fig. 6 describes to increase MDC concentration, to the inhibition chemotaxis influence figure (measuring with flat fluorescent) by the migration of MCP-1 inductive THP-1 monocyte.Solid circles represents that MDC suppresses the effect of chemotactic, and chemotaxis is derived by MCP-1.Empty circles is illustrated in the control experiment, only uses minimum medium when (RPMI adds 0.2%BSA (RBSA), no MCP-1), and MDC suppresses the effect of chemotactic.Zero point on the X-axis, cell was replied MCP-1 and RBSA's corresponding to without any MDC the time.

Fig. 7 describe to increase MDC concentration fibroblast proliferation influenced figure (with per minute counting (cpm) metering).Solid circles is represented the proliferative response from the MDC of Chinese hamster ovary celI recombinant production (reference example 10F) with purifying.Empty circles is represented the reaction with the MDC (reference example 11) of chemosynthesis.

Fig. 8 is a structure of figuring mammals expression vector pDC1.

Detailed Description Of The Invention

Set forth the present invention with following embodiment, relevant people cDNA called after MDC cDNA, coded new C-C chemokine called after MDC (for " being derived from the chemokine of scavenger cell ").In more detail, embodiment 1 describes separate part MDC cDNA from human macrophage cDNA library.Embodiment 2 describes to use from the cDNA of embodiment 1 and makes probe, separates other cDNA from the cDNA library, and one of these cDNA contain the MDC encoding sequence of total length.In addition, embodiment 2 proposes the feature of the derivation acidic amino acid sequence of the compound MDC cDNA nucleotide sequence and the chemokine (MDC) of encoding thus.In embodiment 3, the experiment of description is presented at the level of MDC genetic expression in different people's tissues.Observe the strongest MDC genetic expression at thymus gland, and the weak expression that can detect is in spleen and lung tissue.It is in the scavenger cell process that embodiment 4 has described at monocyte maturation in more detail, MDC expression of gene when inducing the HL60 cytodifferentiation to become the cell type of macrophage-like.

Because MDC genetic expression detects in thymus gland and spleen in embodiment 3, in situ hybridization research can further be expressed in these tissues the MDC gene and position.In addition, in situ hybridization has shown the dependency of MDC gene high expression between intestinal tissue and segmental colitis.These hybridization in situ experiment are described in embodiment 5.

Embodiment 6 describes MDC as recombinant production and the cutting of fusion rotein and the method for reorganization MDC purifying of gst fusion protein in prokaryotic cell prokaryocyte.Embodiment 7 describe the MDC protein expression that is used to recombinate substituting DNA structure body construction process and the method for producing MDC with this structure body transform bacteria host is described.

Embodiment 8 provides experimental program to carry out purifying to describe the reorganization MDC that produces in embodiment 7.Embodiment 9 and 10 provides experimental program to carry out the MDC recombinant production respectively in yeast and cells of mamma animals.In addition, embodiment 10 provides the scheme of purification of Recombinant MDC.Embodiment 11 describes with polypeptide synthesis method and produces MDC and MDC polypeptide analog.

Embodiment 12-17 provides scheme to measure the MDC biologic activity.For example, embodiment 12 provides MDC to basophilic leukocyte, the assay method of mastocyte and eosinophil influence.Embodiment 13 describes MDC to monocyte/macrophage, the assay method of neutrophil leucocyte and granulocytic chemical attractants and cell activation characteristic.

Embodiment 14-17 provides the case side that measures the MDC biologic activity in the body.Embodiment 14 provides MDC to suppress the assay method of tumor growth.Embodiment 15 and 16 provides scheme to measure respectively through abdominal cavity and hypodermic MDC activity.

Embodiment 18 provides scheme to produce and MDC plays the monoclonal antibody that specific immune reacts.

Remaining embodiment provides other MDC activation measurement.For example, embodiment 19 provides the calcium current assay method, to measure MDC inducing cell activatory ability.Embodiment 20 provides method to measure the influence of the anti-HIV propagation of MDC.The influence of the anti-fibroblast proliferation of embodiment 21 explanation MDC.Embodiment 22 provides external MDC method to the influence of other cell type propagation.Embodiment 23 provides the influence of the anti-fibroblast proliferation of method mensuration MDC in the body.Embodiment 24 provides method identification MDC regulatory factor.

Embodiment 1

The separation of portion C-C chemokine cDNA

The Partial cDNA of new C-C chemokine is separated by laxative remedy.PolyA ⁺RNA always is derived from the scavenger cell of peripheral blood lymphocytes and gathers in the crops.Two strands, (San Diego CA) produces flat terminal cDNA with external heredity copy test kit, lactation expression vector pRc/CMV (Invitrogen) is preceding inserting, BstXI modification is connected to cDNA goes up [seeing Te Qiaoerke etc., nature, 374:549-552 (1995)].(stratagene, La Jolla CA) transform through electroporation method with plasmid cDNA blue XL1 intestinal bacteria, plant (3000 transformant of every approximately plate) on 986 flat boards that contain 100 μ g/ml Pyocianils.After 37 ℃ of grow overnight, the bacterium of every plate scrapes and forms 986 bacterium storehouses.(Pu Luomaige, Madison WI), according to the specification sheets of producer, are separated plasmid DNA from 986 bacterium storehouses one by one with a large amount of little preparation dna purification systems.

Further identify by laxative remedy in the plasmid DNA storehouse of purifying, is used to separate one dna clone: the plasmid DNA from each storehouse is used to transform blue XL1 Bacillus coli cells, plants plate, by last method grow overnight.Each transformant of random choose, grow overnight in the 3ml LB substratum that adds Pyocianil, the plasmid purification little preparation purification system of Wizard (Pu Luomaige), do following change: 250mg diatomite (sigma chemical company, the St. Louis MO) is added in the DNA binding resin that producer provides.The plasmid DNA of purifying automatic sequencer model 373 (order-checking CA) is gone up in Applied Biosystems, Inc., Foster city, and used primer is JHSP6:

This primer of 5 ' GACACTATAGAATAGGGC 3 ' (SEQ ID NO:3) can be hybridized with the part of the contiguous cloning site of plasmid vector pRc/CMV carrier.

The Nucleotide of each cDNAs and the aminoacid sequence of deriving and Nucleotide and peptide database are relatively, and be similar to known inflammatory reporter molecule with which clone who determines proteins encoded.Sequence is compared to was undertaken by the special national center biotechnology service of information network portion of Bryce (e-mail: " blast@ncbi.nlm.nih.gov ") on December 14th, 1994, with Ai Erzi Qiu Er etc., the molecular biology magazine, the permutation algorithm of 215:403-410 (1990).Sequential analysis show a clone's the part of isolating scavenger cell cDNA, be called pMP390, contain gene order and preceding confirmed chemokine gene, comprise people MCP-3 gene and rat MIP-1 β gene, the identity of the 60-70% that has an appointment.

The 2.85kb cDNA of pMP390 inserts fragment subclone (Stratagene, La Jolla CA) in pBluescript SK-carrier, from just order-checking fully.The a whole set of deletant that begins from poly-A tail digests formation with the going of Pu Luomaige-base system (promega ' s Erase-a-BaseSystem) (Madison WI).Disappearance plasmid recirculation.The clone advances in the intestinal bacteria, and purifying checks order with the following M13 that represents T3.1 and T7.1 primer:

(SEQ?ID?NO：4)

M13：5′GTAAAACGACGGCCAGT3′

(SEQ?ID?NO：5)

T3.1：5′AATTAACCCTCACTAAAGGG3′

(SEQ?ID?NO：6)

T.7.1:5 ' this pMP390 cDNA complete sequence of GTAATACGACTCACTATAGGGC3 ' is equivalent to the nucleotide segment (the derivation aminoacid sequence that is equivalent to SEQ ID NO:2-6-69 position) of the 73-2923 position of SEQ ID NO:1.The sequence of carrying out the most original comparison with database sequence is equivalent to SEQ ID NO:173-610 position Nucleotide.

Embodiment 2

Contain the other cDNA clone's of complete MDC encoding sequence separation

With the isolating pMP390 cDNA clone of embodiment 1, from same human macrophage cDNA library, separate other cDNA clone, these other cDNA contain 5 other ' sequence, the aminoacid sequence of the complete chemokine from scavenger cell of encoding.

At first, from 986 plasmid DNA storehouses, get 40 with the PCR screening, to differentiate the interested storehouse that contains other cDNA clone from scavenger cell cDNA library (embodiment 1).PMP390 cDNA sequence according to embodiment 1 acquisition, make up synthetic oligonucleotide PCR primer 390-1F (being called SEQ ID NO:7) and 390-2R (SEQ ID NO:8), 211 base-pair sequences of the chemokine gene of encoding by pMP390 with the amplification part:

390-1F：5′TCTATCTAGAGGCCCCTACGGCGCCAACATGGAAG

390-2R:5 ' CACCGGATCCTCATTGGCTCAGCTTATTGAGAA3 ' primer 390-1F is corresponding to the 91-116 position Nucleotide of SEQ ID NO:1, the recognition site of preceding restricted restriction endonuclease XbaI, and 4 additional bases are convenient to this enzyme cutting; The 301-279 position Nucleotide complementation of primer 390-2R and SEQID NO:1 is added with the recognition site of enzyme BamH I, and 4 additional bases are positioned at flank.Be convenient to obtain segmental clone as XbaI and BamHI point of contact.

Each selected plasmid storehouse with 50 μ l PCR reaction mixtures, is contained 0.2 μ g plasmid DNA; 1.5mM magnesium chloride; 50mM Repone K; The 10mM Tutofusin tris, pH8.4; 0.2mM each dNTP; Each primer 10 μ g/ml and 0.5 μ l Taq polysaccharase (5U/ μ l) (Bai Linge mannheim Biochemics Inc. (BMB), Indianapolis, IN).Be reflected at 94 ℃ of incubations 4 minutes, 94 ℃ of sex change are 15 seconds then, and 60 ℃ of annealing 15 seconds and 72 ℃ of extensions 30 seconds circulate 30 times.

The PCR reaction product by 2% sepharose (Life Technologies, Inc., Gaithersburg, MD), electrophoresis [holy nurse Brooker etc., molecular cloning: laboratory manual, second edition in the liquid in 0.5X TBE is slow, the cold spring port, New York: cold spring harbor laboratory (1989)], observe with ethidium bromide.In 40 plasmid storehouses of screening, (the PCR fragment of expection comprises chemokine gene sequence 211bp to the segmental strong band of PCR of 230 base pairs that are equivalent to expect of 6 generations, and the XbaI and the BamHI restriction site of flank), show to have the one or more plasmids that contain the gene order relevant with pMP390.

For separating above relevant clone, from 6 male plasmid storehouses, select three equal portions electroporations to import in the intestinal bacteria XL-IBlue cell, cell is laid on the flat board, presses the method grow overnight that embodiment 1 describes.The clone transfers on the nitrocellulose filter, prepares hybridization (holy nurse Brooker etc., the same) by following standard step.

The radiolabeled MDC probe of screening film is pressed method preparation: the 2.85kb dna fragmentation that contains MDC cDNA is cut with restriction enzyme digestion from pMP390, in the TAE damping fluid, use agarose gel electrophoresis purifying (holy nurse Brooker etc., ditto) electroelution, phenol and chloroform extracting, ethanol sedimentation.Purifying fragment (250ng) is with random primer dna marker test kit (BMB), by the method mark of manufacturer's recommended.Label probe is crossed G-50 and is revolved (Quick Spin) rotary column (BMB) purifying fast.

Film and count per minute (cpm) are 5 * 10 ⁷Probe, in the solution of 40-50ml, 42 ℃ of incubations 16 hours, solution contains 50% methane amide, 5 * Denhardt ' s solution, and (1 times of SSC is a 0.15M sodium-chlor to 5 * SSC, the 15mM Trisodium Citrate), the 50mM sodium phosphate, the salmon sperm dna (Sigma, St. Louis MO) that pH6.5 and 0.1mg/ml shear.After the hybridization, film 0.2 * SSC in give a baby a bath on the third day after its birth time, in 0.2%SDS, washed 30 minutes for 55 ℃.Be the development results of hybridization, the film of washing kodak (Rochester, NK) XAR-5 is on the radiography sheet, (DuPont, DE)-80 a ℃ exposure is spent the night with Lightening Plus intensifying screen.

PCR is used to screen 50 hybridization bacterial clones.50 PCR reactions that contain 390 1F and 390-2R primer are set up as stated above, and template DNA is used from the bacterium from 50 clones and replaced.During beginning, 94 ℃ of sex change of reaction solution 8 minutes.Then, carry out 35 amplification cycles by last method.A mono-clonal produces the product of 230 base pairs of expection; The plasmid called after pMP390-12 that contains this clone.

Interested other MDC cDNA is with the special probe that pMP390 is inserted segmental 5 ' end, clone hybridization identification.Probe is pressed the method preparation: contain 211 bases of pMP390 cDNA (the 91-298 position Nucleotide of SEQ ID NO:1) coding region and be close to the dna fragmentation of 163 bases of 3 ' non-coding region, produce with PCR as stated above, make template and synthetic oligonucleotide 390-1F (SEQ ID NO:7) and 390-4R (SEQ IDNO:9) with the pMP390 cDNA of 60ng and make primer:

390-4R:5 ' AATGGATCCACAGCACGGAGGTGACCAAG3 ' primer 390-4R contains the BamHI restriction site and is complementary to the sequence of the Nucleotide of SEQ ID NO:1461-442 position.

The PCR product is the electrophoresis purifying as stated above, and 50ng purifying fragment is crossed G-50 fast rotational post (BMB) purifying with random primer dna marker preparation box (BMB) mark.Film is surveyed with this fragment as stated above, in 0.4 * SSC and 0.2%SDS 48 ℃, washes totally three times 30 minutes.Radioautograph is carried out as stated above.5 hybridization clones are detected called after MP390A, MP390B, MP390C, MP390D and MP390E.

These 5 clones and the separated and breeding of clone that transforms with pMP390-12, (Pu Luomaige, Madison WI), are added and are carried out plasmid purification by embodiment 1 described diatomite with Wizard micropreparation dna purification system.Plasmid DNA checks order on the automatic sequencer of applying biological system model 373, uses synthetic primer 390-3R (SEQ ID NO:10):

The 266-246 base complementrity of 390-3R:5 ' AGTCAAGCTTAGGGCACTCTGGGATCGGCAC3 ' primer 390-3R and SEQ ID NO:1 contains Hind III restriction restriction endonuclease point of contact and at 4 additional base pairs of 5 ' end.Designing this primer can anneal with primer 390-2R upstream and SEQ ID NO:1216 position nucleotides downstream, 216 sites have an intron [to see Dai Nuofu etc. in the genomic dna of chemokine of the present invention of encoding, Journal of Immunology, 152:1182-1189 (1994)].

Among six clones, pMP390-12 and pMP390B clone contain the additional 5 ' encoding sequence of maximum, 72 Nucleotide of the extra prolongation in sequence upstream of each cDNA that is obtained before being cloned in clone pMP390.The compound dna sequence dna is at this called after MDC cDNA, by producing after pMP390 and the pMP390-12cDNA combined sequence.The compound cDNA sequence of these 2923 base pairs of chemokine MDC and the aminoacid sequence of inferring are listed in respectively in SEQ ID NOs:1 and 2.

Aminoacid sequence of inferring and known chemokine sequence manually relatively show a kind of novel C-C chemokine of MDCcDNA sequence encoding, and length is the amino acid identity (Fig. 1 and table 1) that 93 amino acid and other C-C chemokine are enjoyed 28-34%

Identity per-cent between the aminoacid sequence of Table I MDC and the former C-C chemokine of identifying
										?MDC	MCP-1	MCP-2	MCP-3	RANTES	MIP-1α	MIP-1β	I-309
MDC		29％	28％	33％	34％	29％	33％	32％
									MCp-1	?29％		62％	72％	34％	38％	34％	33％
MCP-2	?28％	62％		59％	30％	36％	33％	34％
									MCP-3	?33％	72％	59％		34％	35％	35％	37％
RANTES	?34％	34％	30％	34％		50％	44％	22％
									MIP-1α	?29％	38％	36％	35％	50％		55％	35％
MIP-1β	?33％	34％	33％	35％	44％	55％		31％
									I-309	?32％	33％	34％	37％	22％	35％	31％

Importantly, four of tool chemokine feature cysteine residues are guarded in MDC.Five additional residues are also conservative fully in 8 sequences that Fig. 1 shows.

