CN1282371A

CN1282371A - Ac (DNA encoding C6 beta-chemokine leukotactin-1 (Lkn-1) isolated from human

Info

Publication number: CN1282371A
Application number: CN98811574A
Authority: CN
Inventors: 拜昂·S·夸恩; 尹秉寿; 郑守一; 朴斗鸿; 白承宰; 李恩京
Original assignee: Ryokugugi K K
Current assignee: Ryokugugi K K
Priority date: 1997-11-27
Filing date: 1998-11-27
Publication date: 2001-01-31
Also published as: WO1999028472A1; KR20010022352A; WO1999028473A1; EP1034272A1; KR100367213B1; KR19990042713A; JP2002505076A

Abstract

The present invention relates to a cDNA coding for a novel protein which belongs to C6 beta -chemokines and attracts subsets of peripheral blood leukocytes, and a process for preparing the said protein by employing expression vector therefor. Open reading frame of the Lkn-1 cDNA encodes 113 amino acids containing a signal peptide of 21 amino acids, and molecular weight of mature protein consisting of 92 amino acids among them is supposed to be 10,162 dalton. A recombinant Lkn-1 which was expressed in E. coli or insect cell employing the said Lkn-1 cDNA and purified, inhibited colony formation and cell proliferation significantly, attracted neutrophils, monocytes and lymphocytes to cause chemotaxis, and bound to CCR1 and CCR3 receptors. Accordingly, it was determined that the recombinant Lkn-1 protein can be used as a potential drug for antibody production, the treatment during HIV-1 infection, the protection of bone marrow stem cells during chemotherapy or radiotherapy and the inhibition of leukemia, etc.

Description

CDNA from people's separated coding C6 beta-chemokine Lkn-1

Background of invention

Invention field

The present invention relates to cDNA and the method for preparing the C6 beta-chemokine from the new C6 beta-chemokine of human body separated coding; Belong to the C6 beta-chemokine and attract the cDNA of new protein of the subgroup of peripheral blood leucocyte more specifically to coding, utilize its expression vector to prepare described method of protein and described proteinic pharmaceutical use.

The description of prior art

The family of the minicell factor that chemokine is made up of low-molecular-weight basic protein, usually have four halfcystines, position according to first and second halfcystines, promptly two halfcystines whether in abutting connection with or two halfcystines between be separated with amino acid between whether, it can be divided into four subfamily CXC (α), CC (β), C (γ) and CX ₃C (referring to, Baggiolini, M. and Dahinden, C.A., today immunology, 15:127 (1994); Kelner, people such as S.G., science, 266:1395 (1994); Bazan, people such as J.F., nature, 385:640 (1997)).The gene cluster of chemokine subfamily is positioned on the identical karyomit(e), and for example the α chemokine gene is positioned human chromosome 4q12-21, and the beta-chemokine assignment of genes gene mapping is on human chromosome 17q11-32 and mouse chromosome 11.

The restraining effect of chemokine biologically active such as HIV-1, immunoregulation effect, leucocyte migration and anti-hemopoietic stem cell splitted restraining effect (referring to, Cocchi, people such as F., science, 270:1811 (1995); Wolpe, people such as S.D., The Journal of Experimental Medicine, 167:570 (1988); Graham, people such as G.J., nature, 344:442 (1990); Broxmeyer, people such as H.E., blood, 76:1110 (1990); Youn, people such as B.-S., Journal of Immunology, 155:2661-2667 (1995)).

Chemokine is also striden the film district proteinic receptors bind of G with activated leukocyte cell with coupling, and some acceptors also can be used as collaborative acceptor (referring to Oh, people such as K.-O., Journal of Immunology, 147:2978 (1991) in the HIV-1 course of infection; Alkabatih, people such as G., science, 272:1955 (1996)).For example, 8 hypotypes at beta-chemokine (CC chemokine) acceptor, be to have 4 hypotype: CCR4, CCR6, CCR7, CCR8 demonstration that a kind of material is had high-affinity among CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, the CCR8, and CCR1, CCR2, CCR3 and CCR5 have the avidity in conjunction with various chemokines.

So far, 9 chemokines that belong to the beta-chemokine subfamily known in the art (referring to, Wilson, people such as S.D., The Journal of Experimental Medicine, 171:1301 (1990); Modi, people such as W.S., human genetics, 84:185 (1990)).In the middle of them, mouse MRP-1 (" mMRP-1 ", MIP (macrophage inflammatory protein matter)-related protein-1 or C10) (referring to, Orlofsky, A. wait the people, cell is regulated, 2:403 (1991)) and mouse MRP-2 (" mMRP-2 ") (referring to Youn, B.-S. wait the people, Journal of Immunology, 155:2661 (1995)) difference with other beta-chemokine is that they have two extra cysteine residues, thereby forms the 3rd disulfide linkage, and their N-terminal district is very long.According to former discovery, they are divided into the C6 beta-chemokine.

People's MRP in these cases, have important reasons need develop and develop people's new MRP, because can infect or protect the potential drug of bone marrow stem cell as treatment HIV-1 in chemotherapy or radiation treatment process.

Brief summary of the invention

The inventor has made great efforts to separate mrp gene from various human cell lines.As a result, the inventor has isolated the new cDNA of the MRP that belongs to C6 β cytokine from the human monocyte cell line, and has determined its nucleotide sequence and the aminoacid sequence of inferring thus.In addition, they have successfully expressed described MRP cDNA in recombination bacillus coli or insect cell, and find that expressed recombinant protein suppresses that colony forms and in vivo with the propagation of vitro inhibition bone marrow stem cell and progenitor cell, and attract the subgroup of peripheral blood leucocyte (lymphocyte, monocyte and neutrophilic granulocyte) in the mode of chemotaxis.So, will be called " Lkn-1 (leukotactin-1) cDNA " and " Lkn-1 of reorganization " from the MRP of isolating described MPR cDNA of human body and reorganization.

So, the aminoacid sequence that first purpose of the present invention provides the new cDNA of coding C6 beta-chemokine (Lkn-1) and infers thus.

The recombinant microorganism that second purpose of the present invention provides the expression vector that contains described Lkn-1 cDNA and utilize this carrier to transform.

The 3rd purpose of the present invention provides the method for preparing the Lkn-1 that recombinates from described microorganism.

The 4th purpose of the present invention provides the method that the Lkn-1 protection medullary cell that utilizes reorganization is not subjected to cytotoxicity cancer therapy drug or not raying murder by poisoning.

Brief description of drawings

From description given below also in conjunction with the accompanying drawings, it is clear that above and other objects of the present invention and feature will become.Wherein:

Fig. 1 (A) has shown the nucleotide sequence (SEQ ID NO:1) of the dna fragmentation of 202bp and the aminoacid sequence (SEQ ID NO:2) of inferring thus.

Fig. 1 (B) has shown the comparison of the 87th to the 110th amino acids sequence (SEQ ID NO:3) of the aminoacid sequence (SEQ ID NO:2) of Lkn-1 and mMRP-2.

Fig. 2 is the figure of demonstration from the result of the RT-PCR (reversed transcriptive enzyme-polymerase chain reaction) of the isolating total RNA of various human cell lines.

Fig. 3 has shown the nucleotide sequence (SEQ ID NO:4) of Lkn-1cDNA and the aminoacid sequence (SEQ ID NO:5) of inferring thus.

Fig. 4 has shown the aminoacid sequence (SEQ ID NO:7) and mMRP-1 (SEQ ID NO:8) of sophisticated Lkn-1, mMRP-2 (SEQ ID NO:9), hMIP-1 α (SEQ ID NO:10), the comparison of those sequences of mMIP-1 α (SEQ ID NO:11) and hMIP β (SEQ ID NO:12).

Fig. 5 (A) shows the figure of Lkn-1 to the influence of the colony formation of the medullary cell in the marrow.

Fig. 5 (B) shows the figure of Lkn-1 to the influence of the colony formation of medullary cell in the spleen.

Fig. 5 (C) shows the figure of Lkn-1 to the influence of the rate of propagation of medullary cell in the marrow.

Fig. 5 (D) shows the figure of Lkn-1 to the influence of the rate of propagation of the medullary cell in spleen.

