JP2002505076A - CDNA encoding C6β-chemokine leukotactin-1 (Lkn-1) isolated from human - Google Patents

Abstract

  (57) [Summary] The present invention relates to a cDNA that belongs to C6β-chemokine and encodes a novel protein that induces a subset of leukocytes in peripheral blood, and a method for producing the protein using its expression vector. The open reading frame of Lkn-1 cDNA encodes 113 amino acids including a 21 amino acid signal peptide, of which the mature protein consisting of 92 amino acids is estimated to have a molecular weight of 10,162 daltons. . Recombinant Lkn-1 expressed and purified in E. coli or insect cells using the Lkn-1 cDNA significantly suppresses colony formation and cell proliferation, attracts neutrophils, monocytes and lymphocytes, and performs chemotaxis. And bound to CCR1 and CCR3 receptors. Therefore, the recombinant Lkn-1 protein can be used as a drug that has a potential for producing antibodies, treating HIV-1 infection, protecting bone marrow stem cells during chemotherapy or radiation therapy, suppressing leukemia, and the like. .

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION 1. Technical Field The present invention relates to a cDNA encoding a novel C6β-chemokine isolated from the human body and a method for producing C6β-chemokine. More specifically, the present invention relates to a cDNA belonging to C6β-chemokine, which encodes a novel protein that induces a subset of peripheral blood leukocytes, a method for producing the protein using its expression vector, and a pharmaceutical method for the protein. For use.

[0002] prior art Chemokines are small cytokine family of basic proteins of a low molecular weight, have in common the four cysteines, the position of the first and second cysteine, i.e., they are adjacent Or CXC (α), CC (β), or an amino acid between two cysteines.
C (γ) and CX ₃ C are classified into four subfamilies (see: Baggiolini, M. and Dahinden, CA, Immunol. Tod).
ay, 15: 127 (1994); Kelner, S .; G. FIG. Et al., Science
, 266: 1395 (1994); Bazan, J. et al. F. Et al., Nature, 3
85: 640 (1997)). Chemokine subfamily genes are grouped and located on the same chromosome, for example, the α-chemokine gene is in human chromosome 4q1
On 2-21, the β-chemokine gene is located on human chromosome 17q11-32 and mouse chromosome 11.

[0003] Chemokines have biological activities such as HIV-suppressing action, immunomodulatory action, leukocyte migration, and hematopoietic stem cell division inhibition action (see: Cocchi, F. et al., Science, 270: 1811). 1995); Wolpe, SD et al.
J. Exp. Med. 167: 570 (1998); Graham, G .; J.
Broxmeyer, H. et al., Nature, 344: 442, 1990; E. FIG.
Et al., Blood, 76: 1110 (1990); -S. J. et al. Immunol. , 155: 2661-2667 (1995)).

[0004] In addition, chemokines bind to G protein-coupled receptors in the transmembrane domain to activate leukocytes, and some of these receptors become co-receptors (co-
(see Oh, K.-O., et al., J. Im.)
munol. , 147: 2978 (1991); Alkbatih, G .; S
science, 272; 1955 (1996)). For example, β-chemokines (C
There are eight subtypes of C chemokine) receptors, namely CCR1, CCR2
, CCR3, CCR4, CCR5, CCR6, CCR7 and CCR8, of which CCR1, CCR2, CCR3 and CCR5 have binding affinities for various chemokines, but have four subtypes of CCR4, CCR6, CCR7 and CCR8. Shows high affinity for one substance.

Up to now, nine chemokines belonging to the β-chemokine subfamily are known (see Wilson, SD et al., J. Exp. Med., 171: 1).
301 (1990); S. Hum. Genet. , 84: 1
85 (1990)). Among them, mouse MRP-1 ("mMRP-1": MI
P (macrophage inflammatory protein) -related protein-1 or C10) (see: Orlofsky, A. et al., Cell Regul., 2: 403 (1991).
)) And mouse MRP-2 ("mMRP-2") (see: Youn, B.-S.
J. et al. Immunol. , 155: 2661 (1995)) have two more cysteine residues than other chemokines in β-chemokines, thus forming a third disulfide bond, and other N-terminal regions due to the very long N-terminal region. It is distinguished from β-chemokines. Based on the above findings, they were classified as C6β-chemokines.