Preceding 24 amino acid of 93 amino acid whose MDC sequences are obviously hydrophobic, and are consistent with the Von.Heijne rule [Nucleotide research, 14:4683-90 (1986)] of decision signal cutting.The polypeptide of these features and Fig. 1 is relatively concentrated and shown: 24 amino acid whose signal Toplink of MDC cDNA coding are cut, and are created in the ripe attitude molecule of MDC that SEQ ID NO:21 position begins with glycine residue.This prediction is described by following examples 10, and the proteic directly order-checking of MDC that produces in the mammalian cell reorganization confirms.MDC compound cDNA sequence shows: on SEQID NO:1 sequence, be predefined for methionine(Met) codon upstream extension 19 Nucleotide and terminator codon downstream extension 2.6kb.

Embodiment 3

What the MDC gene was expressed in people's tissue determines

Carry out rna blot analysis and determine the tissue of MDC genetic expression.

Radiolabeled pMP390 5 ' fragment of mentioning among the embodiment 2 (MDC cDNA section of the ripe attitude of MDC that is equivalent to encode generally acknowledged, add 163 bases of 3 continuous ' non-coding region), contain as probe in detecting and to organize RNA trace (Clontech more from the RNA of different health adult tissues, Palo Alto, CA).Before using probe is boiled sex change, hybridize according to the specification sheets of producer.Carried out radioautograph in 5 days with ℃ exposure of 2 intensifying screens-80.

Observing maximum MDC genetic expression is thymus gland, has detectable than weak expression at spleen and lung tissue.Express even lower from the MDC of small intestine, at brain, colon, heart, kidney, liver, ovary, pancreas, placenta, prostate gland, skeletal muscle, testis or peripheral blood leucocyte do not detect expression.

Embodiment 4

MDC genetic expression in the scavenger cell ripening process

So want the checklist karyocyte to be divided into MDC genetic expression in the scavenger cell process because the cDNAs of coding MDC separates from human macrophage cDNA library.

A.

Cultivate in serial tissue culturing plate from the person monocytic cell of single donor, after 0,2,4 or 6 day from a plate collecting cell.Generally referring to Elst etc., Journal of Immunology, 140:1618-1624; Te Qiaoerke etc., the same.Under these conditions, 4-6 days monocytes are divided into scavenger cell [Stamford Lay Buddhist nun etc., journal of biological chemistry, 265:9682-9687 (1990)].

Isolation of RNA (per pass 10 μ g) from the cell that each time point is collected is prepared the RNA trace, detects with above-mentioned radiolabeled pMP390 fragment.Among the monocytic RNA from fresh separated, do not detect hybridization signal, and cultivate the cell that is divided into scavenger cell after 6 days, produce very strong signal.The cell of cultivating 4 days produces more weak signal, and the signal that the cell of cultivating 2 days produces, and has only film to be extended after the time shutter just as seen.

B.

For further confirming the expression of the MDC in the human macrophage of differentiation, the monoclonal antibody western engram analysis of culture supernatant by the anti-MDC of following examples 18 described generations.The human macrophage of several plates is at macrophage colony stimulating factor (0.5ng/ml, R﹠amp; DSystem, Minneapolis Minnesota) exists down, is grown on the plastics and breaks up 8 days.

Remove the scavenger cell substratum of differentiation, heavily add same substratum or contain low-density lipoprotein (LDL, Sigma), and the LDL of oxidation (press Ma Erdun etc., journal of biological chemistry, the method for 266:13901 (1991), incubation is at 5 μ M CuSO ₄5H ₂Oxidation among the O) or dexamethasone (6nM, sigma chemical company).Each processing is got nutrient solution after carrying out three days, and adding hydrochloric acid accent pH is 6.8, and mistake heparin-dextran CL-6B post (Pharmacia, Piscataway, NJ).With the 20mM Tutofusin tris that contains 0.2M sodium-chlor, pH8 washes post, contains the eluant solution of the 20mM Tutofusin tris pH8 of 0.6M sodium-chlor.The material of wash-out is fractional separation in 18% acrylamide SDS-PAGE glue (NOVEX), and electroblotting is (Millipore, Bedford MA) to pvdf membrane.With standard technique (holy nurse Brooker etc.), film is closed, clean and with the monoclonal antibody reactive of anti-MDC.In the nutrient solution of each analysis, MDC albumen is detected under about 0.5 μ g/ml concentration, confirms the expression of MDC in the human macrophage of differentiation thus.

The expression of MDC is also analyzed in human epithelial cell system.Colonic epithelium T84 clone (ATCC#CCL-248) is cultivated in DMEM/F12 substratum (GIBCO, Gaithersburg MD), and lung epithelial A549 clone (ATCC#CCL-185) is cultivated in the F12 substratum.MDC mRNA that exists in the screening cell and the MDC albumen in the substratum are undertaken by the above-mentioned method that is used for scavenger cell.Arbitrary method does not all detect the expression of MDC in these clones.

In addition, the sample of T84 clone tumor necrosis factor alpha (5ng/ml, PeproTech, Rocky Hill, New Jersey), tumour necrosis factor-β (1ng/ml, R﹠amp; D Systems), or interferon-(200U/ml PeproTech), share separately or with the reorganization MDC (from the CHO transfectional cell, seeing embodiment 10) of 100ng/ml, handles one day.A549 clone sample was handled 0,1,3,5 or 7 day with 50ng/ml PMA (sigma chemical company).When screening, can detect the expression of MDC mRNA to these processing nones of T84 or A549 cell with above-mentioned RNA blotting.

C.

The further detection that the MDC gene is expressed in scavenger cell is carried out with 1%DMSO (sigma chemical company) or 50ng/ml PMA (Sigma) handler clone HL60.Induce HL60 cytodifferentiation myeloblast sample phenotype with the DMSO processing, and PMA induces HL60 to be divided into scavenger cell pedigree [Pa Lusai etc., blood, 58:836-843 (1981)].RNA is from untreated cell or handle with DMSO or PMA the cell of a day or three days and separate electrophoresis (per pass 10 μ g) and trace.The Northern trace of RNA is surveyed 3 described radiolabeled pMP390 5 ' fragments with embodiment.

PMA handled after three days, and the HL60 cell is expressed MDC mRNA significantly, although expression level is significantly than the scavenger cell expression level low (see on) of cultivation after 6 days.Handle after one day or untreated cell is not seen expression.In addition, handled one or three day, do not have detectable MDC to express and derived with DMSO.

Embodiment 5

In situ hybridization

Because MDC genetic expression detects, carry out in situ hybridization to locate the information source of these tissues in thymus gland and spleen.In addition, in situ hybridization is used for determining the MDC genetic expression intestinal tissue inflammation dependency relevant with Crohn disease.

For producing radiolabeled in situ hybridization probe, dna fragmentation (the 91-301 position Nucleotide of the SEQ ID NO:1) subclone that contains the MDC coding region is in pBluescript SK-carrier.Use T3 and T7RNA polymerase (BMB), by the explanation of producer, ³⁵S-UTP is incorporated in the rna transcription basis that is complementary to every chain of gene.

Normal people's spleen, thymus gland and colon's sample, and colon's sample of Crohn disease patient, (Philadelphia PA) obtains from national disease research exchange.Tissue donor is as follows: normal thymus: 19 years old male sex Caucasian, die from motor-driven traffic accident, and get tissue during postmortem; Normal spleen: 51 years old negro male, die from hematencephalon, get tissue during postmortem; Normal colon: black women, get tissue during operation; No. 1, the colon of Crohn disease: the women, the race is not quite clear, and 46 years old, patients of ulcerative colitis was got tissue during operation; No. 2, the colon of Crohn disease: 18 years old male sex, the race is not quite clear, and Crohn disease is got tissue during operation.

Except the analysis of front, the inflammatory tonsilla tissue that cuts when a patient does tonsillectomy uses the non-encoding part of MDC cDNA of the Nucleotide of the 677-1042 position that is equivalent to SEQ ID NO:1 to detect.This part pcr amplification through MDC cDNA clone produces, and used primer is 390-7F (SEQ ID NO:26) and 390-8R (SEQ ID NO:27):

390-7F：5′-TAT?TGG?ATC?GGT?TCT?AGC?TCC?GTG?TTC?TCC?3′

390-8R:5 '-CCA AGA ATT CCT GCA GCC ACT TTC TGG GCT C 3 ' fragment subclone is in pBluescript SK-carrier, to produce above-mentioned rna probe.

The described tissue sample of leading portion is pressed legal system and is done in situ hybridization fully.Tissue sample is embedded in that (Elkhart IN), cuts 6 microns slabs with cryostat (cryostat) 2800E (Leica) for Miles, Inc. in " OCT " compound.Tissue slice stick to Vectabond (the Vector laboratory, Burlingame, CA) on the slide glass of bag quilt, be fixed in 4% Paraformaldehyde 96 4 ℃ 20 minutes, dehydration of alcohol is with 70% methane amide and 70 ℃ of sex change of 2 * SSC.

Hybridization has justice or an antisense strand with radiolabeled proper probes, the slide glass incubation in the hybridization aqueous solution 55 ℃ carried out in 16 hours, the aqueous solution contains 50% methane amide, 0.3M sodium-chlor, 20mM Tutofusin tris pH7.5,10 % T 500,1 * Denhardt ' s solution, 100nM=sulphur threitol and 5mM EDTA.After the hybridization, slide glass room temperature incubation one hour in 4 * SSC and 10mM DTT.Room temperature in 2 * SSC then; 1 * SSC60 ℃; Room temperature is washed slide glass in 0.1 * SSC at last.The sample ethanol dehydration is coated with Kodak NTB2 photographic emulsion then, dry air 2 hours, and 4 ℃ were exposed 11 days, developed, and redyed with phenodin/Yihong.

Observe the hybridization demonstration MDC gene of antisense strand (arbitrary probe) and express in the whole cortex cell of normal human thymocyte, folliculus has weak hybridization signal.MDC expresses in thymus gland and may show that MDC has T lymphocyte growth effect.Expression and localization is in red myelocyte in normal people's spleen, and signal seldom detects in white pulp.As if high expression level is positioned at the dermatotome in the tonsilla of inflammation, although inflammatory cell has soaked into whole tissue sample.

At epithelium, lamina propria shows hybridization in wear spot and the smooth muscle cell from Crohn disease patient's colon sample.On the contrary, normal people's colon does not show hybridization in above-mentioned cell.Observed MDC expresses type and scavenger cell specific gene at Crohn disease patient's colon, and the expression of platelet activation factor acetolysls enzyme (PAF-AH) [your gram of Trajet etc., the same] is closely related.This result, 4 data presented show that scavenger cell is expressed MDC cDNA in vivo in the pathogenicity bo inflammation in conjunction with the embodiments.In addition, identify at the MDC of colon's sample of Crohn disease disease to show: the MDC level (as at patient blood, faecal samples, and/or intestines damaged in) with patient's morbid state or clinical prognosis the diagnosis dependency is arranged.

Embodiment 6

The production of reorganization MDC

For producing reorganization MDC albumen, coding generally acknowledges it is the sequence pcr amplification of the ripe attitude of albumen, and (Pharmacia, Piscataway is NJ) in the carrier to be cloned into pGEX-3X.Design pGEX carrier can produce comprise by the glutathione-S-transferase (GST) of vector encoded and by the dna fragmentation encoded protein in insertion carrier cloning site-kind of fusion rotein.

The Standard PC R condition that embodiment 2 the describes MDC cDNA fragment that is used to once more increase is with primer 390-2R and 390-FX ₂(SEQ ID NO:11):

5′TATCGGATCCTGGTTCCGCGTGGCCCCTACGGCGCCAAC

ATGGAA 3 ' primer 390-FX ₂Containing BamH I restriction site, then is the sequence [normal etc., european journal of biological chemistry, 151:217 (1985)] of coding zymoplasm cleavage site, then is the 92-115 base of SEQ ID NO:1.The zymoplasm cleavage site is as follows: LEU-VAL-proline(Pro)-arginine-glycine-proline(Pro), the first two residue of the ripe attitude of glycine and proline(Pro) MDC wherein.With the arginine-glycine key of Thrombin treatment recombination fusion protein expection cleavage of fusion proteins, from gst fusion protein, discharge sophisticated chemokine.

PCR product agarose gel electrophoresis purifying with the enzymic digestion of Bam HI endonuclease, is cloned on the Bam HI site of pGEX-3X.This pGEX-3X/MDC construct is transformed in the intestinal bacteria XL-1 Blue cell (Stratagene, La Jolla CA), separates each transformant, cultivates.Plasmid DNA from different transformant is purified, and part checks order with automatic sequencer, and used primer GEX5 (SEQ ID NO:12) can be hybridized with the nearly BamH I of pGEX-3X carrier cloning site:

The required MDC fragment that the sequence alleged occurrence that GEX5:5 ' GAAATCCAGCAAGTATATAGCA 3 ' obtains with this primer inserts with proper orientation.

It is that the XL-1Blue bacterium that transforms (interpolation Pyocianil) in the LB substratum is cultivated down at 37 ℃ that the inducing of GST-MDC fusion rotein obtained, it to wavelength 600nM 0.4 proper density, then further descended incubation 4 hours at 0.25-1mM isopropyl ss-D-thiogalactoside (sigma chemical company, St. Louis MO).

The fusion rotein that produces with insoluble inclusion body in bacterium is by the laxative remedy purifying.The cell centrifugation results; Use 0.15M sodium-chlor, the 10mM Tutofusin tris, pH8,1mM EDTA cleans; With 0.1mg/ml N,O-Diacetylmuramidase (sigma chemical company) room temperature treatment 15 minutes.Ultrasonic clarification split product, cell debris 12,000xg formed precipitation in centrifugal 10 minutes.The precipitation that contains fusion rotein is resuspended in the 50mM Tutofusin tris, among pH8 and the 10mM EDTA, spreads on 50% glycerine centrifugal 30 minutes of 6000xg.Precipitation is resuspended in no Mg ⁺⁺And Ca ⁺⁺Standard phosphate buffered saline(PBS) (PBS) in.Fusion rotein still is in insoluble state, is about the 80-90% of protein mass, in sex change SDS-polyacrylamide gel, moves with relative molecular weight 33KD.Press coomassie dyeing and judge protein yield, every liter of culture of Escherichia coli 100mg approximately.

Fusion rotein is digested by zymoplasm, from sophisticated MDC albumen cutting GST.Digestive system (among the 0.5ml PBS, 20-40 μ g fusion rotein, 20-30 unit's zymoplasm (4000U/mg Sigma)) room temperature incubation 16-48 hour is gone up reaction product isolated in the SDS-PAGE glue of sex change.Gel is immersed in manifests GST and MDC protein band in the 0.4M Repone K, respectively with the fragment migration of about 26KD and 7KD.

The segmental characteristic of 7KD SDS-PAGE confirms with the partial amino-acid series analysis.At first, albumen downcuts from glue, and electroelution is in the Tutofusin tris alkali and 20mM glycine of 25mM, and (the applying biological system, the Foster city is CA) on the pvdf membrane in to collect the ProSpin post.Last sample carries out the automatic sequence analysis, and (CA), the result produces the sequence information of 15 residues for the model 473A of applying biological system, Foster city, and this sequence is held (SEQ ID NO:2, amino-acid residue 1-15) corresponding to the expection N of the predetermined ripe attitude of MDC exactly.

Embodiment 7

The structure of bacterium MDC expression vector

The predetermined proteic MDC cDNA of ripe MDC of coding partly is cloned in the plasmid that contains pectinose promotor and pelB leader sequence and [is seen the shellfish top grade, science, 240:1041-43 (1988)].