Fig. 6 (A) shows the figure of Lkn-1 to the inhibition effect of the dependent dose of the rate of propagation of medullary cell in the marrow.

Fig. 6 (B) shows the figure of Lkn-1 to the inhibition effect of the dependent dose of the rate of propagation of medullary cell in the spleen.

Fig. 7 (A) shows the figure of Lkn-1 to the inhibition effect of the dependence time of the rate of propagation of CFU-GM in the marrow.

Fig. 7 (B) shows the figure of Lkn-1 to the inhibition effect of the dependence time of the rate of propagation of BFU-E in the marrow.

Fig. 7 (C) shows the figure of Lkn-1 to the inhibition effect of the dependence time of the rate of propagation of CFU-GEMM in the marrow.

Fig. 7 (D) shows the figure of Lkn-1 to the inhibition effect of the dependence time of the rate of propagation of CFU-GM in the spleen.

Fig. 7 (E) shows the figure of Lkn-1 to the inhibition effect of the dependence time of the rate of propagation of BFU-E in the spleen.

Fig. 7 (F) shows the figure of Lkn-1 to the inhibition effect of the dependence time of the rate of propagation of CFU-GEMM in the spleen.

Fig. 8 (A) shows that the Lkn-1 of reorganization and RANTES (regulated by activation, normal T cell expressing and secretion) attract lymphocytic chemotactic figure.

Fig. 8 (B) is that the Lkn-1 and the hMIP-1 α that show reorganization attract monocytic chemotactic figure.

Fig. 8 (C) shows the Lkn-1 of reorganization and the chemotactic figure that IL-8 attracts neutrophilic granulocyte.

Fig. 9 (A) shows the relative fluorescence of measuring in the lymphocyte that is added a series of RANTES and reorganization Lkn-1 stimulation.

Fig. 9 (B) shows the relative fluorescence of measuring in the lymphocyte that is added a series of reorganization Lkn-1 and RANTES stimulation.

Fig. 9 (C) shows the relative fluorescence of measuring in the monocyte that is added a series of hMIP-1 α and reorganization Lkn-1 stimulation.

Fig. 9 (D) shows the relative fluorescence of measuring in the monocyte that is added a series of reorganization Lkn-1 and hMIP-1 α stimulation.

Fig. 9 (E) shows the figure of the relative fluorescence of measuring in the neutrophilic granulocyte that is added a series of IL-8 and reorganization Lkn-1 stimulation.

Fig. 9 (F) shows the relative fluorescence of measuring in the neutrophilic granulocyte that is added a series of reorganization Lkn-1 and IL-8 stimulation.

Figure 10 (A) shows the relative fluorescence of measuring in the HOS clone of the expression CCR1 that is added a series of all ingredients stimulations.

Figure 10 (B) shows the relative fluorescence of measuring in the HOS clone of the expression CCR3 that is added a series of all ingredients stimulations.

Figure 10 (C) is the figure of the acrometron that shows that the calcium depend on Lkn-1 and hMIP-1 α concentration is replied.

Figure 10 (D) is the figure of the acrometron that shows that the calcium of the concentration depend on Lkn-1, eotaxin and RANTES is replied.

Detailed description of the present invention

In order to separate the Lkn-1 gene from the human cell line, the inventor has at first cloned can be for the preparation of the extron of the Lkn-1 gene of probe. That is, utilize HindIII digestion human gene group DNA, separate at Ago-Gel, with³²The mMRP-2 cDNA of P-mark (referring to Youn, the people such as B.S., Journal of Immunology, 155:2661 (1995)) is as probe, utilize the Southern engram analysis obtain can with the dna fragmentation of the 7.0kb of mMRP-2 hybridization. Then, in order to isolate the exon sequence of Lkn-1 cDNA from the dna fragmentation of the HindIII of 7.0kb digestion, utilize HindIII catapepsis human gene group DNA, and separate at Ago-Gel, to obtain the dna fragmentation of 7.0kb. With the fragment insertion vector that obtains like this, and the carrier that will obtain like this imports the host. After conversion, select the colony of demonstration and described mMRP-2 cDNA Probe Hybridization. At the dna fragmentation of isolating 7.0kb from this colony with after utilizing A1uI digestion, cloned can with the dna fragmentation of the 202bp of mMRP-2 cDNA hybridization. Then, determine the nucleotide sequence of this fragment, this sequence area branches away part introne and the extron from described dna fragmentation.

Then, for the Lkn-1 cDNA that makes new advances from the human cell line clone, prepared the permission that as above confirms from the Lkn-1 PCR primer of Lkn-1 exon sequence amplification 100bp dna fragmentation, and utilize respectively the total RNA that separates from various human cell lines to carry out RT-PCR as template, thereby filter out the clone with Lkn-1 mRNA. Select a clone of so selecting, the person monocytic cell THP-1 clone of interleukin 4 (IL-4) activation, and prepare its cDNA library. On the other hand, the total RNA with described THP-1 clone also carries out RT-PCR with described Lkn-1PCR primer as template, the 100bp-DNA fragment of preparation Lkn-1 extron. Probe Hybridization with the 100bp-DNA fragment of the cDNA library of the THP-1 clone that as above makes up and Lkn-1 extron. As a result, obtained to show the Lkn-1cDNA of positive reaction.

The amino acid sequence of determining and inferring of the nucleotide sequence of described Lkn-1 cDNA shows that Lkn-1 cDNA is a new DNA, the open reading frame coding of Lkn-1 cDNA contains 113 amino acid of 21 amino acid whose signal peptides, and infers that the molecular weight that contains 92 amino acid whose ripe Lkn-1 protein is 10162 dalton. Find that Lkn-1 does not have potential N-glycosylation site, and belong to and have the C6 beta-chemokine family that also has four cysteine residues that usually occur except beta-chemokine family such as two cysteines in mMRP-1 and mMRP-2.

In expression vector, insert as above clone's new Lkn-1 cDNA, and it is imported host cell such as Escherichia coli or insect cell to express the Lkn-1 of restructuring.

On the other hand, studied restructuring Lkn-1 to stem cell and the bone marrow suppression activity that originates from the CFU-GM of people's marrow and spleen, proved that restructuring Lkn-1 suppresses the formation of colony and the propagation of myeloid cell to rely on concentration and the mode of time with external in vivo. In addition, the Lkn-1 that observes restructuring is neutrophil cell, monocyte and lymphocyte and cause chemotaxis and induce calcium current in described cell in the attractive peripheral blood consumingly. Particularly find the Lkn-1 and the receptors bind of RANTES and hMIP-1 α of restructuring, and not with the receptors bind of IL-8 (although its strong attraction neutrophil cell and cause chemotaxis, as IL-8). In addition, the acceptor of the Lkn-1 of discovery restructuring is CC-chemokine receptor 1 (CCR1) and CCR3. The Lkn-1 of restructuring is than hMIP-1 α or the stronger CCR1 antagonist (known hMIP-1 α and RANTES can be combined with CCR1) of RANTES, and it is the CCR3 antagonist stronger than RANTES, although it is than the receptor antagonist a little less than the eotaxin.

The reorganization Lkn-1 of the present invention that shows feature above-mentioned can be used for antibody producing, the treatment that HIV-1 infects, the protection of bone marrow stem cell and leukemic inhibition or the like in chemotherapy or radiation treatment process.

Further illustrate the present invention in the following embodiments, but can not regard these embodiment as limitation of the scope of the invention.The clone of the exon of embodiment 1:Lkn-1 genomic dna

For clone with from people's mMRP-2 homologous genomic dna, utilize BamHI, EcoRI, HindIII, PstI, XbaI and XhoI to digest total human gene group DNA, fractional separation on 1.0% sepharose, and with ³²The mMRP-2 cDNA of p-mark utilizes the Southern engram analysis to obtain the HindIII fragment of the positive signals of 7.0kb as probe.

