KR20010022352A - A cDNA ENCODING C6 β-CHEMOKINE LEUKOTACTIN-1(LKN-1) ISOLATED FROM HUMAN - Google Patents

Abstract

PURPOSE: 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. CONSTITUTION: 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 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

A cDNA ENCODING C6 β-CHEMOKINE LEUKOTACTIN-1 (LKN-1) ISOLATED FROM HUMAN}, which encodes a cDNA encoding the С6 β-chemokine Lｋｎ-1 isolated from humans (Ｌｅｕｋｏｔａｃｔｉｎ-1).

Chemokines are members of the cytokine family of low molecular weight basic proteins, which have four cysteines in common, and whether the first two cysteines are adjacent to each other, or whether an amino acid is located between two cysteines. Thus, it is classified into four subfamily of CXC (α), CC (β), C (γ) and CX ₃ C (Baggiolini M. et al., Immunol. Today, 15: 127, 1994). Kelner SG, et al., Science, 266: 1395, 1994; Bazan JF et al., Nature, 385: 640, 1997). The chemokine subfamily genes are clustered on the same chromosome, for example, the α-chemokine gene is on human chromosome 4q12-21 and the β-chemokine gene is on human chromosome 17q11-32 and mouse chromosome 11 Located.

These chemokines have been reported to exhibit biological activities such as HIV-inhibitory activity, immunomodulatory activity, leukocyte migration or inhibiting the division of hematopoietic stem cells (Cocci F. et al., Science, 270: 1811, 1995; Wolpe SD et al., J. Exp. Med., 167: 570, 1998; Graham GJ et al., Nature, 344: 442, 1990; Broxmeyer HE et al., Blood, 76: 1110, 1990; and Youn BS et al., J. Immunol., 155: 2661, 1995)

In addition, chemokines bind to transmembrane domain G protein-coupled receptors to activate leukocytes, some of which are reported to be used as coreceptors in HIV-1 infection. (Oh K.-O. et al., J. Immunol., 147: 2978, 1991; Alkbatih G. et ak., Science, 272; 1955, 1996). For example, there are eight receptor subtypes of β-chemokine (CC chemokine), namely CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR8, of which CCR1, CCR2, CCR3 and CCR5 are present in various chemokines. Although exhibiting binding capacity, CCR4, CCR6 and CCR8 have been found to exhibit high affinity for one chemokine.

To date, nine chemokines belonging to the β-chemokine subfamily have been identified (Wild SD et al., J. Exp. Med., 171: 1301, 1990; Modi WS et al., Hum. Genet., 84 : 185, 1990), among them murine macrophage inflammatory protein-related protein-1, mMRP-1 (Orlafsky A. et al., Cell Regul., 2: 403, 1991) and murine MRP -2 (mMRP-2) (Youn B.-S. et al., J. Immunol., 155: 2661, 1995) has two more cysteine residues than other chemokines belonging to the β-chemokine subsequence. Tertiary disulfide bonds are possible, and the N-terminal region is quite long. These MRPs were classified as C6 β-chemokines.

Currently, only MRP-1 and MRP-2 have been reported for human MRPs, but MRPs that can be applied for the treatment of HIV-1 infection or for the protection of bone marrow stem cells during chemotherapy or radiation therapy. Because of their potential utility, isolation and characterization of new human MRPs is urgently needed.

Summary of the Invention

Accordingly, the present inventors have made efforts to isolate novel MRP genes from various human cell lines. As a result, the present inventors have isolated new MRP cDNAs belonging to C6 β-chemokine from human mononuclear cell lines, and their base sequences and amino acid sequences inferred therefrom. In addition, the electric MRP cDNA was expressed in recombinant E. coli or insect cells. In addition, the expressed recombinant protein inhibits the proliferation and colony formation of myeloid stem cells in vitro and in vivo, and a subset of peripheral blood leukocytes (lymphocytes, monocytes). And neutrophils) were confirmed to have a chemotaxis to complete the present invention. Thus, hereinafter, the electric MRP cDNA isolated from human was named "Lkn-1 (leukotactin-1) cDNA", and recombinant MRP was named "recombinant Lkn-1".

After all, the first object of the present invention is to provide cDNA of novel C6 β-chemokine (Lkn-1) and amino acid sequences inferred therefrom.

It is a second object of the present invention to provide an expression vector comprising the aforementioned Lkn-1 cDNA and a recombinant microorganism transformed therefrom.

It is a third object of the present invention to provide a method for preparing recombinant Lkn-1 from an electrotransformed recombinant microorganism.

A fourth object of the present invention is to provide a method of protecting myeloid cells from anticancer agents and radiation using recombinant Lkn-1.

The present invention relates to a method for producing cDNA and C6 β-chemokine of novel C6 β-chemokine isolated from humans. More specifically, the present invention relates to the preparation and use of electrical proteins with expression vectors of cDNA belonging to C6 β-chemokine and encoding novel proteins that attract subsets of peripheral blood leukocytes.

1A shows the nucleotide sequence of the 202 bp DNA fragment (SEQ ID NO: 1) and the amino acid sequence inferred therefrom (SEQ ID NO: 2).

FIG. 1B shows a comparison of the amino acid sequence of Lkn-1 (SEQ ID NO: 2) with the 87th to 110th amino acid sequences of mMRP-2 (SEQ ID NO: 3).

Figure 2 is a photograph showing the results of reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA isolated from various human cell lines.

3 shows the nucleotide sequence of Lkn-1 cDNA (SEQ ID NO: 4) and the amino acid sequence inferred therefrom (SEQ ID NO: 5).

4 shows the amino acid sequence of mature Lkn-1 (SEQ ID NO: 7), mMRP-1 (SEQ ID NO: 8), mMRP-2 (SEQ ID NO: 9), hMIP-1α (SEQ ID NO: 10), and mMIP-1α (SEQ ID NO: 11). ) Or the amino acid sequence of hMIP-1β (SEQ ID NO: 12).