More particularly, MDC cDNA presses embodiment 2 described PCR method amplifications, makes template and synthetic Oligonucleolide primers 390-2R and 390-Pel (SEQID NO:13) with about 0.1 μ g pMP390-12:

390-Pel：5′ATTGCCATGGCCGGCCCCTACGGCGCCAACAT

GGAA 3 ' primer 390-Pel contains a NcoI restriction site, then is two cytosine(Cyt) residues, is the 92-115 bit base of SEQ ID NO:1 then.

The PCR product agarose gel electrophoresis purifying of 232bp of expection, with NcoI and BamHI digestion and arabinose operon and pelB leader sequence (the shellfish top grade, ditto) be cloned into together the pUC19 carrier (New England's biology laboratory is doubly tieed up dish, MA) in.The construct that obtains, called after P2-390, the fusion of coding pelB leader sequence (by vector encoded) partly is sophisticated MDC albumen.The sequence of this construct confirms that with the automatic sequence assay method with primer Ara1 (SEQ D NO:28) and Ara2 (SEQ ID NO:29), primer can be annealed with carrier cloning site neighbouring part.

Ara1：5′GCG?ACT?CTC?TAC?TGT?TTC?TC?3′

Ara2:5 '-CAC AGG AAA CAG CTA TGA CC 3 ' uses standard operation method, plasmid P2-390 to transform in the into intestinal bacteria MC1061 strain, selects the amicillin resistance clone who produces MDC.The clone cultivate 3 liters of fermentation containers (Applikon, the Foster city, CA) in, derived when the generation of MDC adds 50% pectinose to final concentration 0.1%.At incubation in the presence of the pectinose after one day, harvested cell.The Western trace shows that MDC exists in the every approximately gram wet cell 4 μ g of level in the cell, and the level that is secreted into substratum is about 1 μ g/ml.

Embodiment 8

Purification of Recombinant MDC from bacterium and substratum

The reorganization MDC of following experimental program purifying embodiment 7 described generations.

Excretory reorganization MDC albumen purifying from bacteria culture medium, as can adopt over the RANTES chemokine that the purification of Recombinant described produces [storehouse that etc., Journal of Immunology, 149:636-642 (1992)], MGSA chemokine [He Luke etc., journal of biological chemistry, 268:541-46 (1993)], method with IP-10 chemokine (in expressed in insect cells) [Seles etc., The Journal of Experimental Medicine, 178:1127-1132 (1993)].

Embodiment 9

The recombinant production of MDC in yeast

Below scheme carry out MDC in yeast recombinant expressed and the reorganization MDC purifying.

The coding region of MDC cDNA is from the pMP390-12 pcr amplification, and used primer is the synthetic oligonucleotide that contains the MDC cDNA sequence that has primer 390-1F and 390-2R.The DNA of coding yeast pre-pro-alpha leader sequence increases the PCR reaction solution from pastoris genomic dna, with the 1-20 bit base that contains α conjugative element gene is a primer, another primer is and this gene 255-235 bit base complementary sequence [Ke Jian and Hess Ke Weizi, cell, 30:933-943 (1982)].Pre-pro-alpha pilot code sequence and MDC encoding sequence fragment are connected in the plasmid that contains yeast alcohol dehydrogenase (ADH2) promotor, merge the Expression of Fusion Protein that ripe MDC polypeptide is formed so that promotor instructs by the pre-pro-alpha factor.At Luo Si and Bu Luoqi, Meth Enz.185:234-279, D.Goeddel, ed., scientific publication company, San Diego, the method for CA (1990) are instructed down, and carrier further comprises the ADH2 transcription terminator in cloning site downstream, yeast " Z-particulate " replication orgin, yeast leu-2d gene, yeast REP1 and REP2 gene, intestinal bacteria β-Nei Xiananmei gene and intestinal bacteria replication orgin.Beta lactamase and leu-2d gene are selected respectively to express in cell and yeast.The leu-2d gene also helps increasing the copy number of plasmid in yeast, induces high-caliber expression.REP1 and REP2 genes encoding relate to the albumen that plasmid copy number is regulated.

The described DNA construct of leading portion is handled [Si Disi etc., Meth.Enz. is the same, pp.280-297] with known method such as lithium acetate and is transformed yeast cell.The ADH2 promotor is induced and is depended on glucose exhaustion [Price etc., gene, 55:287 (1987)] in the growth medium.The pre-pro-alpha sequence influences fusion rotein and secretes from cell.Simultaneously, yeast KEX2 albumen cuts down [skin top grade, institute of NAS newspaper, 81:5330-5334 (1984)] to the pre-pro sequence from sophisticated MDC chemokine.

In addition, use business-like expression system, (CA), it is recombinant expressed in yeast to carry out MDC by the explanation of producer for Invitrogen, San Diego as the Pichia expression system.This system also relies on the sequence-directed secretion of pre-pro-alpha, is driven but insert segmental alcohol oxidase enzyme (AOX1) promotor of transcribing by methanol induction.

Excretory MDC is purifying from the yeast growth substratum, as uses from the method (seeing embodiment 8 and 10) of bacterium and mammalian cell supernatant purifying MDC.

Embodiment 10

MDC is recombinant production in mammalian cell

It is recombinant expressed in mammalian cell to follow these steps to MDC.

A. expression vector 390HXE's is synthetic

It is synthetic that embodiment 2 described PCR methods pressed in the brachymemma translation of MDC cDNA, makes template and synthetic oligonucleotide 390RcH and 390RcX with pMP390-12 and make primer.

(SEQ?ID?NO：14)

390RcH：5′GACCAAGCTTGAGACATACAGGACAGAGCA

(SEQ?ID?NO：15)

390 RcX:5 ' TGGATCTAGAAGTTGGCACAGGCTTCTGG primer 390RcH contains Hind III restriction site, then is the 1-20 bit base of SEQ ID NO:1; Primer 390 RcX contain the XbaI restriction site, then are the 403-385 bit base complementary sequences with SEQ ID NO:1.

The PCR product agarose gel electrophoresis purifying of 423bp of expection is cloned in the pRc/CMV carrier of HindIII/XbaI digestion ((In Vitrogen, San Diego CA) allow in mammalian cell directly the carrier of expression).The plasmid that obtains, called after 390HXE contains the 1-403 bit base of SEQID NO:1.The sequence of inserting confirms that with the automatic sequencing method used primer is DC03 (SEQ ID NO:16) and JHSP6.

DC03:5 ' CGA AAT TAA TAC GAC TCA CT 3 ' primer DC03 can anneal with the sequence of the contiguous cloning site of pRc/CMV carrier.

B. expression vector 390HmX's is synthetic

Another MDC cDNA construct produces with PCR, makes template with pMP390-12, and primer is 390RcH and 390mycRX (SEQ ID NO:17).

390mycRX:5 ' TGGATCTAGATCAATTCAAGTCCTCCTCGCTGATCAGCTTCTGCTCTTGGCTCAGC TTATTGAGAAT 3 ' primer 390mycRX contains the XbaI restriction site, be complementary to coding " myc " epitope sequences [Fu Erkesi etc., biotechnology, 13:422-427 (1992)] sequence and be complementary to the sequence of the 298-278 bit base of SEQID NO:1.This reaction amplifies the 354bp fragment of expection, and this fragment contains the 1-298 bit base of SEQ ID NO:1 and merges " myc " epi-position at the MDC carboxyl terminal.This epi-position can be used for conveniently doing immunoprecipitation, and affinity purification and western blotting detect the MDC-myc fusion rotein.Fragment cloning to pRc/CMV to produce the 390HmX plasmid.The sequence primer DC03 that inserts, the automatic sequencing method confirms.

C.MDC expresses in 293T and NS0 cell

Two kinds of transfection schemes are used to express two MDC cDNA constructs of above-mentioned A. and the inferior part of B.: transient transfection is to human embryo kidney (HEK) 293T clone and be stably transfected into rat bone marrow tumour NS0 clone (ECACC85110503).

The transient transfection of 293T cell is with old and Ou Keyama, biotechnology, and 6:632-638 (1988) and molecular cytobiology, the calcium phosphate precipitation method of 87:2745-2752 (1987) is carried out.Cell and supernatant be collection in four days after transfection.The preparation of Northern trace is the total RNA of 4 μ g from each cell lysate, detects with the radiolabeled MDC fragment of PCR preparation.The template of labeled reactant is the aforesaid PCR fragment that produces with primer 390-1F and 390-4R (seeing embodiment 2) amplification pMP390.This fragment of about 30ng is used for the PCR reaction, and reaction solution contains as follows: 1.5mM magnesium chloride, 50mM Repone K, 10mM Tutofusin tris, pH8.4,0.2mM deoxyadenylic acid triphosphoric acid, 0.2mM deoxythymidine triphosphate, 0.2mM deoxyguanosine triphosphate, 1 μ M deoxycytidine triphosphate, 50 μ Ci α ³²P-deoxycytidine triphosphate (DuPont/New England Nuclear, Boston MA), 2.5U Taq polysaccharase and 10 μ g/ml each primer 390-1F and 390-2R.Reaction solution is then pressed 15 circulations of embodiment 2 described methods amplifications 94 ℃ of heat denatured 4 minutes.Probe is crossed G-25 fast rotational post (BMB) purifying.Hybridization conditions is stated in embodiment 2.Film then in 0.5 * SSC, wash with 42 ℃ of 2%SDS in washed 30 minutes.Radioautograph was carried out 16 hours with an intensifying screen at-80 ℃.MDC DNA construct high expression level in transfectional cell detects not come out in non-transfected cell.

Be the stable transfection body, the NS0 cell grows into 80% and converges density in D-MEM (Gibco), and centrifugal collection is cleaned with PBS.20 μ g plasmid DNA are cut into linearity with ScaI restriction enzyme (BMB), be added on the cell, 0.4cm gap (gap) cuvette (BioRad, HerculesCA) in incubation 15 minutes on ice.Cell electroporation is with the second pulse of 1.5 kilovolt of 3 microfarad.Cell dilution in 20ml D-MEM, 5%CO ₂Cultivated 24 hours in 37 ℃, plant in 96 orifice plates with the different extent of dilution of D-MEM that contains 800 μ g/mlgeneticin and screen.The hole enlarged culturing in selective medium that contains single anti-medicine clone.Total RNA presses the described Northem blotting of leading portion and analyzes.The information of MDC only in transfectional cell series as seen.

MDC is purifying from the mammalian cell culture supernatant, as adopting purification of Recombinant TCA3 chemokine [Weir is inferior etc., Journal of Immunology, 145:2745-2750 (1990)], or the described method of the inferior part of following F.

D.MDC expresses in Chinese hamster ovary celI

With primer 390 RcH and 390RcX (SEQ ID NOs:14 and 15), by the described method of the inferior part of above-mentioned A, pcr amplification MDC cDNA clone's 1-403 bit base.Fragment cloning is between the HindIII and XbaI site of expression vector pDC1, and pDC1 is the redundant organism of pUC19, contains the promotor that drives the cytomegalovirus (CMV) that inserts fragment expression.More particularly, the pDC1 carrier is shown among Fig. 8, derived from pRc/CMV and pSV2-dhfr (ATCC carrier #37146).Carrier pDC1 and lactation expression vector pRc/CMV (Invitrogen, San Diego) are similar, and except at the neomycin phosphotransferase gene position, it is selection marker that pDC1 carries mouse dihydrofolate dehydrogenase (dhfr) gene.Hit gene transcription under the control of CMV strong promoter at pDC1.See Stern Burger etc., Journal of Virology, 49:190-199 (1984).In addition, the polyadenous glycosidation sequence from bovine growth hormone gene [Ginnifer Goodwin and rood are graceful, journal of biological chemistry, 267:16330-16334 (1992)] is connected on 3 of target gene ' end.The cell that dhfr expression cassette [saab Raman Buddhist nun etc., molecular cytobiology, 1:854-864 (1981)] allows to lack the dhfr gene that function is arranged is selected pDC1.

Use CaCl ₂The standard technique of incubation and heat-shocked (holy nurse Brooker etc.), XL-1 cyanobacteria (Stratagene) transforms with pDC1/MDC.Transformant is grown in the LB substratum that contains 100 μ g/ml Pyocianils.From the plasmid DNA that independent transformed clone comes, (Madison WI) is separated, and its sequence confirms with primer 390-IF and 390-2R automatic sequencing with Pu Luomaige (promega) Wizard Maxiprep system.Plasmid is linear through restrictive diges-tion with nuclease (BoehringerMannheim) in the PvuI, and this enzyme only cuts once in the carrier sequence.

Chinese hamster ovary (CHO) clone that is used for MDC production is DG-44, and disappearance dhfr gene produces.See E Luobai etc., cell, 33:405 (1983).During electroporation, 10 ⁷These Chinese hamster ovary celIs clean with PBS, be resuspended among the 1ml PBS and plasmid that 25 μ g are linearized mixes, transfer in the 0.4cm cuvette.Suspension Biorad Gene Pulser (Richmond, CA) at 290 volts, electroporation under 960 microfarads.Transformant is grown in and contains 10% dialysis foetal calf serum (FBS) (UT), (Gaithersburg selects in MD) for Cat.NO.12000, Gibco to lack the α-substratum of xanthoglobulin and thymus pyrimidine for Hyclone, Logan.From the cytomixis of a hundreds of transformed clone, containing 20nM methotrexate (Sigma, St. Louis, stock and inoculating in α-substratum MO) again.Be separated in this and take turns the clone who survives in the selection, enlarged culturing in the α-substratum that contains the 20nM methotrexate.

E. the purifying of the MDC that uses as protein sequencing

The CHO of transfection is cloned in α-substratum, is grown on the plastics tissue culture ware to converge density, the substratum P5 substratum replacing that contains 0.2%-1.0%FBS at that time to about 90%.The P5 substratum is made up of the listed component of following table 2 (with the powder that is pre-mixed available from Hyclone, Logan UT), adds following extra component:

(1) the 3g/l sodium bicarbonate (Sigma, the St. Louis, MO);

(2) 2 μ g/l Sodium Selenites (Sigma);

(3) 1% soybean hydrolyzates (Quest Intemational, Naarden, TheNetherlands);

(4) 1 * ferrous sulfate/EDTA solution (Sigma);

(5) 1.45ml/l EX-CYTEVLE solution (Bayer, Kankakee, IL);

(6) 10 μ g/ml Recombulins (Nucellin, EliLily, Indianapolis, IN);

(7) 0.1% Pluronic F-68 (Sigma);

(8) 30 μ g/ml glycine (Sigma);

(9) 50 μ M thanomins (Sigma);

(10) 1mM Sodium.alpha.-ketopropionate (Sigma);

Table 2
				Component	Powder #5gm/L
Inorganic salt	Sodium-chlor	4.0
			Repone K	0.4
	Sodium phosphate dibasic, anhydrous	0.07102
			Sodium dihydrogen phosphate-water	0.0625
	Sal epsom, anhydrous	0.1
			Cupric sulfate pentahydrate	0.00000125
	Iron vitriol	0.000417
			Zinc Sulphate Heptahydrate	0.0004315
	Nine water iron nitrates	0.00005
			Calcium chloride, anhydrous	0.11661
	Magnesium chloride, anhydrous	0
			Amino acid	The L-L-Ala	0
The L-arginine hydrochloride	0.15
		One water altheine		0.075
The L-aspartic acid	0.04
		One water L-cysteine hydrochloride		0.035
Two hydrochloric acid L-Gelucystines	0.12
		L-L-glutamic acid		0.02
L-glutaminate	0.5846
		Glycine		0.02
One water hydrochloric acid L-Histidine	0.04
		The L-Isoleucine		0.15
The L-leucine	0.15
		The L-lysine hydrochloride		0.1

	The L-methionine(Met)	0.05
			The L-proline(Pro)	0.05
	The L-phenylalanine	0.05
			The L-Serine	0.075
	The L-Threonine	0.075
			The L-tryptophane	0.02
	Two water L-tyrosine disodiums	0.075
			The L-Xie Ansuan	0.125
	VITAMIN	Vitamin H			0.001
The D-calcium pantothenate			0.0025
		Choline chloride 60		0.015
Folic acid			0.005
		The i-inositol		0.175
Nicotinamide			0.005
		Pyridoxal hydrochloride		0.005
Pyridoxine hydrochloride			0.005
		Riboflavin		0.001
Hydrochloric acid sulphur ammonium			0.005
		Cyanogen cobalt ammonium		0.001
Other			D-glucose		1.0
	Xanthoglobulin sodium	0.005
			Thymus pyrimidine	0.005
	Two hydrochloric acid butanediamine	0.000081
			Sodium.alpha.-ketopropionate	0.11004
	Linolic acid	0.0001
			The DL-alpha-lipoic acid	0.0002
	Phenol red, sodium salt	0.0086022

After cultivating extra two days, branch fraction and the mixing of isopyknic acetone such as each supernatant.Sedimentary albumen centrifugation, fractional separation in 18% Tutofusin tris glycine glue (NOVEX), trace to pvdf membrane (Millipore, Bedford, MA).