In order to prepare the segmental inferior library of described HindIII, utilize the human gene group DNA of HindIII complete digestion 100Fg, at 20V, fractional separation is 16 hours on 1% sepharose.Then, utilize Gene-clean test kit (Bio101, the U.S.) to isolate and be inserted into dephosphorylation pBluescript SK through HindIII digestion near the dna fragmentation of 7.0kb ⁺Carrier (Stratagene, the U.S.).Utilize the recombinant vectors of preparation like this, assist and come Transformed E lectro-max (Gibco-BRL, the U.S.) competent cell, and on solid medium, cultivate with electroporation.Utilize about 2 * 10 of mMRP-2 probe and formation ⁵Individual colony hybridization.As a result of, find to have only a colony to show the positive signal of hybridization, and contain the DNA of the 7.0kb of insertion.

In order to separate exon sequence, isolate the pBluescript SK that contains the DNA that has inserted 7.0kb from the described colony of the positive signal of hybridization that shown from the dna fragmentation of described 7.0kb ⁺Carrier utilizes AluI digestion, and fractional separation on 1.5% sepharose clones dna fragmentation with the 202bp of mMRP-2 cDNA hybridization by the Southern engram analysis.

Determine the nucleotide sequence (SEQ ID NO:1) of the dna fragmentation of described 202bp, find intron and translate into amino acid whose a part of exon (referring to, Fig. 1 (A)).The result, find that aminoacid sequence (the SEQ ID NO:2) demonstration of a part of exon and the 87th to the 110th amino acid of mMRP-2 have 50% homology, and in Lkn-1, second halfcystine (being expressed as " * ") among mMRP-1 and the mMRP-2 in common two other halfcystines be guard (referring to: Fig. 1 (B)).In Fig. 1 (B), the frame line is presented at amino acid conservative among Lkn-1 and the mMRP-2.The clone of embodiment 2:Lkn-1 cDNA

In order to clone Lkn-1 cDNA from the human cell line, at first prepare the PCR primer of the Lkn-1 exon that allows amplification 100bp, i.e. forward primer 5 '-TTCCTCACCAAGAAGGGG-3 ' (SEQ IDNO:13) and reverse primer 5 '-CTTTTTCATGCAATCCTG-3 ' (SEQ ID NO:14) according to the disclosed aminoacid sequence of Fig. 1 (B) (SEQ ID NO:2).Then, utilize among the embodiment 1 the 202bp-DNA fragment of preparation respectively, with the 100Fg/ milliliter by total RNA of the total RNA of total RNA, the macrophage system U937 (ATCC CTL1593) of people's monokaryon THP-1 clone (ATCC TIB202) of 24 hours of IL-4 activation and HL-60 clone (ATCC CCL240) as template carry out PCR (referring to: Fig. 2).

In Fig. 2, swimming lane 1 to 4 has shown respectively and utilizes the 202bp-DNA fragment as positive control, total RNA of THP-1 clone, the result of the PCR that total RNA of U937 clone and total RNA of HL-60 clone carry out, swimming lane M has shown the 100bp ladder as the big tick marks of DNA.Just as shown in Figure 2, produced Lkn-1 mRNA by IL-4 activated T HP-1 clone.

In order to prepare THP-1 cDNA library,, utilize Time Saver cDNA test kit (Pharmacia Biotech from by IL-4 activated T HP-1 clone separation of human mRNA, the U.S.) carry out reverse transcription, obtain double-stranded cDNA, and connect with BstXI adapter (Invitrogen, the U.S.).Then, carry out the fractional separation on the sepharose so that isolate 0.5kb or bigger cDNA, isolating cDNA is inserted into utilize BstXI digestion PRc/CMV carrier (Invitrogen, the U.S.) with preparation cDNA library.With this cDNA library and 100bp-DNA fragment hybridization as the Lkn-1 exon of the top pcr amplification of probe.As a result of, be separated to a cDNA clone of positive signals.The nucleotide sequence of embodiment 3:Lkn-1 cDNA

Fig. 3 has shown the cDNA clone's who obtains among the embodiment 2 nucleotide sequence (SEQ IDNO:4) and the aminoacid sequence (SEQ ID NO:5) of inferring thus.In Fig. 3, underscoring be signal peptide, the sequence in the frame line is the aminoacid sequence (SEQ ID NO:2) of the Lkn-1 that shows of Fig. 1 (B), it can be used as probe in THP-1 cDNA library screening, and (* * *) is expressed as translation stop codon.

As shown in Figure 3, the open reading frame of Lkn-1 cDNA (SEQ ID NO:6) coding contains 21 amino acid that form signal peptide and forms 92 amino acid whose 113 amino acid that molecular weight is inferred to be 10162 daltonian mature proteins.In addition, in the aminoacid sequence of inferring (SEQ ID NO:5) of Lkn-1, do not find the N-glycosylation site.

Fig. 4 has shown aminoacid sequence and the mMRP-1 (SEQ ID NO:8) of ripe Lkn-1 (SEQ ID NO:7), mMRP-2 (SEQ ID NO:9), hMIP-1 α (SEQ ID NO:10) (referring to: Youn, B.S. wait the people, Journal of Immunology, 155:2661 (1995)), mMIP-1 α (SEQ IDNO:11) (referring to: Kown, B.S. and Weissman, S.M., American Academy of Sciences's annual report, 86:1963 (1989)) and hMIP-1 β (SEQ ID NO:12) (referring to Sherry, B. wait the people, the comparison of those The Journal of Experimental Medicine, 168:2251 (1988)).In Fig. 4, frame line and () are represented four conserved cysteine residue and two other cysteine residues respectively.As shown in Figure 4, show: (1) before two cysteine residues of common four cysteine residues, Lkn-1 has long N-terminal district in beta-chemokine family; (2) Lkn-2 belongs to as in mMRP-1 and mMRP-2, also has the C6 beta-chemokine family of two cysteine residues in addition except having four common cysteine residues of beta-chemokine family.In addition, the aminoacid sequence (SEQ ID NO:7) of finding sophisticated Lkn-1 shows with mMRP-1 (SEQ ID NO:8) or mMRP-2 (SEQ ID NO:9) to have only 43% homology, has only 40% homology with hMIP-1 α (SEQ ID NO:10), though Lkn-1 is as people's mMRP-2 counterpart and isolating.Embodiment 4: the expression embodiment 4-1 of reorganization Lkn-1: the expression of reorganization Lkn-1 in intestinal bacteria

In order only to express sophisticated Lkn-1 protein as the signal peptide that does not have deduction of recombinant protein, the Lkn-1 cDNA that utilizes clone among the embodiment 2 is as template, carry out the pcr amplification of the open reading frame of sophisticated Lkn-1 with following PCR primer and Pfu polysaccharase (Stratagene, the U.S.):

Forward primer:

5’-CGAATTCCATATGCAGTTCACAAATGATGCAGAG-3’

(SEQ?ID?NO：15)

Reverse primer:

5’-CGCCGCTCGAGTTGAGTAGGGCTTCAGC-3’

(SEQ?ID?NO：16)

The dna fragmentation that utilizes NdeI/XhoI digestion so to amplify, and the clone enters plasmid pET21a (Novagen, the U.S.).With recombinant plasmid called after pET21a-Lkn-1 and the importing intestinal bacteria XL-1Blue that makes up like this.This recombinant plasmid pET21a-Lkn-1 allows the N-terminal at sophisticated Lkn-1 to have the another one methionine residues and has the expression of the reorganization Lkn-1 of other 6 Histidines at C-terminal.

With transformant called after ' intestinal bacteria (XL-1 Blue) hMRP-2 ' for preparing like this, and be preserved in international preservation agent authorized, American type culture collection (ATCC, Rockville with preserving number ATCC98166 on September 10th, 1996, MD20852, the U.S.).