Figure 5a is a graph showing the effect of Lkn-1 on the colonization of myeloid cells in the bone marrow.

Figure 5b is a graph showing the effect of Lkn-1 on the colonization of myeloid cells in the spleen.

5c is a graph showing the effect of Lkn-1 on the proliferation rate of myeloid cells in bone marrow.

5D is a graph showing the effect of Lkn-1 on the proliferation rate of myeloid cells in the spleen.

Figure 6a is a graph showing the concentration-dependent inhibitory effect of Lkn-1 on the proliferation of myeloid cells in bone marrow.

Figure 6b is a graph showing the concentration-dependent inhibitory effect of Lkn-1 on the proliferation of myeloid cells in the spleen.

7A is a graph showing the time dependent inhibition of Lkn-1 on the proliferation rate of CFU-GM in bone marrow.

7B is a graph showing the time-dependent inhibition of Lkn-1 on the proliferation rate of BFU-E in bone marrow.

7C is a graph showing the time-dependent inhibition of Lkn-1 on the proliferation rate of CFU-GEMM in bone marrow.

7D is a graph showing the time-dependent inhibition of Lkn-1 on the proliferation rate of CFU-GM in the spleen.

7E is a graph showing the time dependent inhibition of Lkn-1 on the proliferation rate of BFU-E in the spleen.

7F is a graph showing the time dependent inhibition of Lkn-1 on the proliferation rate of CFU-GEMM in the spleen.

8A is a graph showing lymphocytes chemically attracted by recombinant Lkn-1 and regulated-upon activation, normal T cell expressed and secreted (RANTES).

8B is a graph showing monocytes chemically attracted by recombinant Lkn-1 and hMIP-1α.

8C is a graph showing neutrophils chemically attracted by recombinant Lkn-1 and IL-8.

9A is a graph showing the relative fluorescence of lymphocytes stimulated by a series of RANTES and recombinant Lkn-1.

9B is a graph showing the relative fluorescence of lymphocytes stimulated by a series of recombinant Lkn-1 and RANTES.

9C is a graph showing the relative fluorescence of monocytes stimulated by a series of hMIP-1α and recombinant Lkn-1.

9D is a graph showing the relative fluorescence of monocytes stimulated by a series of recombinant Lkn-1 and hMIP-1α.

9E is a graph showing the relative fluorescence of neutrophils stimulated by a series of IL-8 and recombinant Lkn-1.

9F is a graph showing the relative fluorescence of neutrophils stimulated by a series of recombinant Lkn-1 and IL-8.

10A is a graph showing the relative fluorescence of HOS cell lines expressing CCR1 stimulated by a series of different substances.

10B is a graph showing the relative fluorescence of HOS cell lines expressing CCR3 stimulated by a series of different substances.

Figure 10c is a graph showing the peak height of the reaction of calcium with the concentration of Lkn-1 and hMIP-1α.

Figure 10d is a graph showing the peak height of the reaction of calcium with the concentration of Lkn-1, eotaxin and RANTES.

In order to isolate the novel Lkn-1 gene from human cell lines, we first cloned the exon region of the Lkn-1 gene to be used for the provision of a probe. In other words, human genomic DNA was digested with HindIII restriction enzymes, isolated on agarose gels, and labeled with ³² P mMRP-2 cDNA (Youn BS et al., J. Immunol., 155: 2661, 1995). Southern blot was performed using the probe as a probe to obtain a 7.0 kb DNA fragment hybridized with mMRP-2. Then, in order to separate the exon sequence of Lkn-1 cDNA from the 7.0 kb HindIII DNA fragment, the human genomic DNA was completely cleaved with HindIII and separated on agarose gel, and then the fragment was inserted into a vector and introduced into the host cell. After incubation, colonies exhibiting hybridization with the mMRP-2 cDNA probe were selected. 7.0 kb DNA fragments were isolated from selected colonies, cleaved with AluI, cloned 202 bp DNA fragments hybridized with mMRP-2 cDNA to determine sequencing, and then intron and exon. The part was confirmed.

Then, to clone the novel Lkn-1 cDNA from human cell lines, we devised a PCR primer of Lkn-1 capable of amplifying 100 bp from the Lkn-1 exon sequence identified above and isolated from various human cell lines. RT-PCR (reverse transcriptase-polymerase chain reaction) was carried out using the total RNA as a template to select cell lines in which mRNA of Lkn-1 was detected. The cDNA library of human mononuclear THP-1 cell line activated with IL-4 (interleukin-4) among the selected cell lines was prepared, while the total RNA of the electric THP-1 cell line was used as a template, and the Lkn 100-bp DNA fragments of Lkn-1 exons were prepared by RT-PCR using -1 PCR primers. As a result of hybridizing the cDNA library of the prepared THP-1 cell line with 100 bp DNA fragment of the probe Lkn-1 exon, Lkn-1 cDNA showing a positive signal was found.

As a result of determining the nucleotide sequence of the Lkn-1 cDNA and the amino acid sequence inferred therefrom, the Lkn-1 cDNA is a novel one that has not been reported so far, and the open reading frame of the Lkn-1 cDNA is 21 amino acids. Including a signal peptide consisting of a total of 113 amino acids are encoded, of which 92 amino acids of the mature Lkn-1 protein molecular weight was estimated to be 10,162 Da. In addition, Lkn-1 cDNA does not have an N-glycosylation site and adds two cysteine residues found in mMRP-1 and mMRP-2 in addition to the four cysteine residues shared by the β-chemokine family. The branch was found to belong to the C6 β-chemokine family.