[0006] Under these circumstances, a novel human MRP can be used as a drug capable of treating HIV-1 infection, or protecting bone marrow stem cells during chemotherapy or radiotherapy, and the like. The quest for and development has been urgently needed.

SUMMARY OF THE INVENTION As a result of our efforts to isolate the MRP gene from various human cell lines, we isolated a novel MRP cDNA belonging to C6β-chemokine from a human monocyte cell line, The nucleotide sequence and the amino acid sequence deduced from it were determined. Furthermore, the MRP cDNA was successfully expressed in recombinant Escherichia coli or insect cells, and the expressed recombinant protein was used for colony formation and in vivo.
It was shown to suppress myeloid stem and progenitor cell proliferation in vitro and in vivo and to attract a subset of peripheral blood leukocytes (lymphocytes, monocytes and neutrophils) in a chemotactic manner. As used herein, the MRP isolated from the human body
The cDNA was referred to as "Lkn-1 (leukotactin-1) cDNA" and the recombinant MR
P is each named "recombinant Lkn-1".

Therefore, a first object of the present invention is to provide a novel cDNA encoding C6β-chemokine (Lkn-1) and an amino acid sequence deduced therefrom.

A second object of the present invention is to provide an expression vector containing the Lkn-1 cDNA and a recombinant microorganism transformed with the vector.

[0010] A third object of the present invention is to provide a method for producing recombinant Lkn-1 from the microorganism.

[0011] A fourth object of the present invention is to provide a method for protecting myeloid cells from cytotoxic anticancer drugs or radiation using recombinant Lkn-1.

DETAILED DESCRIPTION OF THE INVENTION In order to isolate the Lkn-1 gene from a human cell line, we first cloned an exon of the Lkn-1 gene that can be used for probe preparation. That is, human genomic DNA is cut with HindIII, separated on an agarose gel, and ³² P-labeled mMRP-2 cDNA (see: Youn, B.-S).
. J. et al. Immunol. , 155: 2661 (1995)) as a probe to obtain a 7.0 kb DNA fragment capable of hybridizing with mMRP-2. Next, in order to isolate the exon sequence of Lkn-1 gDNA from the 7.0 kb HindIII digested DNA fragment, the human genomic DNA was completely digested with HindIII, separated on an agarose gel, and separated into 7.0 kb DNA fragment. Was isolated. The obtained fragment was inserted into a vector, and the obtained vector was introduced into a host cell. After the transformation, colonies showing hybridization with the mMRP-2 cDNA probe were selected. 7.0k from colony
After isolation of the DNA fragment of b and digestion with AluI, a 202 bp DNA fragment capable of hybridizing with the mMRP-2 cDNA was cloned. Thereafter, the nucleotide sequence of the fragment was determined. It differs from the above DNA fragment in part of the intron and exon.

[0013] Then, in order to clone a novel Lkn-1 cDNA from a human cell line, PCR primers of Lkn-1 capable of amplifying a 100 bp DNA fragment from the Lkn-1 exon sequence confirmed above were prepared. RT-PCR was performed using total RNA isolated from various human cell lines as a template to determine the mRNA of Lkn-1.
Each cell line with NA was selected. One of the cell lines sorted in this way,
A human monocyte THP-1 cell line activated with interleukin-4 (IL-4) was selected and its cDNA library was prepared. On the one hand, the total RNA of the THP-1 cell line was used as a template and the Lkn-1 PCR primer was used
A 100 bp DNA fragment of the Lkn-1 exon was prepared by performing PCR. The THP-1 cell line cDNA library constructed as described above was hybridized with a 100 bp DNA fragment of Lkn-1 exon as a probe, and as a result, Lkn-1 cDNA showing a positive reaction was obtained.