Cultivate described transformant to express Lkn-1 and to obtain inclusion body, inclusion body is dissolved in 20 milliliters of sex change damping fluids (6 mol Guanidinium hydrochlorides, 20 mmoles/rise Tris-HCl, pH7.9,500 mmoles/rise NaCl, 4 mmoles/liter just-the octyl group glycopyranoside) and centrifugal acquisition supernatant liquor.Then, utilize activatory Ni-post (Novagen, the U.S.) and heparin-agarose post (Pharmacia fine chemicals company, the U.S.) to carry out the reorganization Lkn-1 of chromatography purification His-mark.Electrophoresis shows that the molecular weight of this reorganization Lkn-1 is about 12 kilodaltons.Embodiment 4-2: the expression of reorganization Lkn-1 in insect cell

In order to express the reorganization Lkn-1 that contains signal peptide, the Lkn-1cDNA that will contain signal peptide sequence is inserted in the PstI restriction site of N-terminal and at the XbaI of C-terminal restriction site, and passes through pcr amplification.Then, separate such amplification PCR products, and be inserted in the PVL1392 carrier (Invitrogen, the U.S.) that digests with PstI/XbaI with construction recombination plasmid PVL1392-Lkn-1.Then, utilize described recombinant plasmid and AcNPV (Autographacalifornia nuclear polyhedron baculovirus) transfection Sf-21 insect cell so that the Lkn-1 cDNA in the described recombinant plasmid is transferred among the AcNPV.According to virus particle is that negative inaccessible feature is separated AcNPV-Lkn-1 virus plaque, and the growth in the Sf-21 insect cell in serum-free Ex-cell 400 substratum (JRH bio-science, the U.S.) that allows is to use after being used for.

The high 5 clone (Invitrogen that in Ex-cell 400 substratum, cultivate, the U.S.) Lkn-1 of express recombinant, and utilize HiTrap-heparin column (pharmacy biotech company, the U.S.) and this Lkn-1 that gives expression to of HiTrap-SP post (pharmacy biotech company, the U.S.) purifying.The Western engram analysis shows that the molecular weight of the Lkn-1 that obtains the reorganization analyzed behind purifying immediately is about 12 kilodaltons.Embodiment 5: the active embodiment 5-1 of the bone marrow depression of reorganization Lkn-1: reorganization Lkn-1 forms at the colony of vitro inhibition medullary cell

Because some beta-chemokines have a bone marrow depression activity external, so we have studied purifying obtains in embodiment 4-1 reorganization Lkn-1 forms colony to the myeloid progenitor that exists in people's marrow influence.That is,, containing on 0.3% nutrient agar of 10%FBS with 5 * 10 in order to form the colony of medullary cell by CFU-GM (colony forming unit-granulocyte-macrophage) ⁴The cells/ml coating utilizes (1.070gm/ centimetre of Ficoll-Hypaque gradient ³Sigma chemical company, the U.S.) the low density human bone marrow cell of centrifugal acquisition, and (reconstituted human granulocyte-macrophage-colony forms the factor, 100U/ milliliter, Immunex company to utilize rhGM-CSF, the U.S.)+rhSLF (recombinant human steel factor, 50 nanograms/milliliter, Immunex company, the U.S.) stimulate above-mentioned human bone marrow cell.On the other hand, in order to form the colony of medullary cell by CFU-GM, BFU-E (burst forming unit-red corpuscle) and CFU-GEMM (colony forming unit-granulocyte-red corpuscle-scavenger cell-megalokaryocyte), containing on the 1% methylcellulose gum substratum of 30%FBS with 5 * 10 ⁴Cells/ml is coated with described medullary cell, and utilizes rhEPO (recombinant human erythropoietin, 1 units per ml, Amgen company, U.S.), rhIL-3 (recombinant human interleukin-3,100 units per ml, Immunex company, U.S.) or rhSLF stimulate.

After stimulation, at 5%CO ₂And 5%O ₂Environment under in the BNP-210 incubator (TabaiESPEC company, the U.S.) culturing cell, the number of counting colony after 14 days (referring to: table 1).In this experiment, reorganization Lkn-1 adds dull and stereotyped with the concentration of 3-50 nanograms/milliliter.

Table 1. reorganization Lkn-1 is to the influence of low-density human bone marrow cell's colony formation

Sample	Concentration	Agar		Methylcellulose gum
		Agar		Methylcellulose gum			??CFU-GM (GM-CSF) ^b	??CFU-GM ??(GM-CSF ??+SLF) ^b	??CFU-GM	????BFU-E ??(EPO，SLF， ??IL-3) ^b	??CFU- ??GEMM
		Contrast ^a		??17±6	??67±2	??66±12	??CFU-GM (GM-CSF) ^b	??CFU-GM ??(GM-CSF ??+SLF) ^b	??CFU-GM	????BFU-E ??(EPO，SLF， ??IL-3) ^b	??CFU- ??GEMM	??94±2	??9±1
Reorganization Lkn-1	??50ng/ml	Contrast ^a		??17±6	??67±2	??66±12	??16±4(-6) ^c	??35±6(-48) ^d	??25±3(-62) ^d	??35±3(-63) ^d	??5±1(-44) ^d	??94±2	??9±1
	??50ng/ml	??25ng/ml	??18±1(+6)	??40±2(-40) ^d	??33±4(-50) ^d	??50±3(-47) ^d	??16±4(-6) ^c	??35±6(-48) ^d	??25±3(-62) ^d	??35±3(-63) ^d	??5±1(-44) ^d	??6±1(-33) ^d
	??6.25ng/ml	??25ng/ml	??18±1(+6)	??40±2(-40) ^d	??33±4(-50) ^d	??50±3(-47) ^d	??18±3(+6)	??52±6(-22) ^d	??45±6(-32) ^d	??65±2(-31) ^d	??7±2(-22) ^d	??6±1(-33) ^d
	??6.25ng/ml	??3.125ng/ml	??17±8(0)	??63±5(-6)	??61±10(-8)	??95±6(+17)	??18±3(+6)	??52±6(-22) ^d	??45±6(-32) ^d	??65±2(-31) ^d	??7±2(-22) ^d	??10±1(+11)

A. the experimental group that in reaction soln, does not add reorganization Lkn-1

B: the somatomedin that is used to stimulate colony formation.

C: with the level (%) of the variation of comparing

D: with compare, the level of variation is significant (p＜0.001).

From top table 1 as seen, the Lkn-1 of reorganization suppresses CFU-GM, BFU-E and CFU-GEMM formation colony significantly in the mode that relies on concentration.With compare, the inhibition level that colony forms is 22-63%.On the other hand, find that the Lkn-1 of reorganization can not suppress to form colony by the independent CFU-GM that stimulates of GM-CSF or by the independent BFU-E that stimulates of EPO, the Lkn-1 that this proof is recombinated is to having the inhibition effect by the jejune progenitor cell of various factors stimulated growth.Embodiment 5-2: the propagation that suppresses medullary cell in the reorganization Lkn-1 body

Assess the biological activity of Lkn-1 in vivo.That is, give the Lkn-1 of C3H/HeJ mouse intravenous injection purifying and measure the absolute number of granular leukocyte macrophage (CFU-GM), erythron (BFU-E) and pluripotent progenitor cells (CFU-GEMM) respectively and their rate of propagation in marrow and spleen: utilize 0.1 milliliter of aseptic no pyrogeneous substance salt solution or the Lkn-1 of the 8 microgram purifying that dilute injects to C3H/HeJ mouse through the tail vein in the salt solution of aseptic no pyrogen.In injection back 24 hours, be similar in embodiment 5-1 the low-density medullary cell of preparation in the marrow of femur and spleen mouse.Then, on 0.3% nutrient agar, be coated with CFU-GM, and utilize 10%PWM mouse boosting cell conditioned medium to stimulate.Equally, separate application BFU-E and CFU-GEMM on 0.9% methylcellulose gum substratum, and utilize the rhEPO, 0.1 mmole of 1 unit/rise hemin and 1% PWM mouse boosting cell conditioned medium stimulates.In this experiment, respectively with 7.5 * 10 ⁴With 1.0 * 10 ⁶The concentration coating marrow and the splenocyte of cells/ml.After stimulation, culturing cell under the same terms described in the embodiment 5-1 is counted the number (referring to Fig. 5 (A) and 5 (B)) of colony after 5 to 7 days at incubation.On the other hand, by means of specific activity is tritium-labeled thymus pyrimidine (50 microcurie/milliliter) kill techniques of 20 Curie/mmole, measure the DNA synthesis phase (promptly, the cell cycle S phase) ratio of phase progenitor cell, percentage ratio with the S phase cell in the cell cycle is determined CFU-GM, the speed of circulation of BFU-E and CFU-GEMM, (referring to Fig. 5 (C) and 5 (D)), described kill techniques is based on the minimizing in external calculating of the colony number of formation when cell contacts than with the cold thymus pyrimidine of a great deal of with the colony number that forms after tritium-labeled hot thymus pyrimidine pulse contacts 20 minutes.Fig. 5 (A) shows that to 5 (D) Lkn-1 has reduced the number of the colony that the marrow ancestral cells forms and their rate of propagation (that is cell cycle speed) in marrow and spleen fast.On the other hand, assessed in marrow, spleen and the blood cell rate of band nuclear, found and compare and be not subjected to tangible influence.