The cloned novel Lkn-1 cDNA was inserted into an expression vector and introduced into recombinant Lkn-1 by introducing it into host cells such as E. coli or insect cells.

On the other hand, the inhibition of myeloid cell proliferation of recombinant Lkn-1 was performed in progenitor cells or myeloid stem cells derived from bone marrow and spleen. In vitro conditions, recombinant Lkn-1 inhibited the concentration and time-dependent colony formation and proliferation of bone marrow cells. In addition, purified recombinant Lkn-1 showed potent chemotaxis against human peripheral blood neutrophils, monocytes and lymphocytes, and induced calcium outflow from these cells. In particular, recombinant Lkn-1 exhibits a strong chemotaxis against neutrophils like IL-8 and binds to receptors of regulated-upon activation, normal T cell expressed and secreted (RANTES) and human macrophage inflammatory protein (hMIP) -1α. It did not appear to bind to the receptor of IL-8. In addition, the receptors of recombinant Lkn-1 were found to be CCR1 and CCR3. Recombinant Lkn-1 is an agonist of the CCR1 receptor that is more potent than hMIP-1α or RANTES, which is known to bind to CCC chemokine receptor 1 (CCR1) receptors, but does not reach eotaxin for the CCR3 receptor. It has been found to be a much more potent agent than RANTES.

The recombinant Lkn-1 of the present invention having the above-described characteristics can be used for antibody production, treatment during HIV-1 infection, bone marrow hepatocyte protection during chemotherapy or radiation therapy, and inhibition of leukemia.

Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Cloning of Exon Sites in Lkn-1 Genomic DNA

To clone genomic genes with sequencing homology with mMRP-2 from humans, human total genomic DNA was digested with BamHI, EcoRI, HindIII, PstI, XbaI and XhoI, and fractionated on 1.0% agarose gels. , Southern blot using a ³² P-labeled mMRP2 cDNA as a probe was performed to obtain a 7.0 kb HinIII fragment showing a positive signal.

To prepare a sublibrary of the HindIII fragment, 100 μg of human genomic DNA was completely cleaved with HindIII, fractionated on 1% agarose gel for 16 hours at 20V voltage, and then DNA near 7.0 kb was Gene-clean. Purified by kit (Bio 101, USA) and inserted into dephosphorylated pBluescript SK ⁺ vector (Stratagene, USA) digested with HindIII. The recombinant vector thus prepared was introduced into an electro-transformable cell (GIBCO-BRL, USA) by electroporation, cultured in a solid medium, and then colonized to about 2 × 10 ⁵ . Was hybridized with the mMRP-2 probe, and only one colony showed a hybridization positive signal, and the colony contained 7.0 kb of insert DNA.

To separate the exon sequence from the 7.0 kb genomic DNA fragment, the pBluescript SK ⁺ vector containing 7.0 kb of insert DNA was isolated from the colonies showing hybridization positive signals, cleaved with AluI, on 1.5% agarose gels. Fractionation was followed by Southern blot analysis to clone a 202 bp DNA fragment that hybridized with mMRP-2 cDNA.

The base sequence (see SEQ ID NO: 1) of the 202 bp DNA fragment was determined to identify the exon moiety translated into intron and amino acid (see FIG. 1A). This exon region has a amino acid sequence from 87 to 110 that is 50% homologous to mMRP and is the second of two additional cysteines that are common to mMRP-1 and mMRP-2. It is confirmed that "is stored" in Lkn-1 (see Figure 1b). In Figure 1b, the squares represent amino acids that are conserved between Lkn-1 and mMRP-2, respectively.

Example 2: Cloning of Lkn-1 cDNA

To clone Lkn-1 cDNA from human cell lines, firstly a PCR primer, ie forward primer 5′-TTCCTCACCAAGAAGGGG-, capable of amplifying 100 bp of Lkn-1 exon from the amino acid sequence shown in FIG. 1B (see SEQ ID NO: 2) 3 ′ (see SEQ ID NO: 13) and reverse primer 5′-CTTTTTCATGCAATCCTG-3 ′ (see SEQ ID NO: 14) were devised, followed by PCR of the 202 bp DNA fragment prepared in Example 1, wherein 100 μg / Total RNA of human mononuclear THP-1 cell line (ATCC TIB202), macrophage cell line U937 (ATCC CRL1593) and HL-60 cell line stimulated for 24 hours with ml of IL-4 (interleukin-4) PCR was performed with the total RNA of ATCC CCL240) as a template (see Figure 2).

In Figure 2, lanes 1 to 4 are positive controls and in turn, lane 2 is the total RNA of THP-1 cell line, lane 3 is the total RNA of U937 cell line, and lane 4 is the total RNA of HL-60 cell line. And M-lane represent 100 bp ladders as DNA size markers, respectively. As shown in Figure 2, it can be seen that the THP-1 cell line activated with IL-4 produces Lkn-1 mRNA.

In this regard, in order to prepare a THP-1 cDNA library, human mRNA was isolated from an IL-4 activated THP-1 cell line, from which a double-chained protein was purified using a Time Saver cDNA kit (Pharmacia Biotech., USA). Reverse transcription with cDNA was followed by attachment of a BstXI adapter (Invitrogen, USA). Subsequently, at least 0.5kb of cDNA was isolated on an agarose gel and inserted into a PRc / CMV (Invitrogen, USA) vector digested with BstXI to prepare a cDNA library. The PCR amplified Lkn-1 exon l 100bp Hybridization was performed using the DNA fragment as a probe. As a result, one cDNA showing a positive signal, that is, Lkn-1 cDNA, was isolated.