As a result of determining the nucleotide sequence of the Lkn-1 cDNA and the amino acid sequence deduced therefrom, the Lkn-1 cDNA is a novel Lkn-1 cDNA.
The open reading frame encodes 113 amino acids including a signal peptide of 21 amino acids, and is a mature Lkn consisting of 92 amino acids.
The molecular weight of the -1 protein was estimated to be 10,162 daltons. Also, Lkn-
1 is unlikely to have an N-glycosylation site and has two cysteine residues similar to mMRP-1 and mMRP-2, in addition to the four cysteine residues commonly found in β-chemokines It was found to belong to the C6β-chemokine family.

By inserting the thus cloned novel Lkn-1 cDNA into an expression vector and introducing it into host cells such as Escherichia coli or insect cells,
Recombinant Lkn-1 was expressed.

On the other hand, the myelocytic inhibitory activity of recombinant Lkn-1 was examined in human bone marrow and spleen-derived progenitor cells and myeloid stem cells. As a result, in vivo and in vitro, recombinant Lkn-1 Inhibited the growth and colony formation of myeloid cells in a concentration-dependent and time-dependent manner. Furthermore, recombinant Lkn-1 strongly attracted human peripheral blood neutrophils, monocytes and lymphocytes, caused chemotaxis and induced calcium efflux in said cells. Notably, recombinant Lkn-1, like IL-8, strongly attracts neutrophils to cause chemotaxis and binds to RANTES and hMIP-1α receptors,
It was found not to bind to the IL-8 receptor. Furthermore, it was confirmed that the receptors for the recombinant Lkn-1 were CCR1 (CC chemokine receptor 1) and CCR3. Recombinant Lkn-1 is a hMIP-1 already known to bind to CCR1.
It was an even more potent agonist at CCR1 than α or RANTES, and at the CCR3 receptor it was less potent than eotaxin, but much more potent than RANTES.

The recombinant Lkn-1 of the present invention having the above-mentioned characteristics can be used for antibody production, treatment for HIV-1 infection, protection of bone marrow stem cells for chemotherapy or radiation treatment, suppression of leukemia, and the like. .

EXAMPLES The present invention will be described more specifically in the following examples. However, the scope of the present invention is not limited by these examples and the like.

Example 1 Cloning of Exon of Lkn-1 Genomic DNA In order to clone genomic DNA homologous to mMRP-2 from human, the whole human genomic DNA was cut with BamHI, EcoRI, HindIII, PstI, XbaI and XhoI. After that, fractionation was performed on a 1.0% agarose gel, and Southern blotting was performed using ³² P-labeled mMRP-2 cDNA as a probe to obtain a 7.0 kb HindIII fragment showing a positive signal.

In order to prepare a sublibrary of the HindIII fragment, 100 Fg of human genomic DNA was completely digested with HindIII, fractionated on a 1% agarose gel at a voltage of 20 V for 16 hours, and then around 7.0 kb. DNA fragment of Gene-cle
The kit was isolated using an kit (Bio101, USA) and inserted into a dephosphorylated pBluescript SK ⁺ vector (Stratagene, USA) that had been cut with HindIII. The recombinant vector thus prepared was introduced into electro-max (GIBCO-BRL, USA) competent cells by electroporation, transformed, and cultured in a solid medium. Approximately 2 × 10 ⁵ colonies formed were hybridized to the mMRP-2 probe. As a result, only one colony showed a hybridization positive signal, and the colony contained the 7.0 kb insert DNA.

In order to isolate an exon sequence from the 7.0 kb genomic DNA fragment, a pBluescript SK ⁺ vector containing a 7.0 kb insert DNA was isolated from the colony that showed a hybridization-positive signal, cut with AluI, After fractionation on a 1.5% agarose gel, Southern blotting and mMR were performed.
A 202 bp DNA fragment hybridized with the P-2 cDNA was cloned.