For these that study Lkn-1 suppress the dose-dependently of effects,, determine the speed of circulation (referring to Fig. 6 (A) and 6 (B)) of CFU-GM, BFU-E and CFU-GEMM as mentioned above similarly except the concentration of Lkn-1 changes to 20 micrograms from 0.1.As Fig. 6 (A) and 6 (B) as seen, proof is in marrow and spleen, CFU-GM, BFU-E and CFU-GEMM from the mouse of having accepted 3 to 10 microgram Lkn-1 are in acyclic or slow recurrent state, and do not change from the speed of circulation of the cell of the mouse of having accepted 0.1 to 1 microgram Lkn-1, and have only the speed of circulation of the CFU-GEMM of spleen to change.The speed of circulation that Lkn-1 reduces whole cell reaches 80 to 90%, and BFU-E and CFU-GEMM are seemingly more responsive to Lkn-1 comparison CFU-GM.

In addition, for these that study Lkn-1 suppress the time-dependent manner of effects, 8 microgram Lkn-1 are injected to C3H/HeJ mouse, respectively different time point detect from CFU-GM, the BFU-E of marrow and spleen and the cell cycle state of CFU-GEMM (referring to: Fig. 7 (A) is to 7 (F)).As Fig. 7 (A) to shown in 7 (F), the inhibition effect of Lkn-1 be depend on the time and in marrow and spleen, be reversible.That is, in 12 hours, Lkn-1 is in acyclic or slow recurrent state CFU-GM, BFU-E and CFU-GEMM, and after 72 hours they is returned to control level.

Lkn-1 suppresses effect to the dosage of proliferation of bone marrow cells in the body and time-dependent manner and reversible and shows strongly: Lkn-1 is not subjected to have the potential clinical application in the murder by poisoning of cytotoxicity cancer therapy drug or the radioactive radiation at the normal hematopoietic cell of protection.Embodiment 6: research is as the reorganization Lkn-1 of chemokine

For whether the Lkn-1 that studies reorganization may cause chemotaxis, utilize (1.077gm/ centimetre of Ficoll-Hypaque gradient ³) the centrifugal peripheral blood lymphocytes that obtains healthy man (PBMC).Then, be attached to the activity of frosting based on them, separating monocytic cell from the PBMC of acquisition like this repeats described separating step 2 times.In this experiment, cell residual behind the monocyte separating step obtains as lymphocyte.Determine the monocyte and the lymphocytic purity of acquisition like this by the microscopic examination of the painted cytospin of Diff-Quick (Baxter scientific company, the U.S.).As a result, find that monocyte and lymphocytic purity are respectively 90% and 88%.

Equally, utilize 3% Dextran T 500 (pharmacy fine chemistry company, the U.S.) to precipitate and utilize the Ficoll-Hypaque gradient centrifugation to obtain red blood cell.The red blood cell that dissolving so obtains in hypotonic solution is to obtain people's neutrophilic granulocyte.Determine the purity of the neutrophilic granulocyte of acquisition like this by morphology.As a result, find that purity is 95%.

Containing among the RPMI 1640 (Gibco, the U.S.) of 0.5% low endotoxin BSA (sigma chemical company, the U.S.) and 20 mmole Hepes respectively with 2 * 10 ⁶Cells/ml and 8 * 10 ⁶Isolating monocyte and lymphocyte above cells/ml suspends.In HBSS with 1 * 10 ⁶Cells/ml suspension neutrophilic granulocyte.

Then, the following level of determining cell migration in 48 hole microchambers (Neuroprobe, the U.S.).Only with buffered soln (contrast) or contain recombinate Lkn-1, hMIP-1 α (PeproTech, the U.S.), RANTES (PeproTech, the U.S.), IL-8 (PeProTech, the U.S.) or eotaxin (PeproTech, the U.S.) damping fluid is filled the following hole of microchamber, the hole above filling with the described cell suspending liquid of 50F1.Become the hole of lower floor and the hole on upper strata by the suitable strainer dispensing orifice that does not have polyvinylpyrrolidone.When utilizing neutrophilic granulocyte and lymphocyte, the diameter in the hole of strainer is 3Fm.When utilizing monocyte, it is 5Fm.At 37 ℃ of incubation described microchambers 1 hour (neutrophilic granulocyte), 2 hours (monocyte) or 4 hours (lymphocyte).From the chamber, take out strainer and washing.Be fixed on the cell on the strainer, and utilize Diff-Quick dyeing.Then, the number of counting cells (referring to: Fig. 8 (A) is to 8 (C)).

At Fig. 8 (A) in 8 (C), will be in the experimental group that chemokine is handled the number of migrating cell be chemotactic index divided by the value representation that the number of the migrating cell in the contrast obtains.Fig. 8 (A) shows that reorganization Lkn-1 and RANTES attract lymphocytic chemotactic figure.Fig. 8 (B) is that reorganization Lkn-1 and hMIP-1 α attract monocytic chemotactic figure.Fig. 8 (C) shows that reorganization Lkn-1 and IL-8 attract the chemotactic figure of neutrophilic granulocyte.

To shown in 8 (C), show that reorganization Lkn-1 is attractive peripheral blood neutrophilic granulocyte, monocyte and lymphocytic strong chemokine as Fig. 8 (A).And, reorganization Lkn-1 show the chemotaxis can attract neutrophilic granulocyte in mode similar in appearance to IL-8 (referring to: Fig. 8 (C)) and with the mode similar in appearance to hMIP-1 α attract monocytic chemotaxis (referring to: Fig. 8 (B)); And it has shown that relying on mode (concentration can up to the Lkn-1 of 10Fg/ milliliter) with concentration attracts the chemotaxis of lymphocytic raising, though the chemotaxis that demonstrates it than the chemotaxis level of RANTES low (referring to: Fig. 8 (A)).Embodiment 7: the analysis embodiment 7-1 that reorganization Lkn-1 influences calcium current: the analysis of calcium current in lymphocyte, monocyte and neutrophilic granulocyte

Whether studied reorganization Lkn-1 can induce calcium current to go out with activated lymphocyte, monocyte and neutrophilic granulocyte by bind receptor.Also detected competitive relation with the antagonist of other anti-acceptor.Utilize MSIII photofluorometer (Photon technology international corporation, the U.S.) to measure the variation of calcium ion in the subgroup (lymphocyte, monocyte, and neutrophilic granulocyte) of isolating peripheral blood leucocyte, thereby determine the activation of acceptor.That is, 37 ℃ with cell and 2FM fura-2AM (sigma chemical company, the U.S.) reaction 45 minutes, washed twice, in containing the HBSS of 0.05%BSA (pH7.4) with 1 * 10 ⁷The concentration resuspending of cells/ml.Stirred, add 2 ml cells suspension in the cover water cuvette, at 37 ℃, make it activation continuously in 340 nanometers and 380 nanometers.Before or after adding 25 nmoles/rise antagonist (reorganization Lkn-1, hMIP-1 α, RANTES, IL-8 or eotaxin), the fluorescence that is sent at 510 nano measurements (referring to Fig. 9 (A) to 9 (F)).