The MDC that is combined on the film detects with the monoclonal antibody (by embodiment 18 described preparations) of the rough anti-MDC that is equipped with.The clone cell of secreting high levels MDC albumen (about 1 μ g/ml) with the solution-treated of 0.5% trypsinase and 5.3mM EDTA (GIBCO), separates on the slave plate, and the α-substratum that is used for adding 10% foetal calf serum (FBS) carries out suspension culture.Spend 8 day time, the P5 substratum that adds 5 times of volumes is in culture.Add polyoxyethylene glycol (MW8000, UnionCarbide, Danbury is CY) to cultivating serum to 20% (weight/volume), albumen precipitates from cultivate serum, in 18% Tutofusin tris glycine glue, separate, the CAPS damping fluid (the 3-[cyclohexylamino]-1-propanesulfonic acid, pH10.4) (Sigma, the St. Louis, MO) in electricity leave on the pvdf membrane (Millipore, Bedford, MA).Downcut the band on the film, with the supernatant of the hybridoma cell line that produces anti-MDC monoclonal antibody (seeing embodiment 18), Western blotting detects MDC.Downcut with the reaction zone of apparent molecular weight 6.4KD migration nubbin from film.

With automatic sequencer (the applying biological system, model 473A, the Foster city, CA), the sequence of measuring albumen n end is: GPYGANMEDS.This sequence is identical with the 1-10 position residue of SEQ ID NO:2, and is consistent with the N end of the predetermined ripe attitude of MDC.

F. make biology and measure the purifying of the MDC of usefulness

Make fairly large incubation growth, the Chinese hamster ovary celI of expressing MDC is grown in the tissue culturing plate to 80% and converges density in α-substratum, handles with trypsinase and EDTA, separates on the cell slave plate, in adding the P5 substratum of 1%FBS, with 3 * 10 ⁵The density of cell/ml is resuspended in the screw socket culturing bottle for 37 ℃.Add the P5/1%FBS substratum when needing, keep cell density 1 * 10 ⁶～3 * 10 ⁶Scope.

After cultivating in 11 days, cell separates from substratum by filtering.Transfer the pH to 6.8 of substratum, substratum cross heparin-dextran CL-6B post (Pharmacia, Piscataway, NJ).With the potassium phosphate buffer of 0.2M sodium-chlor, pH7, after the cleaning, the post linear gradient eluant solution of 0.2-0.7M sodium-chlor.Isolating fraction is analyzed with SDS-PAGE, and coomassie dyeing determines which partly contains MDC.In 0.6M sodium-chlor, MDC wash-out from the post.

Mix the fraction that contains MDC, (MA) ultra-filtration concentrates for Amicon, Beverly in stirring chamber (Stirred-cell Chamber) with the filter membrane that can filter (cutoff) molecular weight 3KD.Add octyl glucoside (final concentration 10mM, Boehringer Mannheim Biochemicals) in spissated MDC, (NJ) fraction is analyzed with SDS-PAGE for Pharmacia, Piscataway, to determine existing of MDC to follow Sephacryl HR100 post.The proteic whole output of MDC is about every liter of culture supernatant 0.1mg, presses coomassie dyeing and judges estimation, and its purity is more than 95%.

Embodiment 11

Method of peptide synthesis is produced MDC and MDC analogue

MDC and MDC polypeptide analog are with the preparation of chemistry of peptides synthesis method, and used technology successfully is used for other chemokines such as IL-8[Croker-Li Weisi etc., journal of biological chemistry, 266:23128-34 (1991)] and the production of MCP-1.It is because the albumen of the such short sequence of object chemokine that these methods have advantage, and they are quick, and are reliable, can optionally import new, non-natural amino acid and other chemical modification object.

For example, the chemosynthesis of MDC and MDC analogue, progressively solid phase method [the Shu Nuoqi etc. of available optimization, Int.J.Pept.Protein Res., 40:180 (1992)] according to Merifield [J.Am.Chem.Soc., 85:2149-2154 (1963)] tertbutyloxycarbonyl chemistry principle, (carry out on CA) in the Foster city at the 430A of applying biological system peptide synthesizer.With anti-phase HPLC purifying protein, the feature of its standard method comprises electrospray mass spectroscopy and nucleus magnetic resonance.

The MDC of chemosynthesis is consistent with the ripe attitude of reorganization MDC, is made up of the 1-69 position residue of SEQ ID NO:2.The MDC that several method is used for chemosynthesis compares with the reorganization MDC that embodiment 10 described CHO transfectional cells produce.Sex change SDS-PAGE (18% Tutofusin tris glycine gels, NOVEX) in, the mobility of the MDC of chemosynthesis with the reorganization MDC identical.In addition, in western blot analysis, mono-clonal and the polyclonal antibody of the MDC that produces with following embodiment 18 described antibacteriums, these albumen tests are similar.In the monoclonal antibody immunity sedimentation analysis with anti-MDC, the MDC of chemosynthesis also shows in the identical mode of reorganization MDC.These studies show that the sex change of chemosynthesis MDC and these structural similitudies of non-sex change structure and reorganization MDC.

Following also chemosynthesis of MDC analogue:

1) " MDC (n+1) " (SEQ ID NO:30) then is that the 1-69 position residue of SEQ IDNO:2 is formed by leucine.

2. " MDC (9-69) " is made up of the 9-69 position residue of SEQ ID NO:2.

3. " MDC-yl " (SEQ ID NO:31) is made up of the 1-69 position residue of SEQ ID NO:2, and following replacement is wherein arranged: 59-60 position residue (Trp-Val) replaces with the Tyr-Leu sequence.

4. " MDC-eyfy " (SEQ ID NO:32) is made up of the 1-69 position residue of SEQ ID NO:2, following replacement is wherein arranged: 28-31 position residue (His-Phe-Tyr-Trp) replaces with the Glu-Tyr-Phe-Tyr sequence, and this sequence is from the aminoacid sequence (26-29 of SEQ ID NO:21 is positioned at residue) of chemokine RANTES.

Expection analogue " MDC (n+1) ", " MDC (9-69) " and " MDC-yl " are the active antagonists of MDC, by suppressing the MDC activity in conjunction with the same receptor of discerning MDC competitively.In addition may be analogue can and natural MDC form heterodimer generation restraining effect.The possible activity of " MDC-eyfy " analogue comprises the MDC restraining effect of aforementioned analogue.On the contrary, " MDC-eyfy " shows as the similar characteristic of natural MDC.Other activity of this analogue may comprise the exemplary functions of chemokine RANTES, as lymphocyte, and the chemotaxis of monocyte or eosinophil.

In addition, can design following monamino acid varient (separately or in conjunction with) especially: (1) non-basic aminoacids is replaced SEQ ID NO:224 and 27 s' alkaline arginine and/or Methionin respectively; (2) charged or polare Aminosaeren (as Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine or halfcystine) is replaced the tyrosine of SEQ ID NO:230 position, replaces the tryptophane of SEQ ID NO:259 position and/or the Xie Ansuan of SEQ ID NO:260 position; (3) alkalescence or uncharged p1 amino acid (as Methionin, arginine, Histidine, glycine, L-Ala) are replaced the L-glutamic acid of SEQ ID NO:250 position.Special analogue with these amino acid changes is included in (SEQ ID NO:25) in the following table:

Met?Ala?Arg?Leu?Gln?Thr?Ala?Leu?Leu?Val?Val?Leu?Val?Leu?Leu?Ala

-24?????????????-20?????????????????-15?????????????????-10

Val?Ala?Leu?Gln?Ala?Thr?Glu?Ala?Gly?Pro?TYr?Gly?Ala?Asn?Met?Glu

-5???????????????????1???????????????5

Asp?Ser?Val?Cys?Cys?Arg?Asp?Tyr?Val?Arg?Tyr?Arg?Leu?Pro?Leu?Xaa

10??????????????????15??????????????????20

Val?Val?Xaa?His?Phe?Xaa?Trp?Thr?Ser?Asp?Ser?Cys?Pro?Arg?Pro?Gly

25??????????????????30??????????????????35??????????????????40

Val?Val?Leu?Leu?Thr?Phe?Arg?Asp?Lys?Xaa?Ile?Cys?Ala?Asp?Pro?Arg

45??????????????????50??????????????????55

Val?Pro?Xaa?Xaa?Lys?Met?Ile?Leu?Asn?Lys?Leu?Ser?Gln

60 65 wherein 24 amino acid can be selected from arginine, glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), Serine, Threonine, phenylalanine, tyrosine, tryptophane, aspartic acid, L-glutamic acid, l-asparagine, glutamine, halfcystine and methionine(Met); Wherein 27 amino acid can be independently selected from Methionin, glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), Serine, Threonine, phenylalanine, tyrosine, tryptophane, aspartic acid, L-glutamic acid, l-asparagine, glutamine, halfcystine and methionine(Met); Wherein 30 amino acid can be independently selected from tyrosine, Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine and halfcystine; Wherein 50 amino acid can be independently selected from L-glutamic acid, Methionin, arginine, Histidine, glycine and L-Ala; Wherein 59 amino acid can be selected from tryptophane, Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine and halfcystine by oneself; Wherein 60 amino acid can be independently selected from Xie Ansuan, Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine and halfcystine.These MDC polypeptide analogs can design especially regulates the binding characteristic of MDC to Chemokine Receptors and/or other molecule (as heparin, glycosaminoglycan, red corpuscle Chemokine Receptors), these characteristics MDC be think when passing its acceptor important.

Also can design do preparation MDC polypeptide analog as the described recombinant technology of front embodiment.More particularly, with the polynucleotide of the technology of knowing such as rite-directed mutagenesis and polymerase chain reaction modification coding MDC, with the interested polypeptide analog of encoding.Generally referring to holy nurse Brooker etc., the same, the 15th chapter.The polynucleotide of modified are recombinant expressed, by the MDC polypeptide analog of the described method purification of Recombinant of front embodiment.

MDC or MDC polypeptide analog are planted the chemical attractants of cell types (as T lymphocyte, monocyte, scavenger cell, basophilic cell, eosinophil, neutrophilic granulocyte, mastocyte, endotheliocyte, epithelial cell, inoblast or other cells) and/or the characteristic of active cells to one or more that relate to inflammatory process, by many other technical Analysis of chemokine of being used for of confirming.In front described one or more kind method purifying of embodiment and isolating natural MDC, the reorganization MDC or MDC polypeptide analog or synthetic MDC or synthetic MDC polypeptide analog, analyze its activity by following described embodiment about MDC.

Embodiment 12

MDC to basophilic cell, mastocyte and

The mensuration of eosinophil effect

Analyze the influence of MDC, as using weber etc., Journal of Immunology, the active method of 154:4166-4172 (1995) described mensuration MCP-1/2/3 to basophilic cell, mastocyte and eosinophil.Use these methods, measure the variation of cell cytosol free ca and the release of preceding inflammatory (proinflammatory) medium (as histamine and leukotriene).The activation of blocking these chemokine mediated kind cells relates to tardy allergic processing, in tardy transformation reactions, and the secretion of pre-inflammatory mediator play an important role [weber etc., the same].

Embodiment 13

MDC is to person monocytic cell/scavenger cell and human neutrophil

Chemical attractants and the analysis of cell activation characteristic

MDC is to person monocytic cell/scavenger cell and human neutrophil impact assessment, as with Dai Wei etc., and Journal of Immunology, the evaluation mouse TCA3 inductive neutrophil leucocyte that 153:5376-5383 (1995) describes and the method for macrophage activation.The activation index of measuring in above-mentioned research comprises because of integrating plain activation to be increased fibrinogenic adhesion, chemotaxis, induced activity nitrogen intermediate, respiratory burst (generation of super-oxide and hydrogen peroxide) and the exocytosis of N,O-Diacetylmuramidase and elastoser in the presence of cytochalasin B.As discussion such as Dai Wei, these are active relevant to the different steps of inflammatory response with white corpuscle.Leukocytic this replying pressed the Spring outstanding person, cell, and the summary of 76:301-314 (1994) relates to the adhesion of white corpuscle to vascular endothelial cell, by the migration of endodermis, to the chemotaxis in chemokine source and the release of inflammatory mediator specific site.Relating to MDC in any of these stages can be clinical intervention, regulates the important target spot of inflammatory response.

With the chemotactic response assay method of a recognized technology, the Boyden chamber method of be revising, tested white corpuscle at room temperature with 20 minutes marks of Calcein incubation on fluorescence.Labeled cell is washed secondary with the RPMI of serum-free, is resuspended among the RPMI of the BSA that contains 2mg/ml, quantitatively is added to then in the last hole of chamber, and (Neuroprobe Inc.Cabin John MD) separates by polycarbonate membrane with following hole in last hole.MDC with the substratum dilution identical with white corpuscle is added to down in the hole with different concns.The chamber was 37 ℃ of following incubations 2 hours.The end part of method is not move the cell that passes film, removes after film and rubber squeegee (policeman) are scraped washing with PBS.The cell that moved film the fluorescent plate reader (Cytofluor, Millipore Inc, Boston, the fluorescent value that reads every hole in MA) is quantitative.

Use the method for recognized technology, carry out a series of experiments to determine the chemotactic characteristic of MDC.During beginning, measure the reaction of person monocytic cell to MCD.MDC has also done detection to the influence of polymorphonuclear leukocyte (granulocyte) chemotactic response.

Verified: C-C chemokine MCP-1 causes monocyte recruitement and activation, but induces the ability of macrophage migration to seem limited.As if MCP-1 can not attract scavenger cell relevant with atomization: when the monocyte differentiation phase, reduce reply [Dai Heermu and Si Tankesi, cytokine, the 7:436-440 (1995)] of cell to MCP-1 gradually.As if the biologic activity of MCP-1 is relevant with the expression of chemokine, and the MCP-1mRNA that exists in monocyte reduces when these cytodifferentiation.

As if the expression type of MCD is opposite with the situation of described MCP-1, and when monocyte was divided into scavenger cell, the quantity of MDC mRNA increased.For determining whether that this expression type is relevant with biological answer-reply to MDC, has compared the influence of MDC to monocyte and macrophage migration.

In chemotaxis and the chemotactic mensuration of inhibition, many dissimilar white corpuscles have been analyzed.With methods known in the art [Dai Heermu etc., American Journal of Pathology, 135:571-580 (1989)], people's monokaryon and polymorphonuclear leukocyte from peripheral blood, have been separated.Secondly, used the human monocyte cell line, THP-1 (from ATCC Rockville, MD) obtain, in the RPMI that contains 10%FBS and penicillin/streptomycin, keep cultivation).THP-1 can cultivate with monocytic form, or can induce with acetate Semen Myristicae phorbol (PMA) processing and to be divided into scavenger cell [Dai Heermu and Si Tankesi, cytokine, 7:436-440 (1995)].Some experiments are divided into scavenger cell with the THP-1 cell of monokaryon, other experiment monokaryon THP-1 cells and acetate Semen Myristicae phorbol (PMA) incubation.The 3rd, the cavy peritoneal macrophages is substantially by knowing Mu La especially, Journal of Immunology, and the method for 150:5025-5032 (1993) obtains.In brief, the first two sky of harvested cell (DIFCO) uses phosphate buffered saline buffer (PBS) lavation that contains 1mM EDTA and 0.1% glucose for animal abdominal injection 3% aseptic thiol-acetic acid (thioglycollate), obtains scavenger cell from the abdominal cavity.Cell centrifugation is washed once, is used for the chemotactic assay of the following stated then.