The base sequence (SEQ ID NO: 1) of the 202 bp DNA fragment was determined, and a part of exons translated into introns and amino acids was confirmed (see FIG. 1A). As a result, the amino acid sequence of a part of the exon (SEQ ID NO: 2) showed 50% homology with the amino acids 87 to 110 of mMRP-2,
It was confirmed that the second cysteine (indicated by *) of the two added cysteines common to P-2 was conserved in Lkn-1 (see: FIG. 1B).
). In FIG. 1B, squares indicate amino acids conserved in Lkn-1 and mMRP-2.

Example 2 Cloning of Lkn-1 cDNA To clone Lkn-1 cDNA from a human cell line, first, based on the amino acid sequence (SEQ ID NO: 2) disclosed in FIG.
PCR primer capable of amplifying 0 bp, ie, forward primer 5
'-TTCCTCACCAAGAAGGGGG-3' (SEQ ID NO: 13) and reverse primer 5'-CTTTTTCATGCAATCCCTG-3 '(SEQ ID NO: 14)
) Was prepared. Thereafter, PCR was performed using the 202 bp DNA fragment prepared in Example 1. At this time, 100 Fg / ml of IL-4 (interleu
kin-4) activated for 24 hours in a human monocyte cell line THP-1 (ATCC TI
B202) total RNA, macrophage cell line U937 (ATCC CTL15
93) and the total RNA of the HL-60 cell line (ATCC CCL240).
Was used as a template for PCR (see FIG. 2).

In FIG. 2, lanes 1 to 4 show 202 as a positive control.
The results of PCR performed using the bp DNA fragment, the total RNA of the THP-1 cell line, the total RNA of the U937 cell line, and the total RNA of the HL-60 cell line, respectively, are shown. Lane M shows a 100 bp ladder as a DNA size marker. As shown in FIG. 2, the THP-1 cell line activated by IL-4 produced Lkn-1 mRNA.

To prepare a THP-1 cDNA library, human mRNA was isolated from a THP-1 cell line activated by IL-4 and Time Saver cD
Reverse transcription was performed using NA kit (Pharmacia Biotech, USA) to obtain a double-stranded cDNA, which was then transferred to a BstXI adapter (Invitro).
gen, USA). Then, on an agarose gel, c of 0.5 kb or more
DNA was isolated and fractionated, and PRc / CMV (Invitrog) digested with BstXI was used.
en, USA) to prepare a cDNA library. A 100 bp DNA fragment of the Lkn-1 exon amplified by the above PCR was used as a probe and hybridized to the cDNA library. As a result, one cDNA clone showing a positive signal was isolated.

Example 3 Determination of Nucleotide Sequence of Lkn-1 cDNA FIG. 3 shows the nucleotide sequence of the cDNA clone obtained in Example 2 (SEQ ID NO:
4) and the amino acid sequence deduced therefrom (SEQ ID NO: 5). In FIG. 3, the underlined portion is a signal peptide, and the sequence surrounded by a square is THP-1 cDNA.
FIG. 1B shows a portion used as a probe during library screening.
Lkn-1 amino acid sequence (SEQ ID NO: 2) and (***) indicate a translation stop codon.

As shown in FIG. 3, the open reading frame of Lkn-1 cDNA (
SEQ ID NO: 6) comprises 21 amino acids forming a signal peptide and a molecular weight of 10
, Encodes a total of 113 amino acids, consisting of 92 amino acids that form the mature protein predicted to be 161 Da. In addition, it was found that the deduced Lkn-1 amino acid sequence (SEQ ID NO: 5) has no N-glycosylation site.