In 9 (F), relative fluorescence is expressed as the relative proportion at 340 nanometers and 380 nanometer activatory fluorescence at Fig. 9 (A).Fig. 9 (A) has shown the relative fluorescence of measuring in the lymphocyte of the Lkn-1 stimulation that is added a series of RANTES and reorganization; Fig. 9 (B) has shown the relative fluorescence of measuring in the lymphocyte that the Lkn-1 that added a series of reorganization and RANTES stimulate; Fig. 9 (C) has shown the relative fluorescence of measuring in the monocyte of the Lkn-1 stimulation that is added a series of hMIP-1 α and reorganization; Fig. 9 (D) has shown the relative fluorescence of measuring in the monocyte that the Lkn-1 that added a series of reorganization and hMIP-1 α stimulate; Fig. 9 (E) has shown the relative fluorescence of measuring in the neutrophilic granulocyte of the Lkn-1 stimulation that is added a series of IL-8 and reorganization; Fig. 9 (F) has shown the relative fluorescence that neutrophilic granulocyte that the Lkn-1 that added a series of reorganization and IL-8 stimulate is measured.Shown in Fig. 9 (A)-9 (F), find that the Lkn-1 that recombinates induces calcium current to go in lymphocyte, monocyte and neutrophilic granulocyte.

On the other hand, when the proteinic acceptor of coupling G contacts the short period of time with the part with binding site identical with receptor signal continuously, described receptor desensitization sense.When the described cell of expressed receptor was subjected to recombinating the stimulation of Lkn-1, such phenomenon just took place.

To shown in 9 (F), find when being subjected to RANTES and then hMIP-1 α stimulates that as Fig. 9 (A) Lkn-1 that recombinates makes the sense (referring to Fig. 9 (B) and 9 (D)) of desensitizing fully of lymphocyte and monocyte.But, when the Lkn-1 that is recombinated stimulates, RANTES or hMIP-1 α not exclusively make lymphocyte and monocyte desensitization sense (referring to Fig. 9 (A) and 9 (C)), and the shared acceptor of this proof reorganization Lkn-1 and RANTES and hMIP-1 α induced more calcium current than RANTES and hMIP-1 α.In addition, the Lkn-1 that recombinates in the further stimulating course of IL-8 does not make neutrophilic granulocyte desensitization sense, though the Lkn-1 of reorganization induced the calcium current in the neutrophilic granulocyte (referring to: Fig. 9 (F)).In addition, in the further stimulating course of reorganization Lkn-1 IL-8 do not make yet neutrophilic granulocyte desensitization sense (referring to: Fig. 9 (E)).So, though proved that clearly it is not in the strong factor of inducing calcium current in the neutrophilic granulocyte as IL-8 to the Lkn-1 that recombinates with the shared acceptor of IL-8.Embodiment 7-2: in the clone of expressing CC-chemokine receptor, analyze calcium current

Studied the clone whether reorganization Lkn-1 can activate the acceptor of expressing CC or CXC chemokine because in embodiment 7-1, analyze find that reorganization Lkn-1 has activated can be by RANTES and hMIP-1 α activated receptors.Except the HOS clone of using express recombinant CCR1, CCR2B, CCR3, CCR4, CCR5 or CXCR4 (referring to the reference reaction reagent program of AIDS research and NIH, the U.S.) replace using outside the isolating white corpuscle subgroup, to analyze similar in appearance to the mode of embodiment 7-1.Known hMIP-1 α combines with CCR1 and CCR5, and RANTES combines with CCR1, CCR3 and CCR5.

Figure 10 (A) has shown the relative fluorescence of measuring in the HOS clone of the expression CCR1 that is added a series of all ingredients stimulations, and Figure 10 (B) shows the relative fluorescence of measuring in the HOS clone of the expression CCR3 that is added a series of all ingredients stimulations.

Shown in Figure 10 (A) and 10 (B), show the Lkn-1 induced strong calcium current of in the HOS clone of expressing CCR1 or CCR3, recombinating.But, in the HOS clone of expressing other acceptor, do not observe reorganization Lkn-1 inductive calcium current.

Equally, shown in Figure 10 (A), after the Lkn-1 that recombinated stimulated, the CCR1-HOS cell no longer was subjected to the other stimulation of hMIP-1 α or RANTES, and stimulated back reorganization Lkn-1 unaffected to the further stimulation of CCR1-HOS cell at hMIP-1 α or RANTES.

Figure 10 (A) shows that reorganization Lkn-1 is that this can obtain replying clearly proof as the calcium of the concentration that depending on reorganization Lkn-1 and hMIP-1 α shown in Figure 10 (C) than hMIP-1 α or the RANTES anti-CCR1 antagonist of intensive more.

In addition, shown in Figure 10 (B), after eotaxin stimulates the CCR3-HOS cell, almost desensitization sense fully of CCR3-HOS cell in eotaxin or the further process that stimulates of reorganization Lkn-1; In the process that stimulates the back with reorganization Lkn-1 and further stimulate with RANTES, the CCR3-HOS cell is desensitization sense fully almost; But after with reorganization Lkn-1 or RANTES irritation cell, eotaxin does not but have influenced to the further stimulation of CCR3-HOS cell.In addition, calcium is replied the Lkn-1 that depends on eotaxin, reorganization and the concentration of RANTES; This point shows that reorganization Lkn-1 is the anti-CCR3 antagonist stronger than RANTES, though it be not than eotaxin stronger (referring to: Figure 10 (D)).

Clearly demonstrate and prove as top institute, the invention provides coding and belonging to the proteinic cDNA of new Lkn-1 of C6 beta-chemokine and the aminoacid sequence of deduction thus.113 amino acid of the open reading frame of Lkn-1 cDNA coding wherein contain 21 amino acid whose signal peptides and contain 92 amino acid whose molecular weight and are inferred as 10162 daltonian mature proteins.Utilize the Lkn-1 of described Lkn-1 cDNA in intestinal bacteria or expressed in insect cells reorganization, and with it purifying, its obviously suppresses colony and forms and cell proliferation, attract neutrophilic granulocyte, monocyte and lymphocyte and cause chemotaxis and can with CCR1 and CCR3 receptors bind.Therefore, determine to utilize reorganization Lkn-1 protein to protect the potential medicine of bone marrow stem cell and inhibition leukemia or the like as the treatment in antibody producing, HIV-1 course of infection, in chemotherapy or radiation treatment process.

Sequence table

(1) total information:

(i) applicant:

(A) title: The Green Cross Corporation; Deng the people

(B) street: 227, Gugal-Ri, Kiheung-Eup

(C) city: Yongin, Kyonggi-Do

(D) state: do not have

(E) country: Korea S

(F) postcode (ZIP): 449-900

(G) phone: 02-584-0131

(H) fax: 02-582-6331

(ii) invention exercise question: from the cDNA of people's separated coding C6 beta-chemokine Lkn-1

(iii) sequence number: 16

(iv) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM PC compatibility

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

(D) software: PatentIn Release#1.0, #1.30 version (EPO)

(2) information of SEQ ID NO:1:

(i) sequence signature

(A) length: 202 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) geometry: linearity

(ii) molecule type: DNA

(iv) antisense: do not have

(vi) primary source:

(A) organism: people

(vii) originate immediately:

(A) library: from people's genome dna library

(xi) sequence description of SEQ ID NO:1: CTGCCATCAG CAGAGAAAGG AAAAAACAGG CTGTGTTGAC TGGGAAATCT 50GAGGAGCAGG GAGGATGGGG CCCCCTGTCT CCATCTGCCC ACACCTCAGT 100TTGTAATCTT TCTCTCCCTT GTTCCCCAGA TTCCTCACCA AGAAGGGGCG 150GCAAGTCTGT GCCAAACCCA GTGGTCCGGG AGTTCAGGAT TGCATGAAAA 200AG 202

(2) information of SEQ ID NO:2:

(i) sequence signature:

(A) length: 24 amino acid

(B) type: amino acid

(D) geometry: linearity

(ii) molecule type: protein

(vi) primary source:

(A) organism; The people

(xi) sequence description of SEQ ID NO:2: Phe Leu Thr Lys Lys Gly Arg Gln Val Cys Ala Lys Pro Ser1 5 10Gly Pro Gly Val Gln Asp Cys Met Lys Lys15 20

(2) information of SEQ ID NO:3:

(i) sequence description:

(A) length: 24 amino acid

(B) type: amino acid

(D) geometry: linearity

(ii) molecule type: protein

(vi) primary source:

(A) organism: mouse

(xi) sequence description of SEQ ID NO:3: Phe Ile Ser Lys Arg Gly Phe Gln Val Cys Ala Asn Pro Ser1 5 10Asp Arg Arg Val Gln Arg Cys Ile Glu Arg15 20

(2) information of SEQ ID NO:4:

(i) sequence signature:

(A) length: 582 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) geometry: linearity

(ii)DNA

(iv) antisense: do not have

(vi) primary source:

(A) organism: people

(vii) originate immediately:

(A) library: from people's DNA library

( xi ) SEQ ID NO：4：CGGAGCCAGG AAGCAGTGAG CCCAGGAGTC CTCGGCCAGC CCTGCCTGCC 50CACCAGGAGG ATGAAGGTCT CCGTGGCTGC CCTCTCCTGC CTCATGCTTA 100TTGCTGTCCT TGGATCCCAG GCCCAGTTCA CAAATGATGC AGAGACAGAG 150TTAATGATGT CAAAGCTTCC ACTGGAAAAT CCAGTAGTTC TGAACAGCTT 200TCACTTTGCT GCTGACTGCT GCACCTCCTA CATCTCACAA AGCATCCCGT 250GTTCACTCAT GAAAAGTTAT TTTGAAACGA GCAGCGAGTG CTCCAAGCCA 300GGTGTCATAT TCCTCACCAA GAAGGGGCGG CAAGTCTGTG CCAAACCCAG 350TGGTCCGGGA GTTCAGGATT GCATGAAAAA GCTGAAGCCC TACTCAATAT 400AATAATAAAC AGACAAAAGA GGCCAGCCAC CCACCTCCAA CACCTCCTGT 450GAGTTTCTTG GTCTGAAATA CTTAAAAAAT ATATATATTG TTGTGTCTGG 500TAATGAAAGT AATGCATCTA ATAAAGGTAT TCAATTTTTT AACTTTGCTT 550GAGTTTTAAG AGGAAATAAA CTAATATAAA AC 582

(2) information of SEQ ID NO:5:

(i) sequence signature:

(A) length: 113 amino acid

(B) type: amino acid

(D) geometry: linearity

(ii) molecule type: protein

(vi) primary source:

(A) organism: people

(xi) sequence description of SEQ ID NO:5: Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu Ile1 5 10Ala Val Leu Gly Ser Gln Ala Gln Phe Thr Asn Asp Ala Glu15 20 25Thr Glu Leu Met Met Ser Lys Leu Pro Leu Glu Asn Pro Val

30??????????????????35??????????????????40Val?Leu?Asn?Ser?Phe?His?Phe?Ala?Ala?Asp?Cys?Cys?Thr?Ser

45??????????????????50??????????????????55Tyr?Ile?Ser?Gln?Ser?Ile?Pro?Cys?Ser?Leu?Met?Lys?Ser?Tyr

60??????????????????65??????????????????70Phe?Glu?Thr?Ser?Ser?Glu?Cys?Ser?Lys?Pro?Gly?Val?Ile?Phe

75??????????????????80Leu?Thr?Lys?Lys?Gly?Arg?Gln?Val?Cys?Ala?Lys?Pro?Ser?Gly85??????????????????90??????????????????95Pro?Gly?Val?Gln?Asp?Cys?Met?Lys?Lys?Leu?Lys?Pro?Tyr?Ser100?????????????????105?????????????????110Ile

(2) information of SEQ ID NO:6:

(i) sequence signature:

(A) length: 342 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) geometry: linearity

(ii) molecule type: DNA

(iv) antisense: do not have

(vi) primary source:

(A) organism: people

(vii) originate immediately:

(A) library: from people's DNA library

(xi) sequence description of SEQ ID NO:6: ATGAAGGTCT CCGTGGCTGC CCTCTCCTGC CTCATGCTTA TTGCTGTCCT 50TGGATCCCAG GCCCAGTTCA CAAATGATGC AGAGACAGAG TTAATGATGT 100CAAAGCTTCC ACTGGAAAAT CCAGTAGTTC TGAACAGCTT TCACTTTGCT 150GCTGACTGCT GCACCTCCTA CATCTCACAA AGCATCCCGT GTTCACTCAT 200GAAAAGTTAT TTTGAAACGA GCAGCGAGTG CTCCAAGCCA GGTGTCATAT 250TCCTCACCAA GAAGGGGCGG CAAGTCTGTG CCAAACCCAG TGGTCCGGGA 300GTTCAGGATT GCATGAAAAA GCTGAAGCCC TACTCAATAT AA 342

(2) information of SEQ IDNO:7:

(i) sequence signature:

(A) length: 92 amino acid

(B) type: amino acid

(D) geometry: linearity

(ii) molecule type: protein

(vi) primary source:

(A) organism: people

(xi) sequence description of SEQ ID NO:7: Gln Phe Thr Asn Asp Ala Glu Thr Glu Leu Met Met Ser Lys1 5 10Leu Pro Leu Glu Asn Pro Val Val Leu Asn Ser Phe His Phe15 20 25Ala Ala Asp Cys Cys Thr Ser Tyr Ile Ser Gln Ser Ile Pro

30??????????????????35??????????????????40Cys?Ser?Leu?Met?Lys?Ser?Tyr?Phe?Glu?Thr?Ser?Ser?Glu?Cys

45??????????????????50??????????????????55Ser?Lys?Pro?Gly?Val?Ile?Phe?Leu?Thr?Lys?Lys?Gly?Arg?Gln

60??????????????????65??????????????????70Val?Cys?Ala?Lys?Pro?Ser?Gly?Pro?Gly?Val?Gln?Asp?Cys?Met

75??????????????????80Lys?Lys?Leu?Lys?Pro?Tyr?Ser?Ile85??????????????????90

(2) information of SEQ ID NO:8:

(i) sequence signature:

(A) length: 95 amino acid

(B) type: amino acid

(D) geometry: linearity

(ii) molecule type: protein

(vi) primary source:

(A) organism: mouse

(xi) sequence description of SEQ ID NO:8: Gly Leu Ile Gln Ile Met Glu Lys Glu Asp Arg Arg Tyr Asn1 5 10Pro Pro Ile Ile His Gln Gly Phe Gln Asp Thr Ser Ser Asp15 20 25Cys Cys Phe Ser Tyr Ala Thr Gln Ile Pro Cys Lys Arg Phe

30??????????????????35??????????????????40Ile?Tyr?Tyr?Phe?Pro?Thr?Ser?Gly?Gly?Cys?Ile?Lys?Pro?Gly

45??????????????????50??????????????????55Ile?Ile?Phe?Ile?Ser?Arg?Arg?Gly?Thr?Gln?Val?Cys?Ala?Asp

60??????????????????65??????????????????70Pro?Ser?Asp?Arg?Arg?Val?Gln?Arg?Cys?Leu?Ser?Thr?Leu?Lys

75??????????????????80Gln?Gly?Pro?Arg?Ser?Gly?Asn?Lys?Val?Ile?Ala85??????????????????90??????????????????95

(2) information of SEQ ID NO:9:

(i) sequence signature:

(A) length: 101 amino acid

(B) type: amino acid

(D) geometry: linearity

(ii) molecule type: protein

(vi) primary source:

(A) organism: mouse

(xi) sequence description of SEQ ID NO:9: Gln Ile Thr His Ala Thr Glu Thr Lys Glu Val Gln Ser Ser1 5 10Leu Lys Ala Gln Gln Gly Leu Glu Ile Glu Met Phe His Met15 20 25Gly Phe Gln Asp Ser Ser Asp Cys Cys Leu Ser Tyr Asn Ser

30??????????????????35??????????????????40Arg?Ile?Gln?Cys?Ser?Arg?Phe?Ile?Gly?Tyr?Phe?Pro?Thr?Ser

45??????????????????50??????????????????55Gly?Gly?Cys?Thr?Arg?Pro?Gly?Ile?Ile?Phe?Ile?Ser?Lys?Arg

60??????????????????65??????????????????70Gly?Phe?Gln?Val?Cys?Ala?Asn?Pro?Ser?Asp?Arg?Arg?Val?Gln