Example 3: Analysis of Lkn-1 cDNA Sequences

The base sequence of Lkn-1 cDNA cloned in Example 2 (see SEQ ID NO: 4) and the amino acid sequence inferred therefrom (see SEQ ID NO: 5) are shown in FIG. In Figure 3, the underlined portions are signal peptides; The square part is the part used as a probe in the screening of the THP-1 cDNA library and the Lkn-1 amino acid sequence of FIG. 1B (see SEQ ID NO: 2); And ( ) Indicates a translation stop codon.

As shown in FIG. 3, the open reading frame of Lkn-cDNA was composed of a total of 113 amino acids consisting of a signal peptide of 21 amino acids and a mature protein of 92 amino acids estimated to have a molecular weight of 10,161 Da. In addition, the inferred Lkn-1 amino acid sequence (SEQ ID NO: 5) did not have an N-glycosylation site.

4 shows the amino acid sequence of mature Lkn-1 (see SEQ ID NO: 7) for mMRP-1 (see SEQ ID NO: 8), mMRP-2 (SEQ ID NO: 9), hMIP-1α (see SEQ ID NO: 10). (Youn BS et al., J. immunol., 155: 2661, 1995), mMIP-1α (SEQ ID NO: 11) (Kwon BS et al., Proc. Natl. Acad. Sci. USA, 86: 1963, 1989) and hMIP-1β (SEQ ID NO: 12) (Sherry B. et al., J. Exp. Med., 168: 2251. 1988). In Fig. 4, the square part represents four conserved cysteine residues, and (.) Represents two additional cysteine residues. As shown in FIG. 4, Lkn-1 has a long N-terminal in front of the first two cysteine residues among the four cysteine residues shared by the chemokine family, and besides the four cysteine residues shared by the β-chemokine family, mMRP-1 And C6 β-chemokine family having two cysteine residues found in mMRP-2.

In addition, the nucleotide sequence of mature Lkn-1 isolated from human (SEQ ID NO: 7) corresponds to mMRP-2, but has only about 43% sequence homology with mMRP-1 or mMRP-22, and hMIP- It was found to have 40% amino acid homology with 1α.

Example 4: Expression of Recombinant Lkn-1

Example 4-1 Expression and Purification of Recombinant Lkn-1 in Escherichia Coli

In order to express only mature Lkn-1 as a recombinant protein except a portion estimated to be a signal peptide, the cloned Lkn-1 cDNA in Example 2 was used as a template, and the following PCR primers and Pfu polymerase (Stratagene, USA) were used. The open reading frame of mature Lkn-1 was PCR:

Forward Primer 5'-CGAATTCCATATGCAGTTCACAAATGATGCAGAG-3 '

Reverse primer 5'-CGCCGCTCGAGTTGAGTAGGGCTTCAGC-3 '

The amplified DNA fragment was digested with NdeI / XhoI restriction enzyme, cloned into plasmid pET21a (Novagen, U.S.A), and the recombinant plasmid was named pET21a-Lkn-1 and introduced into E. coli XL-1 Blue. The recombinant plasmid pET21a-Lkn-1 prepared as described above expresses recombinant Lkn-1 having one methionine residue at the N-terminus of mature Lkn-1 and six histidines added at the C-terminus.

The transformant thus prepared was named 'Escherichia coli (XL-1 Blue) hMRP-2' and was deposited on September 10, 1996 under the accession number ATCC 98166 to the American Type Culture Collection (ATCC).

After culturing the transformants to express Lkn-1, the inclusion bodies were obtained and dissolved in 20 ml denatured buffer (6M guanidine-HCl, 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 4 mM n-octylglucopyranoside). The supernatant obtained by centrifugation was chromatographed using activated Ni-column (Novagen, USA) and heparin agarose column (Pharmacia Fine Chemiclas, USA), and histidine-tagged recombinant Lkn-1. ) Was isolated and purified. The molecular weight of the recombinant Lkn-1 was found to be about 12 kDa by electrophoresis.

Example 4-2 Expression and Purification of Recombinant Lkn-1 in Insect Cells

In order to express recombinant Lkn-1 including a signal peptide, a PstI restriction enzyme recognition site was inserted at the N-terminus of the Lkn-1 cDNA containing the signal peptide sequence and an XbaI restriction enzyme recognition site was inserted at the C-terminus, and the Lkn- 1 cDNA was PCR amplified. Subsequently, the amplified PCR product was isolated and inserted into vector PVL 1392 (Invitrogen, U.S.A.) digested with PstI / XbaI restriction enzymes to prepare recombinant plasmid PVL 1392-Lkn-1. Then, the Lkn-1 cDNA inserted into the recombinant recombinant plasmid was transferred to AcNPV by transfecting Sf-21 insect cells with the recombinant recombinant plasmid and AcNPV (Autographa california nuclear polyhedrosis baculovirus). Subsequently, AcNPV-Lkn-1 virus plaques were isolated on the basis of occlusion-negative, which is a characteristic of the virion, and the serum-free Ex-Cell 400. SF-21 insect cells were grown in media (JRH Biosciences, USA) and used as stock.

Recombinant Lkn-1 was expressed in High five cell lines (Invitrogen, USA) cultured in Ex-Cell 400 medium, and expressed Lkn-1 was expressed in Hitrap-Heparin column (Pharmacia biotech., USA) and Hitrap-SP column (Pharmacia). Biotech., USA) and Western blot showed that the molecular weight of recombinant Lkn-1 was estimated to be about 12 kDa.

Example 5 Myelosuppression Function of Recombinant Lkn-1

Example 5-1 Inhibition of Colony Formation of Bone Marrow Cells by Recombinant Lkn-1 in Laboratory Conditions

Since some β-chemokines have myelosuppressive activity under laboratory conditions, colony by myeloid progenitor cells in which recombinant Lkn-1 purified in Example 4-1 is present in human bone marrow The purpose of this study was to investigate the effect on formation.