FIG. 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) (
See: Youn, B .; S. J. et al. Immunol. , 155: 2661, (1
995)), mMIP-1α (SEQ ID NO: 11) (see: Kwon, BS and Weissman, SM, Proc. Natl. Acad. Sci., US).
A, 86: 1963 (1989)) and hMIP-1β (SEQ ID NO: 12) (see: Sherry, B. et al., J. Exp. Med., 168: 2251, (19).
88)) shows a comparison with the amino acid sequence. In FIG. 4, the boxes and (•) indicate four conserved cysteine residues and two added cysteine residues, respectively. As shown in FIG. 4, (1) Lkn-1 has a long N-terminal region before the first two cysteine residues among four cysteine residues common to the β-chemokine family. And (2) Lkn-2 has mMRP-1 and mMRP in addition to the four cysteine residues common to the β-chemokine family.
-2 was found to belong to the C6β-chemokine family which also has the two cysteine residues found in -2. In addition, the amino acid sequence of mature Lkn-1 (SEQ ID NO: 7) shows that either Lkn-1 was isolated as the human equivalent of mMRP-2 or mMRP-1 (SEQ ID NO: 8) or mMRP- 2 (SEQ ID NO: 9
) Shows only about 43% homology and hMIP-1α (SEQ ID NO: 10) shows only 40% homology.

Example 4 Expression of Recombinant Lkn-1 Example 4-1 : Expression of Recombinant Lkn-1 in Escherichia coli Only mature Lkn-1 protein is used as a recombinant protein except for a portion presumed to be a signal peptide. For expression, mature Lkn was first used using the Lkn-1 cDNA cloned in Example 2 as a template and the following PCR primers and Pfu polymerase (Stratagene, USA).
-1 open reading frame was amplified by PCR: forward primer: 5′-CGAATTCCATATGCAGTTCACAAA
ATGATGCAGAG-3 '(SEQ ID NO: 15) Reverse primer: 5'-CGCCGCTCGAGTTGAGTAGGGCT
TCAGC-3 '(SEQ ID NO: 16) The DNA fragment thus amplified was digested with NdeI / XhoI and the plasmid pE
After cloning into T21a (Novagen, USA), the constructed recombinant plasmid was named pET21a-Lkn-1 and introduced into Escherichia coli XL-1 Blue. Recombinant plasmid pET21a-Lkn-1 contains the N of mature Lkn-1.
-To express recombinant Lkn-1 with one methionine residue at the terminal and six histidines at the C-terminal.

[0030] The transformant prepared as described above was used in Escherichia coli (
XL-1 Blue) hMRP-2 ', and on September 10, 1996, ATCC (American Type Culture Coll.) Of the International Depository.
section, Rockville, MD 20852, USA)
-98166.

After culturing the transformant to express Lkn-1, an inclusion body was obtained and
ml of denaturing buffer solution (6 M guanidine-HC1, 20 mM Tris-HC
1, pH 7.9, 500 mM NaCl, 4 mM n-octylglucopyranoside), and centrifuged. The obtained supernatant was subjected to an activated Ni-column (No.
vagen, USA) and a heparin-agarose column (Pharmacia).
Chromatography was performed using Fine Chemicals, USA) to purify the histidine-tagged recombinant Lkn-1. As a result of electrophoresis, it was found that the molecular weight of the recombinant Lkn-1 was about 12 kDa.

Example 4-2 : Expression of recombinant Lkn-1 in insect cells In order to express recombinant Lkn-1 containing a signal peptide, PstI restriction was applied to the N-terminal of Lkn-1 cDNA containing a signal peptide sequence. An XbaI restriction enzyme site was inserted into the enzyme site and the C-terminus, and Lkn-1 cDNA was amplified by PCR. Subsequently, the PCR product thus amplified was isolated and inserted into a PVL1392 vector (Invitrogen, USA) cut with PstI / XbaI to construct a recombinant plasmid PVL1392-Lkn-1. Then, the recombinant plasmid and AcNPV (nuclear polyhedrosis baculovirus: Autograp ha california nuclear polyhedrosis b
The Lkn-1 cDNA was transferred to AcNPV in the recombinant plasmid by transfecting both Sac-21 cells into Sf-21 insect cells. Then, virion was occlusive-n.
AcNPV-Lkn-1 viral plaques were isolated and serum-free Ex-Cell400 medium (JRH Biosc)
genes, USA) in Sf-21 insect cells for later use.