75??????????????????80Arg?Cys?Ile?Glu?Arg?Leu?Glu?Gln?Asn?Ser?Gln?Pro?Arg?Thr85??????????????????90???????????????????95Tyr?Tyr?Lys

100

(2) information of SEQ ID NO:10:

(i) sequence signature:

(A) length: 67 amino acid

(B) type: amino acid

(D) geometry: linearity

(ii) molecule type: protein

(vi) primary source:

(A) organism: people

(xi) sequence description of SEQ ID NO:10: Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser1 5 10Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile Ala Asp Tyr15 20 25Phe 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

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

60??????????????????65

(2) information of SEQ ID NO:11:

(i) sequence signature:

(A) length: 66 amino acid

(B) type: amino acid

(D) geometry: linearity

(ii) molecule type: protein

(vi) primary source:

(A) organism: mouse

(xi) sequence description of SEQ ID NO:11: Ala Pro Tyr Gly Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser1 5 10Tyr Ser Arg Lys Ile Pro Arg Gln Phe Ile Val Glu Val Phe15 20 25Glu Thr Ser Ser Leu Cys Ser Gln Pro Gly Val Ile Phe Leu

30??????????????????35??????????????????40Thr?Lys?Arg?Asn?Arg?Gln?Ile?Cys?Ala?Asp?Ser?Lys?Glu?Thr

45??????????????????50??????????????????55Trp?Val?Gln?Glu?Tyr?Ile?Thr?Asp?Leu?Glu

60??????????????????65

(2) information of SEQ ID NO:12:

(i) sequence signature:

(A) length: 67 amino acid

(B) type: amino acid

(D) geometry: linearity

(ii) molecule type: protein

(vi) primary source:

(A) organism: people

(xi) sequence description of SEQ ID NO:12: Ala Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cys Phe Ser1 5 10Tyr Thr Ala Arg Lys Leu Pro Arg Asn Phe Val Val Asp Tyr15 20 25Tyr 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

45??????????????????50??????????????????55Ser?Trp?Val?Gln?Glu?Val?Tyr?Tyr?Asp?Leu?Glu

60??????????????????65

(2) information of SEQ ID NO:13:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) geometry: linearity

(ii) molecule type: DNA

(vii) originate immediately:

(B) clone: primer

(xi) sequence description of SEQ ID NO:13:

TTCCTCACCA?AGAAGGGG

(2) information of SEQ ID NO:14:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) geometry: linearity

(ii) molecule type: DNA

(vii) originate immediately:

(B) clone: primer

(xi) sequence description of SEQ ID NO:14:

CTTTTTCATG?CAATCCTG

(2) information of SEQ ID NO:15:

(i) sequence signature:

(A) length: 34 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) geometry: linearity

(ii) molecule type: DNA

(vii) originate immediately:

(B) clone: primer

(xi) sequence description of SEQ ID NO:15:

CGAATTCCAT?ATGCAGTTCA?CAAATGATGC?AGAG

(2) information of SEQ ID NO:16:

(i) sequence signature:

(A) length: 28 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) geometry: linearity

(ii) molecule type: DNA

(vii) originate immediately:

(B) clone: primer

(xi) sequence description of SEQ ID NO:16:

CGCCGCTCGA?GTTGAGTAGG?GCTTCAGC

Claims

1. the cDNA of people C6 beta-chemokine Lkn-1, its nucleotide sequence is expressed as follows (SEQ ID NO:6): ATG AAG GTC TCC GTG GCT GCC CTC TCC TGC CTC ATG CTT 39ATT GCT GTC CTT GGA TCC CAG GCC CAG TTC ACA AAT GAT 78GCA GAG ACA GAG TTA ATG ATG TCA AAG CTT CCA CTG GAA 117AAT CCA GTA GTT CTG AAC AGC TTT CAC TTT GCT GCT GAC 156TGC TGC ACC TCC TAC ATC TCA CAA AGC ATC CCG TGT TCA 195CTC ATG AAA AGT TAT TTT GAA ACG AGC AGC GAG TGC TCC 234AAG CCA GGT GTC ATA TTC CTC ACC AAG AAG GGG CGG CAA 273GTC TGT GCC AAA CCC AGT GGT CCG GGA GTT CAG GAT TGC 312ATG AAA AAG CTG AAG CCC TAC TCA ATA TAA 342

2. people C6 beta-chemokine Lkn-1, its aminoacid sequence is expressed as follows (SEQ IDNO:5): Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu Ile1 5 10Ala Val Leu Gly Ser Gln Ala Gln Phe Thr Asn Asp Ala Glu15 20 25Thr Glu Leu Met Met Ser Lys Leu Pro Leu Glu Asn Pro Val

75??????????????????80Leu?Thr?Lys?Lys?Gly?Arg?Gln?Val?Cys?Ala?Lys?Pro?Ser?Gly85??????????????????90??????????????????95Pro?Gly?Val?Gln?Asp?Cys?Met?Lys?Lys?Leu?Lys?Pro?Tyr?Ser

100?????????????????105?????????????????110

3. the cDNA of people C6 beta-chemokine Lkn-1 has wherein lacked the 1st to the 63rd nucleotide sequence.

4. the expression vector that comprises the cDNA of claim 1.

5. the expression vector PVL 1392-Lkn-1 that comprises the cDNA of claim 1.

6. the method for preparing the Lkn-1 that recombinates comprises with the described expression vector transformed host cell of claim 4, cultivates transformant, and the Lkn-1 that obtains reorganization from culture.

7. method according to claim 6, wherein said host cell are intestinal bacteria or insect cell.

8.-plant the Lkn-1 of reorganization, it is to be prepared by the method that comprises the steps: with the described expression vector transformed host cell of claim 4, cultivate transformant, from culture, obtain the Lkn-1 of reorganization.

9. the Lkn-1 of reorganization according to claim 8, its suppresses colony and forms.

10. the Lkn-1 of reorganization according to claim 8, neutrophilic granulocyte, monocyte and lymphocyte in its strong attractive peripheral blood and cause chemotaxis.

11. the Lkn-1 of reorganization according to claim 8, it and CCR1 and CCR3 receptors bind.

12. reorganization Lkn-1 according to claim 11, it and RANTES (being activated the sequence of the normal T cell expressing of adjusting) and hMIP-1 α (human macrophage inflammatory protein-1 α) share acceptor, and do not share acceptor with IL-8 (interleukin 8).

13. an expression vector, it comprises the described cDNA of claim 3.

14. comprise the expression vector pET21a-Lkn-1 of the described cDNA of claim 3.

15. intestinal bacteria (XL-1 Blue) hMRP-2 (ATCC 98166) with the described expression vector conversion of claim 14.

16. the method for the Lkn-1 of preparation reorganization, this method comprises the steps: to utilize the described expression vector transformed host cell of claim 13, the Lkn-1 that cultivates transformant and obtain to recombinate from culture.

17. method according to claim 16, wherein said host cell are intestinal bacteria or insect cell.

18. the Lkn-1 of a reorganization, it is to be prepared by the method that comprises the steps: with the described expression vector transformed host cell of claim 13, and the Lkn-1 that cultivates transformant and from culture, obtain to recombinate.

19. the Lkn-1 of reorganization according to claim 18, it suppresses colony and forms.

20. the Lkn-1 of reorganization according to claim 18, neutrophilic granulocyte, monocyte and lymphocyte in its strong attractive peripheral blood and to cause chemotaxis.

21. the Lkn-1 of the described reorganization of claim 18, it and CCR1 and CCR3 receptors bind.

22. the Lkn-1 of reorganization according to claim 21, it and RANTES (being activated the sequence of the normal T cell expressing of adjusting) and hMIP-1 α (human macrophage inflammatory protein-1 α) share acceptor, and do not share acceptor with IL-8 (interleukin 8).

23. the protection medullary cell is not poisoned or the radiating method by the cytotoxicity cancer therapy drug, comprises with people C6 beta-chemokine Lkn-1 to needing shielded individual administration.

24. method according to claim 23, wherein said people C6 beta-chemokine Lkn-1 is the Lkn-1 of the described reorganization of claim 8.

25. method according to claim 23, wherein people C6 beta-chemokine Lkn-1 is the Lkn-1 of the described reorganization of claim 18.