In other words, to form colonies of bone marrow cells by colony forming unit-granulocyte-macrophage (CFU-GM), centrifugation under a Ficoll-Hypaque gradient (Ficoll-Hypaque gradient, 1.070 mg / cm 3, Sigma Chemicla Co., USA) The low density human bone marrow cells obtained by smearing in a 0.3% agar culture medium containing 10% FBS at 5 × 10 ⁴ cells / ㎖, rhGM-CSF (recombinant human granulocyte-macrophage-colony stimulating factor, 100U / ㎖, Immunex Corporation, USA) and rhSLF (recombinant human steel factor, 50 ng / ml, Immunex Corporation, USA). On the other hand, to form colonies of bone marrow cells by CFU-GM, BFU-E (burstforming unit -erythoid) and CFU-GEMM (colony forming unit-granulocyte-erythroid-macrophage-megakaryote), electric bone marrow cells 30% Smear 5 × 10 ⁴ cells / ml in a 1% methylcellulose culture medium containing FBS, rhEPO (recombinant human erythropoietin, 1U / ml, Amgen Corporation, USA), rhIL-3 (recombinant human interleukin-3, 100U / Ml, Immunex Corporation, USA) or rhSLF.

After the above stimulus was applied, the cells were cultured in a BNP-210 incubator (Tabai ESPEC Corp., USA) under conditions of 5% CO ₂ and 5% O ₂ , and colony counts were calculated after 14 days (see Table 1). At this time, recombinant Lkn-1 was added at 3-50ng / ml per plate.

Effect of Recombinant Lkn-1 on Colony Formation of Bone Marrow Cells in Low Density Humans sample density Agar Methylcellulose CFU-GM [GM-CSF] ^b CFU-GM [GM-CSF + SLF] ^b CFU-GM BFU-E [EPO, SLF, IL-3] ^b CFU-GEMM Control 17 ± 6 67 ± 2 66 ± 2 94 ± 2 9 ± 1 Recombinant Lkn-1 500ng / ml 16 ± 4 (-6) ^c 35 ± 6 (-48) ^d 25 ± 3 (-62) ^d 35 ± 3 (-63) ^d 5 ± 1 (-44) ^d 25ng / ml 18 ± 1 (+6) 40 ± 2 (-40) ^d 33 ± 4 (-50) ^d 50 ± 3 (-47) ^d 6 ± 1 (-33) ^d 6.25ng / ml 18 ± 3 (+6) 52 ± 69 (-22) ^d 45 ± 6 (-32) ^d 65 ± 2 (-31) ^d 7 ± 2 (-22) ^d 3.125ng / ml 17 ± 8 (0) 63 ± 5 (-6) 61 ± 10 (-8) 95 ± 6 (+17) 10 ± 1 (+11)

a: Experimental group without adding recombinant Lkn-1 to the reaction solution

b: growth factor used to promote colony formation

c: amount of change in% compared to the control group

d: Significant change in% of the values compared to the control group (p <0.001)

As shown in Table 1, the recombinant Lkn-1 was found to significantly inhibit colony formation by CFU-GM, BFU-E and CFU-GEMM in a concentration-dependent manner. The degree of inhibition was an amount corresponding to 22-63% of the control. On the other hand, recombinant Lkn-1 did not appear to inhibit the colony formed by CFU-GM stimulated with GM-CSF alone or BFU-E stimulated with EPO alone. It was predicted to have an inhibitory effect on immature blasts in response to factor-induced stimulation.

Example 5-2 Inhibition of Proliferation of Bone Marrow Cells in In Vivo Conditions

In order to measure the biological activity of Lkn-1 in vivo, purified Lkn-1 was first injected intravenously into C3H / HeJ mice. At this time, the absolute number of granulocyte macrophage is CFU-GM, the absolute number of erythroid progenitor cells is BFU-E, and the absolute number of multipotential progenitor cells is CFU-GEMM. The proliferation rate was measured in the bone marrow and spleen, respectively. That is, by injecting sterilized saline without 0.1 ml of pyrogenic material and dilutions of 8 µg of purified recombinant Lkn-1 (diluted in sterile saline without pyrogen) into the tail vein of C3H / HeJ mice. After 24 hours, low-density myeloid cells were taken from the bone marrow and spleen of the mouse femur in the same manner as in Example 5-1. CFU-GM was then plated in 0.3% agar culture medium and stimulated with 10% PWM mouse splenocytes culturing medium. On the other hand, BFU-E or CFU-GEMM were plated in 0.9% methyl cellulose culture medium, respectively, and stimulated with 1% PWM mouse splenocyte conditioned medium containing 1 unit of rhEPO and 0.1 mmol / L hemin. At this time, the bone marrow cells were plated at 7.5 × 10 ⁴ cells / ml, the splenocytes at 1.0 × 10 ⁶ cells / ml, and after stimulation, the cells were cultured in the same manner as in Example 5-1, and 5 to 7 days later. Colony numbers were calculated (see FIGS. 5A and 5B).

On the other hand, the proliferation rate, ie, the cycling rates of CFU-GM, BFU-E and CFU-GEMM, was measured by the cell rate of the S-phase state of the cell cycle: In other words, in vitro In the following conditions, after a 20-minute pulse exposure of radioisotope ³ H-labeled thymidine to a cell, the decrease in colony counts was compared with cells treated with thymidine that had not been radioisotope labeled. Based on the ratio of progenitors of the DNA synthesizer (S phase), the method of inactivation (20 Ci / mmol) and ³ H labeled thymidine (50 μCi / ml) killing technique ( ³ H thymidine kill technique) was used. Measurements were made (see FIGS. 5C and 5D).

As shown in Figures 5a to 5 (D), it can be seen that Lkn-1 significantly reduced colony formation and proliferation rate (cell cycle rate) caused by myeloid hair cells in bone marrow and spleen, while nucleus ( The cellularity of nucleated was found to be not significantly affected compared to the control.