[0033] Recombinant Lkn-1 was obtained from High five cultured in Ex-Cell400 medium.
Lkn-1 expressed in an e-cell line (Invitrogen, USA) and thus expressed Lkn-1 was applied to a HiTrap-Heparin column (Pharmacia Bi).
otech. , USA) and HiTrap-SP columns (Pharmacia).
Biotech. , USA). Western blot analysis performed immediately after purification showed that the molecular weight of the recombinant Lkn-1 was about 12 kDa.

Example 5 Bone Marrow Cell Inhibitory Activity of Recombinant Lkn-1Example 5-1 : In vitro conversion of bone marrow cells by recombinant Lkn-1
Inhibition of colony formation Some β-chemokines have myelocytic inhibitory activity (myelos) in vitro.
upactivity, so that it is present in human bone marrow.
Against myeloid progenitor cells
The effect of the recombinant Lkn-1 purified from Example 4-1 on colony formation was examined.
. That is, CFU-GM (granulocyte macrophage colony forming unit: co
lonely forming unit-granulocyte-macrop
hedge) to form colonies of bone marrow cells.
aque gradient (Ficol-Hypaque gradient, 1.070 g)
m / cm^Three, Sigma Chemical Co. , USA) and centrifuged to obtain the obtained low-density human bone marrow cells in 0.3% agar containing 10% FBS.
5 × 10 on medium^FourCells were applied at a concentration of cells / ml, and rhGM-CSF (recombinant human granulocyte macrophage colony stimulating factor: recombinant human)
 granulocyte-macrophage-colony stimu
rating factor, 100 U / ml, Immunex Corp
oration, USA) and rhSLF (recombinant human steel factor: reco
mbinant human steel factor, 50ng / ml,
 (Immunex Corporation, USA). on the other hand,
CFU-GM, BFU-E (red blood cell burst forming unit: burstform
ing unit-erythroid) and CFU-GEMM (granulocyte erythrocytes)
Macrophage megakaryocyte colony forming unit: colony forming
unit-granulocyte-erythroid-macrophag
e-megakaryocyte) to form colonies of bone marrow cells.
First, 5 × 10 5 bone marrow cells were placed in 1% methylcellulose medium containing 30% FBS. ^Four Cells were applied at a concentration of cells / ml, and rhEPO (recombinant human erythropoietin, 1 U / m) was applied.
1, Amgen Corporation, USA), rhIL-3 (recombinant
Human interleukin-3: recombinant human inte
rleukin-3, 100U / ml, Immunex Corporation
ion, USA), or rhSLF.

After stimulation, a BNP-210 incubator (Tabay ESPEC COR)
p. , USA) in a 5% CO ₂ and 5% O ₂ environment and colonies were counted 14 days later (see Table 1). At this time, add 3-50 ng / ml to the plate.
Of recombinant Lkn-1 was added.

[0036]

[Table 1] a: Experimental group in which recombinant Lkn-1 was not added to the reaction solution b: Growth factor used to stimulate colony formation c: Change (%) compared to control d: Significant change compared to control (%) (P <0.001)

As can be seen in Table 1 above, recombinant Lkn-1 is concentration-dependently dependent on CFU-GM,
Colony formation by BFU-E and CFU-GEMM was significantly inhibited. The level of inhibition of colony formation was between 22% and 63% as compared to the control. On the other hand, it was found that the recombinant Lkn-1 could not inhibit the colony formation by CFU-GM stimulated only by GM-CSF or BFU-E stimulated only by EPO. This indicates that recombinant Lkn-1 has an inhibitory effect on immature progenitor cells that can be stimulated by various growth factors.