Chemotactic activity is measured, cell with above-mentioned preparation, basically press Dai Heermu and Si Tankesi, Cytometry, the described method of 19:366-369 (1995) is with 96-pore chamber (NeuroprobeInc.Cabin John, MD) and the cell fluorescence dye, (molecular probe, Eugene OR) carry out Calcein.Used polycarbonate membrane is (the Neuroprobe Inc.) of no PVP during mensuration; The fenestra size that is used for dissimilar cells is: monocyte and THP-1 cell are 5 μ m, and polymorphonuclear leukocyte is 3 μ m, and the cavy scavenger cell is 8 μ m.

The cell of 50,000 Calcein signs is resuspended in the RPMI substratum that contains 2mg/ml BSA, puts into the hole.MDC or other test substances with the RPMI dilution that contains BSA (as, MDC final concentration be 25,50,100,250ng/ml), put into down in the hole.Then 37 ℃ of incubations are 2 hours, wipe the still not migrating cell on film, allow the film dry air then.The contrast of these mensuration is: contain the negative contrast of RPMI of BSA, (ZAS is by [Dai Heermu and Li Weisi, American Journal of Pathology for 50ng/ml MCP-1 and 1% zymosan activation serum, 126:464-474, (1987)] described method preparation) as positive control.(Cytofluor, Millipore Inc.Bedford MA) go up quantitatively the mobility of cell, and the cell count of migration is represented with flat fluorescent at the fluorescent plate reader.

Suppress in the determination of activity, at the cell suspension in hole on the chamber in the MDC of different concns (0.005,0.05,0.5,5.0 and 5ng/ml).The following hole of chamber is only filled with to substratum or substratum add chemokine, MCP-1 or zymosan activation serum (ZAS).Move to the cell count and the cell count that increases the migration of MDC concentration of MCP-1 or ZAS when relatively lacking MDC, estimate inhibiting rate.The quantitative strictness of the preparation of cell and mensuration is by above-mentioned chemotactic measuring method operation.The cell count of migration is represented with flat fluorescent.

As shown in Figure 2, MDC can not induce the monocyte migration from THP-1, but suppresses monocytic migration in 10～100ng/ml concentration more significantly.Other C-C chemokine as MCP-1 and RANTES, is typically induced maximum monocyte chemotaxis in this concentration range.

As shown in Figure 3, concentration is that the MDC of 0.001～100ng/ml is to the unclean influence of granulocyte mobility.Except lacking the influence of granulocyte chemotaxis, MDC is similar to other aforesaid C-C chemokine.

Scavenger cell and monokaryon THP-1 cell are represented replying with Fig. 4 of MDC.Scavenger cell (solid circles) moves to MDC in dose-dependent mode, and optimal activity is 50ng/ml.Scavenger cell has reflected the desensitization of cell to the reduction of the MDC chemotactic response of higher concentration (100ng/ml), and this is the features [Fu Erke and row Nader, Infect Inmunol.32:464-468 (1981)] of most of chemokines when high density.But, monokaryon THP-1 cell (empty circles) does not move to MDC.

MDC further uses the experiment confirm of activated cavy peritoneal macrophage to the chemotactic activity of scavenger cell.Concentration is that the MDC of 100～500ng/ml induces the cavy scavenger cell with dose-dependently migration (Fig. 5).Induce the required concentration of cavy macrophage migration to be about to work 10 times of concentration of people's cell (Fig. 4).To the required concentration of high biological activity of other species human chemokine, MCP-1 has similar difference, and this result is by knowing Mu La especially, Journal of Immunology, 150:5025-5032 (1993) report.

These experimental results show: the biologic activity of MDC and monocyte are related to the differentiation of scavenger cell.Compare [knowing Mu La especially, Journal of Immunology, 150:5025-5032 (1993)] with MCP-1, MDC induces scavenger cell rather than monocytic chemotaxis.

MDC attracts the ability of scavenger cell to show: this chemokine may work in the focus accumulation of induced tissue scavenger cell.Tissue macrophages is important in the granuloma that is accumulated in of Special Areas in forming, pulmonary macrophage surrounded [Adam Si of working with wrapping up foreign matter or relative nondestructive bacteria pathogeny such as mycobacterium when granuloma formed, American Journal of Pathology, 84:164-191 (1976)].

As sacroiliitis, the accumulation of having understood scavenger cell is deleterious, has destructiveness in some cases.MDC can promote the scavenger cell chemotaxis to show that MDC inhibitor of the present invention has treatment effectiveness, accumulates in tissue with prevention, reduction or elimination scavenger cell.

The monocytic chemotactic assay result of the personnel selection that Fig. 2 represents hints that MDC can suppress cell migration.When (MDC is 0ng/ml) lacked MDC as shown in Figure 6, monokaryon THP-1 cell was to the migration of MCP-1.But, when cell and MDC exist, the chemotactic response (solid circles) of MCP-1 is reduced.Concentration is that the MDC of 0.005-0.5ng/ml suppresses the chemotactic response of monocyte to MCP-1.Although MDC suppresses the chemotactic response of monocyte to MCP-1, when existing or lack MDC, from seeing to the cell count that only is substratum (empty circles, RPMI contains BSA) migration, MDC does not have significantly influence to chemotactic kinetics or random migration.

In the treatment effectiveness of handling several chronic inflammatory morbid state (atherosclerosis, sacroiliitis, pulmonary fibrosis), in these morbid state, monocytic chemotaxis has seemed important etiologic agent to MDC to the active MDC of demonstration of the inhibition of monocyte chemotaxis.Strengthen the activity of MDC in above disease and can cause reducing the transport property of monocyte, thereby reduce the severity of disease symptoms tissue.

Embodiment 14

The inhibiting mensuration of MDC tumor growth in vivo

MDC suppresses the analysis of tumor growth characteristic, as revising from Lan Ning etc., and Journal of Immunology, the method for the inhibition tumor characteristic of the described analysis of 153:4625-4635 (1995) mouse TCA3.The coding MDC the transfection of cDNA electroporation come in from the J558 of myelomatosis clone (American type culture collection, Rockville, MD).Transfection body and function standard technique such as ELISA (enzyme-linked immunosorbent assay) that screening MDC produces, the monoclonal antibody of the anti-MDC that employing embodiment 18 carefully states.Agglomerate from 10,000,000 cells that produce the MDC clone is subcutaneously injected in the bottom right tetrad of BALB/c mouse.In order to compare, 10,000,000 non-transfected cells are expelled in the contrast mouse.Relatively the speed of two groups of tumour formation and incidence determine that MDC suppresses the effect of tumor growth.The characteristics of relevant with tumour cell subsequently cellular infiltration thing are recognized with Histological method.In addition, reorganization MDC (20ng) and non-conversions J558 cytomixis, injection (20ng/ days) is to forming in the tumour of above cell, with analysis to the MDC of external source influence to tumour cell.

Embodiment 15

Peritoneal injection is measured

Press Lip river etc., Journal of Immunology, the described method of 153:4616-4624 (1994) arrives in the peritonaeum inner chamber of mouse or other animal (as rabbit or cavy) by the MDC that injects the 1-1000ng purifying, determines in the body MDC to be worked the cell of replying.After the injection, white corpuscle separates from peripheral blood and peritoneal cavity, with Diff Quick test kit (Baxer, McGraw, IL) dyeing identification.Leukocytic overall picture is to estimate the kinetic determination that different cell types occur at different time.In independent experiment, neutralizing antibody and the MDC of anti-MDC (embodiment 18) inject together, are because due to the activity of MDC to confirm leukocytic infiltration.

Embodiment 16

Activity in vivo mensuration-subcutaneous injection

Measure in the body of MDC chemoattractant characteristic, adopt Mei La etc., The Journal of Experimental Medicine, the described scheme of 178:1913-1921 (1993).Reorganization MDC (10-500pmol/ point) is expelled to the subcutaneous of suitable Mammals such as dog or rabbit.At 4-24 hour, rules of organization were estimated the cellular infiltration of injection point.Antibody mediated immunity histochemical method alleged occurrence MDC with direct anti-MDC.Characteristics Baxer ' the s Diff Quick test kit dyeing identification of cellular infiltration thing.

Embodiment 17

Spinal cord suppresses determination of activity

The MDC spinal cord suppresses active mensuration, injection MDC in mouse or other Mammals (as rabbit, cavy), as with maze etc., Journal of Immunology, the inhibiting method of spinal cord of the described mensuration of 149:1004-1009 (1992) MIP-1 α is carried out.The reorganization MDC intravenous injection of single dose 0.2-10 μ g is (Jackson Lab, Bar Harbor ME) in C3H/HeJ mouse.The cycle efficiency (cycling rate) that is determined at marrow sample progenitor cell in femur marrow and the spleen is determined the bone marrow depression effect of chemokine.Growth of progenitor cells and splitted restraining effect are clinically with relevant to the processing of accepting chemotherapy or radiocurable patient.The spinal cord protective effect that above chemokine is handled is by Da Luopu etc., blood, and 79:2221 (1992) illustrates in preclinical models.

To chemokine derivative interleukin 8 (IL-8) and the identical mode of platelet factor 4 (PF-4), the external test method also is used to measure the influence that MDC suppresses spinal cord with aforementioned.See Dai Li etc., journal of biological chemistry, 270:2382 (1985).In brief, for estimating the grain-huge precursor cell of biting, when lacking (contrast) or having MDC, normal people's medullary cell of low density (being less than 1.077g/cm) is planted plate in containing 10% foetal calf serum (HyClone, Logan, UT), the recombinant human GM-CSF (R﹠amp of 100 units/ml; D Systems, Mineapolis, MN), 50ng/ml recombinant human steel gray (Steel) factor (Immunex Corp, Seattle, WA), in 0.3% the nutrient agar.For estimating that red is granulocyte medullary system megakaryocyte colony forming unit (CFU-GEMM) and bunch formation unit (BFU-E) of red system, have recombinant human erythropoietin (1-2units/ml) and share with the 50ng/ml steel factor, cell is grown in the substratum that contains 0.9% methylcellulose gum.After 37 ℃ of hypoxemia (5%) are cultivated 14 days, obtained colony on several plates.Share of GM-CSF and steel factor or erythropoietin and steel factor enables to detect big colony (common＞1000 cell/clones), these colonies are from granulocyte medullary system colony forming unit (CFU-GM), the morning of CFU-GEMM and BFU-E, the more subclass that does not become to collect.

Embodiment 18

The antibody of anti-people MDC

A. monoclonal antibody

Reorganization MDC, the method for pressing embodiment 6 described cutting GST-MDC fusion roteins produces, and is used for immune mouse, to produce monoclonal antibody.In addition, different mouse are with being equivalent to the chemical synthesising peptide section immunity that the ripe attitude N of MDC holds (SEQ ID NO:21-12 position residue).(the Foster city, CA) synthetic, the method by producer is introduced is connected on key hole _ keyhole limpet hemocyanin (Pierce) peptide at the model 473A of applying biological system peptide synthesizer.During start injection, about 10 μ gMDC albumen or connection peptides Freund's complete adjuvant emulsification, subcutaneous injection.Behind the At intervals of two to three weeks, the MDC albumen Freund's incomplete adjuvant emulsification of additional content, subcutaneous injection.It is preceding that in the end perfusion strengthens (prefusion boost), takes out serum sample from immune mouse.These serum are analyzed with the western blotting, to confirm itself and the proteic reactivity of MDC.For perfusion strengthens, mouse is used in the MDC liquid injection among the PBS, kills mouse after four days and takes out spleen.Spleen is placed among the RPMI1640 of 10ml serum-free, be immersed in interpolation 2mM L-L-glutamic acid, 1mM Sodium.alpha.-ketopropionate in the grinding of two sheet glass slide frosted ends, 100units/ml penicillin and 100 μ g/ml Streptomycin sulphate (RPMI) (Gibco, Canada) spleen among the serum-free RPMI1640 forms single cell suspension.Cell suspension filters with aseptic 70 order Nitex cell nutsche filters (strainer) (BectonDickinson, Parsippany, New Jersey), and centrifugal 200g washed secondary in 5 minutes, resuspended being deposited among the 10ml serum-free RPMI.Take from the thymocyte of the Balb/c mouse of not doing experiment for three times, be used as trophoderm with the similar methods preparation.NS-1 myeloma cell, before merging contain 10% foetal calf serum (FBS) (Logan kept its logarithmic phase three days among RPMI Utah) for Hyclone laboratory, Inc, 200g, centrifugal collection in 5 minutes is washed the precipitation secondary by the described method of leading portion.

With splenocyte (2 * 10 ⁸) and 4 * 10 ⁷The NS-1 cytomixis, centrifugal, inhale and remove supernatant.Rap centrifuge tube migratory cell precipitation, the PEG1500 (50% in 75mM N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid (Hepes)) that adds 37 ℃ of 2ml (BoehringerMannheim), stir more than 1 minute, then add 14ml serum-free RPMI, more than 7 minutes.Add other 16ml RPMI, cell is used centrifugal 10 minutes of 200g.After removing supernatant liquor, precipitation is resuspended in and contains 15%FBS, 100 μ M xanthoglobulin sodium, 0.4 μ M aminopterin, 16 μ M thymidines (HAT) (Gibco), 25 untis/ml interleukin-6s (Boehringer Mannheim) and 1.5 * 10 ⁶Among the 200ml RPMI of thymocyte/ml, plant plate in 10 flat 96 well culture plates of Corning (Corning, Corning New York).

Merge the back the 2nd, 4 and 6 day, and from the hole of merging plate, removed 100 μ l substratum, heavily add 100 μ l fresh cultures.At the 8th day, existing by following detection can be in conjunction with the ELISA method screening fused cell of the mouse IgG of MDC.The Immulon4 plate (Dynatech, Cambridge, MA) with being diluted in the 25mM Tutofusin tris, the MDC of every hole 100ng among the pH7.5,37 ℃ of bags were by 2 hours.Coating buffer is removed in suction, and every hole adds 200 μ l confining liquids [being diluted in 0.5% fishskin gelatin (Sigma) among the CMF-PBS], 37 ℃ of incubations 30 minutes.Inhale deblocking liquid, add 50 μ l culture supernatant.37 ℃ of incubations are after 30 minutes, give a baby a bath on the third day after its birth time with the PBS that contains 0.05% polysorbas20 (PBST), add 50 μ l to be diluted in link coupled goat anti-mouse igg (fc) horseradish peroxidase (Jackson's immune Research, Xi Geluofu, Pennsylvania) among the PBST at 1: 7000.Plate by last method incubation, is washed 4 times with PBST, added 100 μ l, contain the substrate solution that 1mg/ml O-Phenylene Diamine (Sigma) and 0.1 μ l/ml, 30% hydrogen peroxide are formed among the pH4.5 at the 100mM Citrate trianion.After 5 minutes, the sulfuric acid that adds 50 μ l 15% stops color reaction.On plate readout instrument (Dynafech), read the A490 value.

The fused cell of selecting is diluted in 96 orifice plates and clones secondary, clone's number in denumerable every hole after 5 days.Monoclonal anti body and function Isostrip system (Boehringer Mannheim, Indianapolis, IN) homotypeization by the hybridoma generation.