In order to determine whether the inhibitory effect of Lkn-1 is concentration-dependent, the above-described periodic rates of CFU-GM, BFU-E and CFU-GEMM are changed except that the concentration of Lkn-1 is changed from 0.1 to 20 µg. Measurements were made in the same manner as the method (see FIGS. 6A and 6B). 6A and 6B, CFU-GM, BFU-E, and CFU-GEMM of mice treated with 3 to 10 μg of Lkn-1 have no periodic rate or a low cycling state in both bone marrow and spleen. Compared to), mice treated with 0.1 to 1 μg of Lkn-1 showed no change in periodicity except for CFU-GEMM in the spleen. In addition, the overall cell cycle rate was reduced to 80 to 90% by Lkn-1 treatment, BFU-E or CFU-GEMM was more sensitive to Lkn-1 than CFU-GM.

In addition, to determine whether the inhibitory effect of Lkn-1 is time-dependent, 8 μg of Lkn-1 was injected into C3H / HeJ and the cell cycle state of CFU-GM, BFU-E, and CFU-GEMM in bone marrow or spleen. Were compared at time intervals (see FIGS. 7A-7 (F)). As shown in Figure 7a to 7 (F), it was confirmed that the inhibitory effect of Lkn-1 is time-dependent and reversible in the bone marrow or spleen. That is, Lkn-1 changes CFU-GM, BFU-E, and CFU-GEMM into nocyclic or slow cycling state after 12 hours, and returns to control value after 72 hours. It was. The inhibitory effect of Lkn-1, which is time-dependent and reversible on the proliferation of bone marrow cells in the above-described in vivo conditions, is clinically used to protect normal hematopoietic cells from anticancer drugs or radiation with Lkn-1. Indicates that it has

Example 6: Investigation of the function of recombinant Lkn-1 as a coin factor

In order to determine the chemotaxis of recombinant Lkn-1, first, peripheral blood mononuclear cells (PBMC) of healthy humans were obtained by centrifugation under a Ficoll-Hypaque gradient (1,077 mg / cm 3). Then, monocytes separation from PBMC was performed as an ability to adhere to the plastic surface, and the cells remaining after two such monocyte separations were obtained as lymphocytes. The purity of mononuclear and lymphocytes obtained as described above was determined by microscopic observation of cytospin stained with Diff-Quik (Baxter Scientific, U.S.A.), resulting in 90% and 80%, respectively.

Human neutrophils are also obtained by precipitating erythrocytes with 2% Dextran T 500 (Pharmacia Fine Chemicals, USA), then centrifuging them under a Ficoll-Hypaque gradient, and obtaining the erythrocytes from a hypotonic solution. Prepared by dissolving at. The purity of neutrophils thus obtained was determined by morphology, which was 95%.

Monocytes and lymphocytes isolated from the above were 2 × 10 ⁶ cells / ml and 8 × 10 ⁶ cells in 0.5% low endotoxin BSA (Sigma Chemical Co., USA) and RPMI1640 (GIBCO-BRL, USA) added with 20 mM Hepes, respectively. Suspension at a concentration of / ml and neutrophils were suspended in HBSS at a concentration of 1 x 10 ⁶ cells / ml.

Er, the degree of cell migration in 48 well microchamber (Neuroprobe, U.S.A.) was measured as follows. The lower well of the microchamber contains only buffer (control) or recombinant Lkn-1, hMIP-1α (PeproTech., USA), RANTES (PeproTech., USA), IL-8 (PeproTech., USA) or eotaxin (eotaxin, PeproTech., USA) is filled with buffer, the upper well is filled with 50 μl of the cell suspension described above, and these two layers are fractionated with a suitable pore filter free of polyvinylpyrrolidone. The pore diameter of the filter used when the cells to be measured were neutrophils and lymphocytes was 3 μm, and the filter diameter used when the monocytes were monocytes was 5 μm. 1 hour (for neutrophils), 2 hours (for monocytes), 4 hours (for lymphocytes) at 37 ° C. The microchamber was left to stand, then the filter was removed from the chamber and washed, followed by cell fixation and Diff-Quick The cell number was calculated by staining with (see FIGS. 8A to 8C).

In FIGS. 8A to 8C, the number of cells transferred to chemokines divided by the number of cells moved to the control group was used as a chemoactic index. Figure 8a is a graph showing the chemotaxis of recombinant Lkn-1 and RANTES for lymphocytes, Figure 8b is a graph showing the chemotaxis of recombinant Lkn-1 and hMIP-1α for monocytes, Figure 8c is a recombinant Lkn for neutrophils It is a graph showing the chemotaxis of -1 and IL-8.

As shown in Figures 8a to 8 (c), recombinant Lkn-1 was found to be a potent chemotactic factor for human peripheral blood neutrophils, monocytes and lymphocytes. In addition, recombinant Lkn-1 exhibits chemotaxis similar to IL-8 for neutrophils (see FIG. 8C), and shows chemotaxis similar to hMIP-1α for monocytes (FIG. 8B); The lymphocytes exhibited lower chemotaxis than RANTES, but up to 10 μg / ml dml Lkn-1 concentration showed improved chemotaxis (see FIG. 8A).

Example 7: Analysis of the degree of calcium flux by recombinant Lkn-1

Example 7-1: Calcium Outflow Analysis in Lymphocytes, Mononuclear Cells, Neutrophil

The aim of this study was to determine whether recombinant Lkn-1 binds to receptors, activates lymphocytes, monocytes and neutrophils to induce calcium efflux from them and how they compete with other agonists for that receptor.

Activation of the receptor was measured by [Ca ²⁺ ] changes in purified peripheral blood leukocyte subsets (lymphocytes, monocytes, neutrophils) with an MSIII fluorimeter (Photon Technology International, USA). That is, cells and 2 μM of fura-2AM (Sigma Chemicla co., USA) were reacted at 37 ° C. for 45 minutes and washed twice to 1 × 10 ⁷ cells / ml in HBSS (pH 7.4) containing 0.05% BSA. Suspended to concentration. 2 ml of this cell suspension is placed in a stirred, water-jacketed cuvette in a 37 ° C. water bath, continuously excited at 340 nm and 380 nm, and the fluorescence emitted is 25 nM agonist (recombinant Lkn-1, Measurements were made at 510 nm before and after addition of hMIP-1α, RANTES, IL-8 or eotaxin (see FIGS. 9A-9F).

Relative fluorescence in FIGS. 9A-9F is expressed as relative ratio of fluorescence excited at 340 nm and 380 nm. 9A shows relative fluorescence measured for a series of RANTES and stimulation of recombinant Lkn-1 applied to lymphocytes; 9B shows relative fluorescence measured for stimulation of a series of recombinant Lkn-1 and RANTES applied to lymphocytes; 9C shows relative fluorescence measured for a series of MIP-1α and recombinant Lkn-1 stimuli applied to monocytes; 9E shows relative fluorescence measured for a series of IL-8 and recombinant Lkn-1 stimuli applied to lymphocytes; 9F shows the relative fluorescence measured for stimulation of a series of recombinant Lkn-1 and UL-8 applied to lymphocytes, respectively. As shown in Figures 9a to 9 (F), it can be seen that recombinant Lkn-1 induces calcium outflow in lymphocytes, monocytes and neutrophils.

On the other hand, when G protein-coupled receptors are continuously exposed to a ligand having the same binding site of the receptor signal within a short time, it is known that the electrical receptor is desensitized. This phenomenon was also observed when the receptor-expressing cells were stimulated with recombinant Lkn-1.

In addition, as shown in FIGS. 9A-9F, in the event of subsequent stimulation by RANTES and hMIP-1α, recombinant Lkn-1 completely desensitizes lymphocytes and monocytes (see FIGS. 9B and 9 (D)). In case of subsequent stimulation by recombinant Lkn-1, RANTES and hMIP-1α, respectively, did not completely desensitize lymphocytes and monocytes (see FIGS. 9A and 9 (C)). This indicates that recombinant Lkn-1 shares receptors with RANTES and hMIP-1α and induces calcium efflux better than RANTES or hMIP-1α. In addition, even though recombinant Lkn-1 induces calcium efflux from neutrophils, neutrophils are not desensitized by subsequent stimulation by IL-8 (see FIG. 9F), and IL-8 is induced after stimulation by recombinant Lkn-1. It can be seen that it does not desensitize neutrophils (FIG. 9E), which does not share receptors with IL-8, even though recombinant Lkn-1, like IL-8, is one potent factor inducing calcium efflux from neutrophils. It does not.

Example 7-2 Calcium Leakage Analysis in Cell Lines Expressing CC Chemokine Receptor

In Example 7-1, recombinant Lkn-1 was found to activate receptors activated by RANTES and hMIP-1α. To test this, a cell line expressing a CC or CXC chemokine receptor was selected from recombinant Lkn-1. Activation was investigated. Here, instead of using a purified leukocyte subset, except for using HOS cell lines expressing recombinant CCR1, CCR2B, CCR3, CCR4, CCR5 and CXCR4 (AIDS Research and Reference Reagent Program of NIH, USA). The experiment was performed in the same manner as in Example 7-1. hMIP-1α binds to both CCR1 and CCR5, and RANTES is known to bind to both CCR1, CCR3, CCR5.

FIG. 10A shows relative fluorescence measured for a series of stimuli of various agents applied to CCR1 expressing HCR cell lines, and FIG. 10B shows relative fluorescence measured for a series of stimuli by various substances applied to HCR cell lines expressing CCR3. It is a graph showing degrees. As shown in Figures 10a and 10 (b), it can be seen that recombinant Lkn-1 strongly induces calcium outflow in HOS cells expressing CCR1 and CCR3. However, no calcium efflux was observed in HOS cells expressing receptors other than CCR1 and CCR3.

In addition, as shown in FIG. 10A, when CCR1-HOS cells are first stimulated with recombinant Lkn-1, there is no response to subsequent recombinant hMIP-1α or RANTES stimulation, whereas CCR1-HOS cells first receive hMIP-1α or When stimulated with RANTES, it was found that the response to the subsequent recombinant Lkn-1 stimulation was not affected. From the results of FIG. 10A, it can be seen that recombinant Lkn-1 is a stronger agonist for CCR1 than hMIP-1α or RANTES, which is also shown in the results of FIG. 10C examining the reaction according to the concentration of recombinant Lkn-1 and hMIP-1α. It is clearly proven.

In addition, as shown in FIG. 10B, when CCR3-HOS cells are first stimulated with eotaxin, a nearly complete desensitization response to subsequent eotaxin or recombinant Lkn-1 stimulation occurs; When CCR3-HOS cells are first stimulated with recombinant Lkn-1, an almost complete desensitization response to subsequent RANTES stimulation occurs; When CCR-HOS cells were first stimulated with recombinant Lkn-1 or RANTES, it was found that subsequent responses to eotaxin stimulation were not affected. In addition, as a result of examining the reaction according to the concentrations of eotaxin, recombinant Lkn-1 and RANTES (see FIG. 10D), it was found that recombinant Lkn-1 was not as strong as eotaxin, but was a potent agonist for CCR3 than RANTES. .