The antibody of anti-MDC with the anti-above-mentioned reorganization MDC in intestinal bacteria or the generation of lactation Chinese hamster ovary celI of western blotting, is further determined its characteristics.For preparing trace, about 3 μ l sedimentation cells (produce the transformed into escherichia coli of MDC, produce the transfection CHO cell of MDC; Unconverted intestinal bacteria (contrast); With untransfected Chinese hamster ovary celI (contrast)) be dissolved in the standard model that contains SDS (sodium lauryl sulphate) and DTT (dithiothreitol (DTT)) and prepare (holy nurse Brooker etc.) in the damping fluid.After boiling, lysate by sex change SDS-PAGE (18% acrylamide, Tutofusin tris glycine gels, NOVEX) fractional separation, electroblotting to pvdf membrane (Millipore, Bedford, MA).MDC monoclonal anti body and function PBS is diluted to 0.7 μ g/ml, according to standard technique (holy nurse Brooker etc.), is used for Western blot.As other contrast, further test the cross reactivity of monoclonal antibody to the Western blot band of organizing lysate entirely of human skin, tonsilla and thymus gland.

The monoclonal antibody that one anti-MDC is arranged, called after monoclonal antibody 191D, the reorganization MDC that it and bacterium and mammalian cell produce has kickback.And this antibody shows organizes very little background response to the bacterium of being tried, CHO mammal cell line or whole people.In addition, this antibody shows as: according to the immunoprecipitation method (holy nurse Brooker etc.) of standard, the energy immunoprecipitation is from the reorganization MDC of CHO.

Produce the hybridoma cell line of monoclonal antibody 191D (called after hybridoma 191D), according to clause (the ATCC preservation date: on June 4th, 1996 of the special treaty in Budapest; ATCC registration number HB-12122), leaves in the American type culture collection (ATCC) 12301 Parklawn DriVe, RockVille MD20852 (U.S.) in.The viability of preserved material should only not be interpreted as when the pass when violating any Governmental Authority according to its patent law granted entitlements.

B. polyclonal antibody

The polyclonal antibody of anti-MDC according to standard scheme (Sheng Mubumuke etc.), produces in rabbit.The above-mentioned reorganization MDC that produces with gst fusion protein dilutes with PBS, and Freund's complete adjuvant emulsification is subcutaneously injected in the rabbit.At interval 3-6 week, be diluted in the MDC Freund's incomplete adjuvant emulsification among the PBS in addition, be subcutaneously injected in the identical rabbit.Immune for the third time back 10 days, from rabbit, extract serum, with 10 times the Tutofusin tris buffer salt solution dilution (TBS-T, Sheng Mubumuke etc.) that contains 0.5% polysorbas20, qualitative by the Western blotting of above-mentioned anti-reorganization MDC.

Embodiment 19

Calcium current is measured

The variation of intracellular calcium concentration, index for the chemokine cell activation, calcium current assay method with recognized technology is monitored in several clones, cell at room temperature incubation contains 1 μ M Fura-2/AM (molecular probe at 1ml, Eugene, OR) in the perfect medium 30 minutes, wash once, with～10 ⁶Cell/ml is resuspended among the D-PBS.

The 2ml suspension cell is placed on and is positioned at photofluorometer (AMINCO-Bowman Series2, Rochester is NY) in interior 37 ℃ of cuvettes that stir down constantly.Cellular calcium concentration represents that with fluorescence this is when changing the excitation wavelength of 340nm and 380nm at interval with 0.5 second, the fluorescent value of measuring in emission wavelength 510nm place.Emission ratios from 340nm to the 380nm excitation wavelength is equivalent to the cellular calcium level.

The clone that this mensuration is measured comprises as follows: the Chemokine Receptors gene V28[Lay Pood that stable transfection is generally acknowledged etc., gene, 163:295-299 (1995)] Human Embryonic Kidney HEK-293 clone; The HEK-293 cell of stable transfection Chemokine Receptors gene C CR5 [Sai Musen etc., biological chemistry, 35:3362-3367 (1995); Also see and own together, common unsettled U.S. Patent application, serial number 08/575,967, be incorporated herein by reference December 20 nineteen ninety-five submission date, open chemokine material and method, comprise CCR5 (taking " 88C " as) at that], people's monokaryon THP-1 clone, people's lung epithelial A-549 clone; Human desmocyte IMR-90 clone.These cells all do not have calcium current when reorganization MDC albumen is replied.As positive control, the HEK-293 transfectional cell has kickback to zymoplasm, and it is effective that prompting is measured.In addition, the THP-1 cell is that (Peprotech, Rocky Hill NJ) have kickback for the chemokine MCP-1 that buys of 25ng/ml and MCP-3 to final concentration.To A-549 or IMR-90 clone, the stimulation that undetermined is other.

Embodiment 20

The effect of HIV inhibition of proliferation

Several CC chemokines relate to the propagation of HIV inhibiting (HIV), and HIV is the risk factor of people's acquired immune deficiency syndrome (AIDS) (AIDS).See that the Cork is wished etc., science, 270:1811 (1995).The activity of the anti-HIV of MDC propagation is measured with described methods such as the Cork are uncommon, in the method, and CD ₄ ⁺The T cell is cultivated in the presence of the MDC of different concns with HTV strain acute infection.After three days, in cell, add the substratum that contains fresh dilution MDC.Infected the back 5～7 days, the HIV level detects box with business-like ELISA, and (Cole spy, Miami FL), detect the HIVp24 antigen of culture supernatant existence and measure.

(Cork is uncommon to have detected the active ability of inhibition that the antibody neutralization of anti-MDC produces by human lymphocyte, the same) in addition, in HIV suppressed to measure, the effect of the analogue that MDC analogue of chemosynthesis (embodiment 11) or recombination method produce was also tested.

Embodiment 21

MDC is to the influence of fibroblast proliferation

Except can attract and activated leukocyte cell, some chemokines such as IL-8 show the cell proliferation that can influence non-white corpuscle [see reach this know you, J.Invest.Dermatol., 92:294-298 (1992)].The inoblast of whole machine body is important to the integrity of great majority tissue.Fibroblast proliferation is absolutely necessary to wound healing with to the reaction of damage, but under the situation of chronic inflammatory disease such as pulmonary fibrosis, also is deleterious [kind grace, inflammation immunology, Elsevier (1983), pp.121-162)].

Body outer cell proliferation is measured and is used to estimate the influence of MDC to fibroblast proliferation.Human fibroblasts (CRL-1635) obtains from ATCC, keeps cultivation in containing 10%FBS and 1% antibiotic DMEM.The operation of proliferation assay and quantitative is pressed this area by Dai Heermu and kind grace, American Journal of Pathology, the aforesaid method of 134:355-363 (1989).In brief, first day, in containing the DMEM of 10%FBS, 2.5 * 10 ³The cells/well kind is to 96 orifice plates.Second day: plant plate after 24 hours, the substratum on the cell changed the DMEM of serum-free into.The 3rd day: substratum was removed from cell, replaced to contain the DMEM that the 0.4%FBS dilution has MDC.The 5th day: every hole added the 3H thymidine of 1 microcurie, continued incubation 5 hours.Cell harvesting to glass fiber filter.Cell proliferation rate is represented with the cpm that is incorporated into the 3H thymidine in the inoblast.The contrast of this mensuration comprises measures used minimum medium, and the DMEM that contains 0.4%FBS makes negative control, and the DMEM that contains 10%FBS makes positive control.

As shown in Figure 7, MDC handles and is lowered into fibrocellular propagation in dosage dependence mode.With observing similar fibroblast proliferation restraining effect from the MDC (solid circles) of Chinese hamster ovary celI purifying and the MDC (empty circles) of chemosynthesis.The effect of the anti-fibroblast proliferation of MDC shows therepic use, handles as pulmonary fibrosis and tumor disease with MDC, and in these diseases, enhancing or cell proliferation out of control are principal characters.

Embodiment 22

Cell proliferating determining

Measure the influence of MDC to epithelial cell, T cell, inoblast, endotheliocyte, scavenger cell and tumor cell proliferation, with well known in the art as Dai Heermu etc., American Journal of Pathology, 3-4 joint in the described method of 134:355-363 (1989) and " immunology modernism " book, the growth factor activity assay method that " lymphocyte function external test " write by Willie and Sang Si (1992).In these methods, the enhancing of cell growth and the release of restraining effect and somatomedin have been measured.

MDC is to the mensuration of epithelial cell and endothelial cell proliferation influence, with above-mentioned to the identical step of inoblast (embodiment 21).

Use peripheral blood lymphocyte, measure influence T cell proliferation.Monocyte is pressed Dai Heermu etc., American Journal of Pathology, and the described method of 135:571-580 (1989) is separated from peripheral blood; Cell is resuspended among the RPMI that contains 10%FBS, is incubated overnight in the plastic culture bottle.Lymphocyte still is suspended in these cultures, and centrifugation medium obtains.100,000 lymphocytes are inoculated in the hole of 96 orifice plates, contain 1 μ g/ml phytoh(a)emagglutinin (PHA) add or do not add 50,125,250 or the substratum (RPMI adds 10%FBS) of 500ng/ml MDC in cultivated 3 days.Cultivating last 18 hours, and adding the 3H-thymidine of 1 microcurie.Harvested cell, proliferation rate is used for fibroblastic method representation by embodiment 21 is described.

Measure the influence of MDC, with pressing the activated cavy peritoneal macrophage that obtains in the foregoing description 13 to macrophage proliferation.Scavenger cell, is planted in 96 orifice plates in containing the RPMI of 10%FBS with the density of 100,000 cells in every hole, and incubation made cell attachment in 2 hours.Remove substratum then, Dai Yijia or do not add 50,125,250 or the fresh culture of the MDC of 500ng/ml.Cell and MDC incubation 3 days are used for lymphocytic method and measure proliferation rate by above-mentioned.

The propagation control of these chemokine mediated cell types when strengthening tissue repair after the damage and limit these cell proliferations in chronic inflammatory reaction such as psoriasis, fibrosis, atherosclerosis and tumor disease, has the treatment meaning.

Embodiment 23

Measure in the body of fibroblast proliferation

Measure MDC in the body to the antiproliferative influence of inoblast, with art-recognized method, as kind grace and square Toon, American Journal of Pathology, the report method of 50:587-591 (1984), this method Pulmonary Fibrosis in Rats model of bleomycin induced.This aspect of model is clear and definite, allows to estimate that in all stage of disease fibroblasts proliferation and collagen synthesize.

In brief, rat is divided into 4 treatment group: 1) contrast gives the physiological saline intratracheal injection; 2) saline injection rat is accepted the 500ng MDC in abdominal injection salt solution every day again; 3) bleomycin is handled, and gives 1.5mg/kg bleomycin (Calbiochem, Palo Alto, CA) intratracheal injection; 4) bleomycin handle rat give again every day abdominal injection 500ng MDC.

Beginning tracheae injection back 4,7,14,21 and 28 days, every group is killed three rats.Get lung, histological examination done by each lobe of the lung sample and collagen content is measured.

Embodiment 24

The MDC regulatory factor is measured

The active regulatory factor of MDC (modulator), the disease that MDC is worked therein or the processing of disease symptoms may be useful.Above regulatory factor or be MDC bonded agonist or antagonist.Following receptors bind is measured provides method identification above MDC regulatory factor.

MDC with detectable marker as ¹²⁵I, ³H, ¹⁴C, vitamin H or europium mark.The cytolemma preparation that contains the MDC acceptor is from having the nature cell of replying to MDC, as human macrophage, Buddhist ripple ester activated THP-1 cell, human fibroblasts, human fibroblast cell line or cavy scavenger cell.(the alternate method is: when above MDC receptor cdna is identified and is cloned, and system recombinant receptor prepared product from the cell of transfection MDC receptor cdna).For example, membrane product is exposed to ¹²⁵Among the MDC of I mark, incubation under appropriate condition (as 37 ℃ 10 minutes).Any way combination is collected in vacuum filtration on filter membrane then ¹²⁵The film of I-MDC, cleaning is removed unconjugated ¹²⁵I-MDC.Then film be put in the liquid scintillation spectrophotometer quantitatively with in conjunction with the relevant radioactivity of MDC.

MDC bonded specificity can repeat the method for front under the situation of the quantity that increases unmarked MDC, measure the level that is attached to acceptor competitively and confirm.These are in conjunction with measuring the regulatory factor that also can be used to discern the MDC receptors bind.

The receptors bind of front is measured also can be modified as follows and carried out: except that mark MDC, MDC regulatory factor undetermined is put in the membrane product.In the measuring method of this change, the relevant marker of film increases level (quantity) and shows that regulatory factor undetermined is a MDC bonded activation factor; The relevant marker of film reduces level (quantity) and shows that regulatory factor undetermined is the inhibitor of MDC receptors bind.This measuring method can be used for from a large amount of compounds or natural product identification MDC bonded specific activators and inhibitor.Screening simultaneously a large amount of regulatory factor candidate compounds apace is special hope.

As the biological function of the above-mentioned MDC that illustrates, show the clinical application that several respects are arranged.

In general, chemokine attraction and activated monocyte and scavenger cell (Ba Geoulini etc., the same), MDC attract scavenger cell especially and suppress monocyte chemotaxis.MDC at pathogenicity bo inflammatory point of fixity expresses like this, can activate the white corpuscle that has existed, or induce white corpuscle still to increase the weight of morbid state at disease site by replenishing other scavenger cell or white corpuscle to disease site.Thereby the chemical attractants activity that suppresses MDC is expected to alleviate deleterious inflammatory process.Meaningfully: the potential benefit of aforesaid method is directly illustrated in the experiment that relates to IL-8, and IL-8 is the C-X-C cell chemotactic factor that attracts and activate neutrophil leucocyte.The antibody of direct anti-IL-8 has extremely strong ability.Inhibition is by the inflammatory diseases of neutrophil leucocyte mediation [Harrar reach etc., Leukoc.Biol.56:559 (1994)].The restraining effect that is expected MDC in rheumatic arthritis or the atherosclerosis, has similar effect in disease such as Crohn disease that the supposition scavenger cell works.

Alternative is that the influence that increases MDC may have useful effect in above disease, in wound healing and vasculogenesis positive interaction is arranged because shown chemokine.Like this, the MDC of external source or MDC agonist may be useful in promoting above disease recovery.

In addition, the bone marrow depression effect of illustrating C-C chemokine MIP-1 α (maze etc., the same) shows that MDC has similar activity.Above activity by MDC or MDC agonist provide produces actual benefit to accepting chemotherapy or radiotherapeutic patient, reduces the harmful effect of treatment to sick human medullary progenitor cell.

Also provable MDC or MDC agonist have clinical importance when the treatment tumour, can suppress the tumour formation (seeing Lan Ning etc., the same) of mouse as showing C-C chemokine TCA3.MDC can suppress tumour directly or indirectly and form and work, as attracting and activating different non-specific response cells to tumor sites or activate special antineoplastic immune.The effect of the anti-fibroblast proliferation of MDC shows that MDC is treating such as pulmonary fibrosis and these treatment of diseases purposes of tumour, and in these diseases, enhancing or cell proliferation out of control are main features.

When the present invention describes by special embodiment, be appreciated that those skilled in the art can expect changing with the method for revising.Therefore, the present invention only is subjected to the restriction of limiting factor in the claims.

The total information of sequence table (1):

(i) applicant: Godiska, Ronald; Gray, Patrick W.

(ii) invention exercise question: macrophage derived chemokine and chemokine analogue

(iii) sequence number: 32

(iv) mailing address:

(A) addressee: Marshall, O ' Toole, Gerstein, Murray﹠amp; Borun

(B) street: 6300Sears Tower, 233South Wacker Drive

(C) city: Chicago

(D) state: Illinois

(E) country: the United States of America

(F) postal region: 60606-6402

(v) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, Version#1.30

(vi) current request for data:

(A) application number:

(B) submission date:

(C) classification:

(vii) request for data formerly:

(A) application number: 08/558,658

(B) submission date: November 16 nineteen ninety-five

(vii) request for data formerly:

(A) application number: 08/479,620

(B) submission date: June 7 nineteen ninety-five

(viii) consignor/proxy's information:

(A) name: Gass, David A.