As described and demonstrated in detail above, the present invention provides a novel C6 β-chemokine Lkn-1 cDNA isolated from human mononuclear cell lines and amino acid sequences inferred therefrom. The Lkn-1 cDNA open reading frame encodes a total of 113 amino acids, including a signal peptide consisting of 21 amino acids, of which the molecular weight of the mature protein consisting of 92 amino acids is estimated to be 10,162 Da. Recombinant Lkn-1 purified by expressing the aforementioned Lkn-1 cDNA in recombinant E. coli or insect cells significantly inhibits colony formation, has chemotaxis that attracts lymphocytes, monocytes, and neutrophils, and the receptors of CCR1 and CCR3 It was confirmed to bind. The recombinant Lkn-1 protein can be used for antibody production, treatment during HIV-1 infection, bone marrow hepatocyte protection during chemotherapy or radiation therapy, and inhibition of leukemia.

Claims (25)

CDNA (SEQ ID NO: 6) of C6 β-chemokine Lkn-1 (leukotactin-1) of human having the following nucleotide sequence. ATG AAG GTC TCC GTG GCT GCC CTC TCC TGC CTC ATG CTT ATT GCT GTC CTT 51 GGA TCC CAG GCC CAG TTC ACA AAT GAT GCA GAG ACA GAG TTA ATG ATG TCA 102 AAG CTT CCA CTG GAA AAT CCA GTA GTT CTG AAC AGC TTT CAC TTT GCT GCT 153 GAC TGC TGC ACC TCC TAC ATC TCA CAA AGC ATC CCG TGT TCA CTC ATG AAA 204 AGT TAT TTT GAA ACG AGC AGC GAG TGC TCC AAG CCA GGT GTC ATA TTC CTC 255 ACC AAG AAG GGG CGG CAA GTC TGT GCC AAA CCC AGT GGT CCG GGA GTT CAG 306 GAT TGC ATG AAA AAG CTG AAG CCC TAC TCA ATA TAA 342 Human C6 β-chemokine Lkn-1 having the amino acid sequence as follows (SEQ ID NO: 5). Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu Ile 1 5 10 Ala Val Leu Gly Ser Gln Ala Gln Phe Thr Asn Asp Ala Glu 15 20 25 Thr Glu Leu Met Met Ser Lys Leu Pro Leu Glu Asn Pro Val 30 35 40 Val Leu Asn Ser Phe His Phe Ala Ala Asp Cys Cys Thr Ser 45 50 55 Tyr Ile Ser Gln Ser Ile Pro Cys Ser Leu Met Lys Ser Tyr 60 65 70 Phe Glu Thr Ser Ser Glu Cys Ser Lys Pro Gly Val Ile Phe 75 80 Leu Thr Lys Lys Gly Arg Gln Val Cys Ala Lys Pro Ser Gly 85 90 95 Pro Gly Val Gln Asp Cys Met Lys Lys Leu Lys Pro Tyr Ser 100 105 110 Ile Human c6 β-chemokine Lkn-1 (leukotactin-1) cDNA of human sequence C6 β-chemokine removed from 1 to 63 bases. An expression vector comprising the cDNA of claim 1. Expression vector PVL 1392-Lkn-1 comprising the cDNA of claim 1. A method for producing recombinant Lkn-1 comprising transforming a host cell with the expression vector of claim 4 and then culturing the transformant to obtain recombinant Lkn-1 from the electric transformant. The method of claim 6, The host cell is characterized in that E. coli or insect cells Method for preparing recombinant Lkn-1. The recombinant Lkn-1 prepared by the manufacturing method comprising transforming the host cell with the expression vector of claim 4, and then culturing the transformant to obtain recombinant Lkn-1 from the electric transformant. The method of claim 8, Characterized by suppressing colony formation Recombinant Lkn-1. The method of claim 8, About human peripheral blood neutrophils, monocytes and lymphocytes Characterized by exhibiting strong chemotaxis Recombinant Lkn-1. The method of claim 8, Characterized by binding to the receptors of CCR1 and CCR3 Recombinant Lkn-1. The method of claim 11, RANTES (regulated-upon activation, normal T cell expressed and secreted) and hMIP-1α (human macrophage inflammatory protein-1α) Does share a receptor but IL-8 (interleukin-8) does. Characterized by Recombinant Lkn-1. An expression vector comprising the cDNA of claim 3. Expression vector pET21a-Lkn-1 comprising the cDNA of claim 3. Escherichia coli (XL-1 Blue) hMRP-2 (ATCC 98166) transformed with the expression vector of claim 14. A method for producing recombinant Lkn-1 comprising transforming a host cell with the expression vector of claim 13 and then culturing the transformant to obtain recombinant Lkn-1 from the electric transformant. The method of claim 16, The host cell is characterized in that E. coli or insect cells Method for preparing recombinant Lkn-1. Recombinant Lkn-1 prepared by the method comprising the step of transforming the host cell with the expression vector of claim 13, followed by culturing the transformant, to obtain a recombinant Lkn-1 from the electric transformant. The method of claim 18, Characterized by suppressing colony formation Recombinant Lkn-1. The method of claim 18, Peripheral blood neutrophils, monocytes or lymphocytes Characterized by strong chemical attraction Recombinant Lkn-1. The method of claim 18, Characterized by binding to the CCR1 or CCR3 receptor Recombinant Lkn-1. The method of claim 21, RANTES or hMIP-1α share receptors and IL-8 Characterized by not sharing Recombinant Lkn-1. A method of protecting myeloid cells from cytotoxic anticancer drugs or radiation by administering human C6 β-chemokine Lkn-1 to humans. The method of claim 23, wherein Human C6 β-chemokine Lkn-1 is the recombinant Lkn-1 of claim 8 Characterized How to protect myeloid cells. The method of claim 23, wherein Human C6 β-chemokine Lkn-1 is the recombinant Lkn-1 of claim 18 Characterized How to protect myeloid cells.