(B) registration number: 38,153

(C) reference/file number: 27866/33318

(ix) telecommunication information:

(A) phone: 312/474-6300

(B) fax: 312/474-0448

(C) information of telex: 25-3856 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 2923 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: protein

(ix) feature:

(A) title/key: CDS

(B) position: 20..298

(ix) feature:

(A) title/key: mat_ peptide

(B) position: 92..298

(xi) sequence description: SEQ ID NO:1:GAGACATACA GGACAGAGC ATG GCT CGC CTA CAG ACT GCA CTC CTG GTT GTC 52

Met?Ala?Arg?Leu?Gln?Thr?Ala?Leu?Leu?Val?Val

-24?????????????-20?????????????????-15CTC?GTC?CTC?CTT?GCT?GTG?GCG?CTT?CAA?GCA?ACT?GAG?GCA?GGC?CCC?TAC????100Leu?Val?Leu?Leu?Ala?Val?Ala?Leu?Gln?Ala?Thr?Glu?Ala?Gly?Pro?Tyr

-10??????????????????-5???????????????????1GGC?GCC?AAC?ATG?GAA?GAC?AGC?GTC?TGC?TGC?CGT?GAT?TAC?GTC?CGT?TAC????148Gly?Ala?Asn?Met?Glu?Asp?Ser?Val?Cys?Cys?Arg?Asp?Tyr?Val?Arg?Tyr

5??????????????????10??????????????????15CGT?CTG?CCC?CTG?CGC?GTG?GTG?AAA?CAC?TTC?TAC?TGG?ACC?TCA?GAC?TCC????196Arg?Leu?Pro?Leu?Arg?Val?Val?Lys?His?Phe?Tyr?Trp?Thr?Ser?Asp?Ser?20??????????????????25??????????????????30??????????????????35TGC?CCG?AGG?CCT?GGC?GTG?GTG?TTG?CTA?ACC?TTC?AGG?GAT?AAG?GAG?ATC????244Cys?Pro?Arg?Pro?Gly?Val?Val?Leu?Leu?Thr?Phe?Arg?Asp?Lys?Glu?Ile

40??????????????????45??????????????????50TGT?GCC?GAT?CCC?AGA?GTG?CCC?TGG?GTG?AAG?ATG?ATT?CTC?AAT?AAG?CTG????292Cys?Ala?Asp?Pro?Arg?Val?Pro?Trp?Val?Lys?Met?Ile?Leu?Asn?Lys?Leu

55 60 65AGC CAA TGAAGAGCCT ACTCTGATGA CCGTGGCCTT GGCTCCTCCA GGAAGGCTCA 348Ser GlnGGAGCCCTAC CTCCCTGCCA TTATAGCTGC TCCCCGCCAG AAGCCTGTGC CAACTCTCTG 408CATTCCCTGA TCTCCATCCC TGTGGCTGTC ACCCTTGGTC ACCTCCGTGC TGTCACTGCC 468ATCTCCCCCC TGACCCCTCT AACCCATCCT CTGCCTCCCT CCCTGCAGTC AGAGGGTCCT 528GTTCCCATCA GCGATTCCCC TGCTTAAACC CTTCCATGAC TCCCCACTGC CCTAAGCTGA 588GGTCAGTCTC CCAAGCCTGG CATGTGGCCC TCTGGATCTG GGTTCCATCT CTGTCTCCAG 648CCTGCCCACT TCCCTTCATG AATGTTGGGT TCTAGCTCCC TGTTCTCCAA ACCCATACTA 708CACATCCCAC TTCTGGGTCT TTGCCTGGGA TGTTGCTGAC ACTCAGAAAG TCCCACCACC 768TGCACATGTG TAGCCCCACC AGCCCTCCAA GGCATTGCTC GCCCAAGCAG CTGGTAATTC 828CATTTCATGT ATTAGATGTC CCCTGGCCCT CTGTCCCCTC TTAATAACCC TAGTCACAGT 888CTCCGCAGAT TCTTGGGATT TGGGGGTTTT CTCCCCCACC TCTCCACTAG TTGGACCAAG 948GTTTCTAGCT AAGTTACTCT AGTCTCCAAG CCTCTAGCAT AGAGCACTGC AGACAGGCCC 1008TGGCTCAGAA TCAGAGCCCA GAAAGTGGCT GCAGACAAAA TCAATAAAAC TAATGTCCCT 1068CCCCTCTCCC TGCCAAAAGG CAGTTACATA TCAATACAGA GACTCAAGGT CACTAGAAAT 1128GGGCCAGCTG GGTCAATGTG AAGCCCCAAA TTTGCCCAGA TTCACCTTTC TTCCCCCACT 1188CCCTTTTTTT TTTTTTTTTT TTTGAGATGG AGTTTCGCTC TTGTCACCCA CGCTGGAGTG 1248CAATGGTGTG GTCTTGGCTT ATTGAAGCCT CTGCCTCCTG GGTTCAAGTG ATTCTCTTGC 1308CTCAGCCTCC TGAGTAGCTG GGATTACAGG TTCCTGCTAC CACGCCCAGC TAATTTTTGT 1368ATTTTTAGTA GAGACGAGGC TTCACCATGT TGGCCAGGCT GGTCTCGAAC TCCTGTCCTC 1428AGGTAATCCG CCCACCTCAG CCTCCCAAAG TGCTGGGATT ACAGGCGTGA GCCACAGTGC 1488CTGGCCTCTT CCCTCTCCCC ACTGCCCCCC CCAACTTTTT TTTTTTTTTT ATGGCAGGGT 1548CTCACTCTGT CGCCCAGGCT GGAGTGCAGT GGCGTGATCT CGGCTCACTA CAACCTCGAC 1608CTCCTGGGTT CAAGTGATTC TCCCACCCCA GCCTCCCAAG TAGCTGGGAT TACAGGTGTG 1668TGCCACTACG GCTGGCTAAT TTTTGTATTT TTAGTAGAGA CAGGTTTCAC CATATTGGCC 1728AGGCTGGTCT TGAACTCCTG ACCTCAAGTG ATCCACCTTC CTTGTGCTCC CAAAGTGCTG 1788AGATTACAGG CGTGAGCTAT CACACCCAGC CTCCCCCTTT TTTTCCTAAT AGGAGACTCC 1848TGTACCTTTC TTCGTTTTAC CTATGTGTCG TGTCTGCTTA CATTTCCTTC TCCCCTCAGG 1908CTTTTTTTGG GTGGTCCTCC AACCTCCAAT ACCCAGGCCT GGCCTCTTCA GAGTACCCCC 1968CATTCCACTT TCCCTGCCTC CTTCCTTAAA TAGCTGACAA TCAAATTCAT GCTATGGTGT 2028GAAAGACTAC CTTTGACTTG GTATTATAAG CTGGAGTTAT ATATGTATTT GAAAACAGAG 2088TAAATACTTA AGAGGCCAAA TAGATGAATG GAAGAATTTT AGGAACTGTG AGAGGGGGAC 2148AAGGTGAAGC TTTCCTGGCC CTGGGAGGAA GCTGGCTGTG GTAGCGTAGC GCTCTCTCTC 2208TCTGTCTGTG GCAGGAGCCA AAGAGTAGGG TGTAATTGAG TGAAGGAATC CTGGGTAGAG 2268ACCATTCTCA GGTGGTTGGG CCAGGCTAAA GACTGGGAGT TGGGTCTATC TATGCCTTTC 2328TGGCTGATTT TTGTAGAGAC GGGGTTTTGC GATGTTACCC AGGCTGGTCT CAAACTCCTG 2388GGCTCAAGCG ATCCTCCTGG CTCAGCCTCC CAAAGTGCTG GGATTACAGG CGTGAATCAC 2448TGCGCCTGGC TTCCTCTTCC TCTTGAGAAA TATTCTTTTC ATACAGCAAG TATGGGACAG 2508CAGTGTCCCA GGTAAAGGAC ATAAATGTTA CAAGTGTCTG GTCCTTTCTG AGGGAGGCTG 2568GTGCCGCTCT GCAGGGTATT TGAACCTGTG GAATTGGAGG AGGCCATTTC ACTCCCTGAA 2628CCCAGCCTGA CAAATCACAG TGAGAATGTT CACCTTATAG GCTTGCTGTG GGGCTCAGGT 2688TGAAAGTGTG GGGAGTGACA CTGCCTAGGC ATCCAGCTCA GTGTCATCCA GGGCCTGTGT 2748CCCTCCCGAA CCCAGGGTCA ACCTGCCTGC CACAGGCACT AGAAGGACGA ATCTGCCTAC 2808TGCCCATGAA CGGGGCCCTC AAGCGTCCTG GGATCTCCTT CTCCCTCCTG TCCTGTCCTT 2868GCCCCTCAGG ACTGCTGGAA AATAAATCCT TTAAAATAGT AAAAAAAAAA AAAAA 2923 ( 2 ) SEQ ID NO：2：

(i) sequence signature:

(A) length: 93 amino acid

(B) type: amino acid

(D) topology: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:2:Met Ala Arg Leu Gln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala-24-20-15-10Val Ala Leu Gln Ala Thr Glu Ala Gly Pro Tyr Gly Ala Asn Met Glu

-5???????????????????1???????????????5Asp?Ser?Val?Cys?Cys?Arg?Asp?Tyr?Val?Arg?Tyr?Arg?Leu?Pro?Leu?Arg

10??????????????????15??????????????????20Val?Val?Lys?His?Phe?Tyr?Trp?Thr?Ser?Asp?Ser?Cys?Pro?Arg?Pro?Gly?25??????????????????30??????????????????35??????????????????40Val?Val?Leu?Leu?Thr?Phe?Arg?Asp?Lys?Glu?Ile?Cys?Ala?Asp?Pro?Arg

45??????????????????50??????????????????55Val?Pro?Trp?Val?Lys?Met?Ile?Leu?Asn?Lys?Leu?Ser?Gln

The information of 60 65 (2) SEQ ID NO:3:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:3:GACACTATAG AATAGGGC 18 (2) SEQ ID NO:4:

(i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:4:GTAAAACGAC GGCCAGT 17 (2) SEQ ID NO:5:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:5:AATTAACCCT CACTAAAGGG 20 (2) SEQ ID NO:6:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:6:GTAATACGAC TCACTATAGG GC 22 (2) SEQ ID NO:7:

(i) sequence signature:

(A) length: 35 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:7:TCTATCTAGA GGCCCCTACG GCGCCAACAT GGAAG 35 (2) SEQ ID NO:8:

(i) sequence signature:

(A) length: 33 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:8:CACCGGATCC TCATTGGCTC AGCTTATTGA GAA 33 (2) SEQ ID NO:9:

(i) sequence signature:

(A) length: 29 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:9:AATGGATCCA CAGCACGGAG GTGACCAAG 29 (2) SEQ ID NO:10:

(i) sequence signature:

(A) length: 31 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:10:AGTCAAGCTT AGGGCACTCT GGGATCGGCA C 31 (2) SEQ ID NO:11:

(i) sequence signature:

(A) length: 45 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:11:TATCGGATCC TGGTTCCGCG TGGCCCCTAC GGCGCCAACA TGGAA 45 (2) SEQ ID NO:12:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:12:GAAATCCAGC AAGTATATAG CA 22 (2) SEQ ID NO:13:

(i) sequence signature:

(A) length: 36 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:13:ATTGCCATGG CCGGCCCCTA CGGCGCCAAC ATGGAA 36 (2) SEQ ID NO:14:

(i) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:14:GACCAAGCTT GAGACATACA GGACAGAGCA 30 (2) SEQ ID NO:15:

(i) sequence signature:

(A) length: 29 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:15:TGGATCTAGA AGTTGGCACA GGCTTCTGG 29 (2) SEQ ID NO:16:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:16:CGAAATTAAT ACGACTCACT 20 (2) SEQ ID NO:17:

(i) sequence signature:

(A) length: 67 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: DNA

(xi) sequence description: the information of SEQ ID NO:17:TGGATCTAGA TCAATTCAAG TCCTCCTCGC TGATCAGCTT CTGCTCTTGG CTCAGCTTAT 60TGAGAAT 67 (2) SEQ ID NO:18:

(i) sequence signature:

(A) length: 99 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory: " Hu MCP-3 "

(ix) feature:

(A) title/key: protein

(B) position: 1..76

(xi) sequence description: SEQ ID NO:18:Met Lys Ala Ser Ala Ala Leu Leu Cys Leu Leu Leu Thr Ala Ala Ala

-20?????????????????-15?????????????????-10Phe?Ser?Pro?Gln?Gly?Leu?Ala?Gln?Pro?Val?Gly?Ile?Asn?Thr?Ser?Thr

-5??????????????????1???????????????5Thr?Cys?Cys?Tyr?Arg?Phe?Ile?Asn?Lys?Lys?Ile?Pro?Lys?Gln?Arg?Leu10??????????????????15??????????????????20??????????????????25Glu?Ser?Tyr?Arg?Arg?Thr?Thr?Ser?Ser?His?Cys?Pro?Arg?Glu?Ala?Val

30??????????????????35??????????????????40Ile?Phe?Lys?Thr?Lys?Leu?Asp?Lys?Glu?Ile?Cys?Ala?Asp?Pro?Thr?Gln

45??????????????????50??????????????????55Lys?Trp?Val?Gln?Asp?Phe?Met?Lys?His?Leu?Asp?Lys?Lys?Thr?Gln?Thr

60??????????????????65??????????????????70Pro?Lys?Leu

The information of 75 (2) SEQ ID NO:19:

(i) sequence signature:

(A) length: 99 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(ix) feature:

(A) title/keyword: misc_ feature

(D) out of Memory: " Hu MCP-1 "

(ix) feature:

(A) title/key: protein

(B) position: 1..76

(xi) sequence description: SEQ ID NO:19:Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu Ile Ala Ala Thr

-20?????????????????-15?????????????????-10Phe?Ile?Pro?Gln?Gly?Leu?Ala?Gln?Pro?Asp?Ala?Ile?Asn?Ala?Pro?Val

-5??????????????????1???????????????5Thr?Cys?Cys?Tyr?Asn?Phe?Thr?Asn?Arg?Lys?Ile?Ser?Val?Gln?Arg?Leu10??????????????????15??????????????????20??????????????????25Ala?Ser?Tyr?Arg?Arg?Ile?Thr?Ser?Ser?Lys?Cys?Pro?Lys?Glu?Ala?Val

30??????????????????35??????????????????40Ile?Phe?Lys?Thr?Ile?Val?Ala?Lys?Glu?Ile?Cys?Ala?Asp?Pro?Lys?Gln

45??????????????????50??????????????????55Lys?Trp?Val?Gln?Asp?Ser?Met?Asp?His?Leu?Asp?Lys?Gln?Thr?Gln?Thr

60??????????????????65??????????????????70Pro?Lys?Thr

The information of 75 (2) SEQ ID NO:20:

(i) sequence signature:

(A) length: 76 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory: " Hu MCP-2 "

(xi) sequence description: SEQ ID NO:20:Gln Pro Asp Ser Val Ser Ile Pro Ile Thr Cys Cys Phe Asn Val Ile1 5 10 15Asn Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr

20??????????????????25??????????????????30Asn?Ile?Gln?Cys?Pro?Lys?Glu?Ala?Val?Ile?Phe?Lys?Thr?Lys?Arg?Gly

35??????????????????40??????????????????45Lys?Glu?Val?Cys?Ala?Asp?Pro?Lys?Glu?Arg?Trp?Val?Arg?Asp?Ser?Met

The information of 50 55 60Lys His Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro65,70 75 (2) SEQ ID NO:21:

(i) sequence signature:

(A) length: 91 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory: " RANTES "

(ix) feature:

(A) title/key: protein

(B) position: 1..68 (xi) sequence description: SEQ ID NO:21:Met Lys Val Ser Ala Ala Ala Leu Ala Val Ile Leu Ile Ala Thr Ala

-20?????????????????-15?????????????????-10Leu?Cys?Ala?Pro?Ala?Ser?Ala?Ser?Pro?Tyr?Ser?Ser?Asp?Thr?Thr?Pro

-5??????????????????1???????????????5Cys?Cys?Phe?Ala?Tyr?Ile?Ala?Arg?Pro?Leu?Pro?Arg?Ala?His?Ile?Lys10??????????????????15??????????????????20??????????????????25Glu?Tyr?Phe?Tyr?Thr?Ser?Gly?Lys?Cys?Ser?Asn?Pro?Ala?Val?Val?Phe

30??????????????????35??????????????????40Val?Thr?Arg?Lys?Asn?Arg?Gln?Val?Cys?Ala?Asn?Pro?Glu?Lys?Lys?Trp

45??????????????????50??????????????????55Val?Arg?Glu?Tyr?Ile?Asn?Ser?Leu?Glu?Met?Ser

The information of 60 65 (2) SEQ ID NO:22:

(i) sequence signature:

(A) length: 91 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory: " MIP-1 β "

(ix) feature:

(A) title/key: protein

(B) position: 1..68

(xi) sequence description: SEQ ID NO:22:Met Lys Leu Cys Val Thr Val Leu Ser Leu Leu Met Leu Val Ala Ala

-20?????????????????-15?????????????????-10Phe?Cys?Ser?Pro?Ala?Leu?Ser?Ala?Pro?Met?Gly?Ser?Asp?Pro?Pro?Thr

-5??????????????????1???????????????5Ala?Cys?Cys?Phe?Ser?Tyr?Thr?Arg?Glu?Ala?Ser?Ser?Asn?Phe?Val?Val10??????????????????15??????????????????20??????????????????25Asp?Tyr?Tyr?Glu?Thr?Ser?Ser?Leu?Cys?Ser?Gln?Pro?Ala?Val?Val?Phe

30??????????????????35??????????????????40Gln?Thr?Lys?Arg?Ser?Lys?Gln?Val?Cys?Ala?Asp?Pro?Ser?Glu?Ser?Trp

45??????????????????50??????????????????55Val?Gln?Glu?Tyr?Val?Tyr?Asp?Leu?Glu?Leu?Asn

The information of 60 65 (2) SEQ ID NO:23:

(i) sequence signature:

(A) length: 92 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory: " MIP-1 α "

(ix) feature:

(A) title/key: protein

(B) position: 1..70

(xi) sequence description: SEQ ID NO:23:Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

-20?????????????????-15?????????????????-10Leu?Cys?Asn?Gln?Phe?Ser?Ala?Ser?Leu?Ala?Ala?Asp?Thr?Pro?Thr?Ala

-5??????????????????1???????????????5???????????????????10Cys?Cys?Phe?Ser?Tyr?Thr?Ser?Arg?Gln?Ile?Pro?Gln?Asn?Phe?Ile?Ala

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

30??????????????????35??????????????????40Leu?Thr?Lys?Arg?Ser?Arg?Gln?Val?Cys?Ala?Asp?Pro?Ser?Glu?Glu?Trp

45??????????????????50??????????????????55Val?Gln?Lys?Tyr?Val?Ser?Asp?Leu?Glu?Leu?Ser?Ala

The information of 60 65 70 (2) SEQ ID NO:24:

(i) sequence signature:

(A) length: 96 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory: " I-309 "

(ix) feature:

(A) title/key: protein

(B) position: 1..73

(xi) sequence description: SEQ ID NO:24:Met Gln Ile Ile Thr Thr Ala Leu Val Cys Leu Leu Leu Ala Gly Met

-20?????????????????-15?????????????????-10Trp?Pro?Glu?Asp?Val?Asp?Ser?Lys?Ser?Met?Gln?Val?Pro?Phe?Ser?Arg

-5??????????????????1???????????????5Cys?Cys?Phe?Ser?Phe?Ala?Glu?Gln?Glu?Ile?Pro?Leu?Arg?Ala?Ile?Leu10??????????????????15??????????????????20??????????????????25Cys?Tyr?Arg?Asn?Thr?Ser?Ser?Ile?Cys?Ser?Asn?Glu?Gly?Leu?Ile?Phe

30??????????????????35??????????????????40Lys?Leu?Lys?Arg?Gly?Lys?Glu?Ala?Cys?Ala?Leu?Asp?Thr?Val?Gly?Trp

45??????????????????50??????????????????55Val?Gln?Arg?His?Arg?Lys?Met?Leu?Arg?His?Cys?Pro?Ser?Lys?Arg?Lys

The information of 60 65 70 (2) SEQ ID NO:25:

(i) sequence signature:

(A) length: 93 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(ix) feature:

(A) title/key: protein

(B) position: 1..69

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory:

Prompting /=" 24 amino acid is selected from arginine, glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), Serine, Threonine, phenylalanine, tyrosine, tryptophane, aspartic acid, L-glutamic acid, l-asparagine, glutamine, halfcystine and methionine(Met) "

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory:

Prompting /=" 27 amino acid is selected from Methionin, glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), Serine, Threonine, phenylalanine, tyrosine, tryptophane, aspartic acid, L-glutamic acid, l-asparagine, glutamine, halfcystine and methionine(Met) respectively "

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory:

/ prompting=" 30 amino acid is selected from tyrosine, Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine and halfcystine respectively "

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory:

/ prompting=" 50 amino acid is selected from L-glutamic acid, Methionin, arginine, Histidine, glycine and L-Ala respectively "

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory:

/ prompting=" 59 amino acids are selected from tryptophane, Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine and halfcystine "

(ix) feature:

(A) title/key: misc_ feature

(D) out of Memory:

/ prompting=" 60 amino acids are selected from L-Ala, Serine, Methionin, arginine, Histidine, aspartic acid, L-glutamic acid, l-asparagine, glutamine and halfcystine "

(xi) sequence description: SEQ ID NO:25:Met Ala Arg Leu Gln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala

-20?????????????????-15?????????????????-10Val?Ala?Leu?Gln?Ala?Thr?Glu?Ala?Gly?Pro?Tyr?Gly?Ala?Asn?Met?Glu

-5??????????????????1???????????????5Asp?Ser?Val?Cys?Cys?Arg?Asp?Tyr?Val?Arg?Tyr?Arg?Leu?Pro?Leu?Xaa

10??????????????????15??????????????????20Val?Val?Xaa?His?Phe?Xaa?Trp?Thr?Ser?Asp?Ser?Cys?Pro?Arg?Pro?Gly25??????????????????30??????????????????35??????????????????40Val?Val?Leu?Leu?Thr?Phe?Arg?Asp?Lys?Xaa?Ile?Cys?Ala?Asp?Pro?Arg

45??????????????????50??????????????????55Val?Pro?Xaa?Xaa?Lys?Met?Ile?Leu?Asn?Lys?Leu?Ser?Gln

The information of 60 65 (2) SEQ ID NO:26:

(i) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: the information of SEQ ID NO:26:TATTGGATCC GTTCTAGCTC CCTGTTCTCC 30 (2) SEQ ID NO:27:

(i) sequence signature:

(A) length: 31 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: the information of SEQ ID NO:27:CCAAGAATTC CTGCAGCCAC TTTCTGGGCT C 31 (2) SEQ ID NO:28:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: the information of SEQ ID NO:28:GCGACTCTCT ACTGTTTCTC 20 (2) SEQ ID NO:29:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: the information of SEQ ID NO:29:CACAGGAAAC AGCTATGACC 20 (2) SEQ ID NO:30:

(i) sequence signature:

(A) length: 70 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:30:Leu Gly Pro Tyr Gly Ala Asn Met Glu Asp Ser Val Cys Cys Arg Asp1 5 10 15Tyr Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys His Phe Tyr Trp

20??????????????????25??????????????????30Thr?Ser?Asp?Ser?CysPro?Arg?Pro?Gly?Val?Val?Leu?Leu?Thr?Phe?Arg

35?????????????????40??????????????????45Asp?Lys?Glu?Ile?Cys?Ala?Asp?Pro?Arg?Val?Pro?Trp?Val?Lys?Met?Ile

The information of 50 55 60Leu Asn Lys Leu Ser Gln65,70 (2) SEQ ID NO:31:

(i) sequence signature:

(A) length: 69 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:31:Gly Pro Tyr Gly Ala Asn Met Glu Asp Ser Val Cys Cys Arg Asp Tyr1 5 10 15 Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys His Phe Tyr Trp Thr

20??????????????????25??????????????????30Ser?Asp?Ser?Cys?Pro?Arg?Pro?Gly?Val?Val?Leu?Leu?Thr?Phe?Arg?Asp

35??????????????????40??????????????????45Lys?Glu?Ile?Cys?Ala?Asp?Pro?Arg?Val?Pro?Tyr?Leu?Lys?Met?Ile?Leu

The information of 50 55 60Asn Lys Leu Ser Gln65 (2) SEQ ID NO:32:

(i) sequence signature:

(A) length: 69 amino acid

(B) type: amino acid

(C) chain: single

(D) topology: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:32:Gly Pro Tyr Gly Ala Asn Met Glu Asp Ser Val Cys Cys Arg Asp Tyr1 5 10 15Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys Glu Tyr Phe Tyr Thr

20??????????????????25??????????????????30Ser?Asp?Ser?Cys?Pro?Arg?Pro?Gly?Val?Val?Leu?Leu?Thr?Phe?Arg?Asp

35??????????????????40??????????????????45Lys?Glu?Ile?Cys?Ala?Asp?Pro?Arg?Val?Pro?Trp?Val?Lys?Met?Ile?Leu

50??????????????????55??????????????????60Asn?Lys?Leu?Ser?Gln65

Applicant or attorney docket WO96/40923

International application no PCT/US96/10114

Explanation about microbial preservation

(detailed rules and regulations 13 two) PCT/RO/134 shows (in July, 1992)

Claims (46)

1. coding has the purifying polynucleotide of polypeptide of macrophage derived chemokine (MDC) aminoacid sequence of SEQ ID NO:2.

2. claim 1 is the polynucleotide of DNA.

3. the DNA of the 20-298 position Nucleotide with SEQ ID NO:1 of claim 2.

4. the purifying polynucleotide of the 1-69 amino acids of coding MDC SEQ ID NO:2.

5. claim 4 is the polynucleotide of DNA.

6. the DNA of the 92-298 position Nucleotide with SEQ ID NO:1 of claim 5.

7. be selected from following purified polynucleotides:

(a) DNA of SEQ ID NO:1;

(b) under rigorous condition can with the polynucleotide of the DNA noncoding strand of SEQ ID NO:1 hybridization;

(c) because the redundancy of genetic code, under stringent condition can with the polynucleotide of the DNA noncoding strand hybridization of SEQ ID NO:1; With

(d) polynucleotide of the polypeptide identical with the dna encoding of SEQ ID NO:1.

8. claim 7 is the polynucleotide of DNA.

9. coding has the purifying polynucleotide of the polypeptide of SEQ ID NO:25 aminoacid sequence.

10. claim 9 is the polynucleotide of DNA.

11. the purified polynucleotides of the 1-69 amino acids of coding SEQ ID NO:25.

12. claim 11 is the polynucleotide of DNA.

13. be selected from following purified polynucleotides:

(a) DNA of claim 12;

(b) under stringent condition can with the polynucleotide of claim 12 hybridization; With

(c) because the redundancy of genetic code, under the condition of strictness can with the polynucleotide of the DNA hybridization of claim 12.

14. claim 13 is the polynucleotide of DNA.

15. comprise the carrier of claim 2,5,8,10,12 or 14 DNA.

16. claim 15 is the carrier of expression vector, wherein DNA can operationally be connected with expression control dna sequence dna.

17. with a kind of mode the DNA stable conversion of claim 2 or 5 or the host cell of transfection that can in described host cell, express MDC.

18. produce the method for MDC, described method is included in the host cell of cultivating claim 17 in the nutritional medium and separates MDC from described cell or substratum.

19. in a kind of mode of MDC polypeptide analog of can in described host cell, expressing by the DNA stable conversion of claim 10 or 12 or the host cell of transfection.

20. produce the method for MDC polypeptide analog, described method is included in the host cell of cultivating claim 19 in the nutritional medium and separates the MDC polypeptide analog from described cell or substratum.

21. have the purified polypeptide of the aminoacid sequence of SEQ ID NO:25.

22. the purified polypeptide of the aminoacid sequence with SEQ ID NO:2 of claim 21.

23. have the purified polypeptide of the 1-69 amino acids of SEQ ID NO:25.

24. have the purified polypeptide of the 1-69 amino acids of SEQ ID NO:2 according to claim 23.

25. be selected from following polypeptide:

(a) comprise the polypeptide of the aminoacid sequence of the 1-70 position that is accredited as SEQ ID NO:30; With

(b) comprise the purified polypeptide of the aminoacid sequence of the 9-69 position that is accredited as SEQ ID NO:2; With

(c) comprise the polypeptide of the aminoacid sequence of the 1-69 position identification that is accredited as SEQ ID NO:31; With

(d) comprise the polypeptide of the aminoacid sequence of the 1-69 position identification that is accredited as SEQ ID NO:32.

26. the polynucleotide of the polypeptide of coding claim 25.

27. claim 26 is the polynucleotide of DNA.

28. comprise the carrier of the DNA of claim 27.

29. the DNA with claim 27 can express the mode stable conversion of described polypeptide or the host cell of transfection with a kind of in described host cell.

30. one kind and claim 21, the antibody of 22,23,24 or 25 polypeptide specific reaction.

31. according to claim 30 is the antibody of monoclonal antibody.

32. produce the hybridoma cell line of the antibody of claim 31.

33. comprise polyclonal antiserum by claim 30 antibody.

34. and MDC plays the antibody of specific reaction.

35. regulate the method for leucocyte chemotaxis in mammalian hosts, it comprises the steps:

(a) use claim 21,22,23,24 or 25 polypeptide for described mammalian hosts, wherein said polypeptide can be regulated leucocyte chemotaxis in described host.

36. the method for claim 35, wherein said white corpuscle is selected from monocyte and scavenger cell.

37. alleviate the method for patient's inflammatory disease, its feature of described disease be at least (i) monocyte is to inflammation site chemotactic or (ii) one of in the fibroblast proliferation in described patient, described method comprises the steps:

(a) give the MDC of patient's administering therapeutic significant quantity.

38. the method for claim 37, the MDC of wherein said treatment significant quantity is the amount that can suppress monocyte chemotaxis.

39. by the method for claim 37, the MDC of wherein said treatment significant quantity is the amount that can suppress fibroblast proliferation.

40. comprise SEQ ID NO:1 nucleotide sequence sequential portion or with the DNA of its complementary noncoding strand,

Described sequential portion comprises at least 18 Nucleotide,

Described DNA is energy and people MDC gene recombination under rigorous condition.

41. the DNA of claim 40, wherein said DNA under described rigorous condition, can not with the human chemokine gene recombination that is selected from MCP-1 gene, MCP-2 gene, MCP-3 gene, RANTES gene, MIP-1 α gene, MIP-1 β gene and I-309 gene.

42. in pharmaceutically acceptable carrier, comprise claim 21, the medicinal compositions of 22,23,24 or 25 polypeptide.

43. the purposes of the composition of claim 42 in treatment inflammatory diseases state.

44. the purposes of claim 43, wherein said inflammatory diseases state is characterized in that in having the patient of described morbid state monocyte is to inflammation site chemotactic.

45. by the purposes of claim 43, wherein said inflammatory diseases state is characterized in that having among the patient of described morbid state, fibroblast proliferation.

46. identify to have the method that MDC regulates active compound, comprise step:

(a) provide the first and second acceptor compositions that comprise the MDC acceptor;

(b) provide the reference composition of the MDC that comprises detectable label;

(c) provide MDC that comprises the possibility certification mark and the test composition that further comprises described compound;

(d) MDC can with the situation of MDC receptors bind under, contact the first acceptor composition with reference composition;

(e) MDC can with the situation of MDC receptors bind under, contact the second acceptor composition with detection composition;

(f) the flush away first and second acceptor compositions are to remove not the MDC with the detectable label of MDC receptors bind;

(g) MDC of mensuration detectable label in the described first and second acceptor compositions; With

(h) identify that containing MDC regulates active compound, wherein MDC regulates active in relevant by the difference of the MDC of detectable label in the described first and second acceptor compositions.

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