Example 5-2 : Inhibition of proliferation of myeloid cells by recombinant Lkn-1 in vivo The biological activity of Lkn-1 was evaluated in vivo. That is, purified Lkn-1 was injected into C3H / HeJ mice by intravenous injection, and granulocyte macrophages (CFU-GM), erythrocytes (BFU-E) and multipotential progenitor cells (CF
U-GEMM) and their proliferation rate in bone marrow and spleen were measured, respectively. Briefly, C3H / HeJ mice were injected in the tail vein with 0.1 ml of pyrogen-free sterile saline or 8 μg of purified Lkn-1 diluted in pyrogen-free sterile saline. Twenty-four hours after the injection, low-density myeloid cells were obtained from the bone marrow and spleen of the femur of the mouse in the same manner as in Example 5-1. Next, CFU-GM was plated on a 0.3% agar medium and stimulated with a 10% PWM mouse spleen cell conditioned medium. Similarly, BFU-E and CFU-GEM
M was plated on 0.9% methylcellulose medium, and 1 unit of rhEP
O, stimulated with 0.1 mmole / L hemin and 1% PWM mouse spleen cell conditioned medium. At this time, bone marrow cells were applied at a concentration of 7.5 × 10 ⁴ cells / ml, and spleen cells were applied at a concentration of 1.0 × 10 ⁶ cells / ml. After the stimulation, the cells were cultured under the same conditions as described in Example 5-1, and the number of colonies was counted 5 to 7 days after the incubation (see FIGS. 5A and 5B). On the other hand, the proliferation rate, that is, the cycle rate of CFU-GM, BFU-E and CFU-GEMM (cyclic)
ng rate) is determined by measuring the specific activity (20 Ci / mmol) and the ratio of progenitor cells in DNA synthesis (ie, S phase of the cell cycle) with the aid of tritiated thymidine (50 μCi / ml) killing technology. It was measured as the percentage of cells in the S phase of the cycle. The technique is based on calculating in vitro the reduction in the number of colonies formed after pulse exposure of cells to hot tritiated thymidine compared to an equivalent amount of cold thymidine (see: FIG. 5C). And 5D). FIGS. 5A-5D show that Lkn-1 is the number of colonies by myeloid stem / progenitor cells and their growth rate (ie, cell cycle rate) in bone marrow and spleen
Was rapidly reduced. On the other hand, when nucleated cellularity in bone marrow, spleen and blood was evaluated, it was confirmed that it was not significantly affected as compared with the control.

In order to examine the dose dependence of the inhibitory effect of Lkn-1, CFU-GM and BFU were used in the same manner as described above except that the concentration of Lkn-1 was changed to 0.1 to 20 μg.
-E and the period rate of CFU-GEMM were measured (see: FIG. 6A and FIG. 6B). As seen in FIGS. 6A and 6B, CFU-GM, BFU-E and CFU-GEMM from mice that received 3-10 μg of Lkn-1 are in an acyclic or hypocyclic state in bone marrow and spleen Was shown. On the other hand, 0.1 μg to 1 μg
In cells from mice that received Lkn-1 in CFU-GEMM of the spleen
Other than the above, no change in the periodicity was found. The total cell cycle rate was 80 by Lkn-1.
% To 90%, BFU-E and CFU-GEMM are more Lkn than CFU-GM.
Seemed to be sensitive to -1.

Further, in order to examine the time dependency of the inhibitory effect of Lkn-1, 8 μg of Lkn-1 was injected into C3H / HeJ mice, and CFU-GM, BF obtained from bone marrow and spleen.
The cell cycle status of UE and CFU-GEMM was tested at different times, respectively (see: FIGS. 7A-7F). As shown in FIGS. 7A to 7F, the inhibitory effect of Lkn-1 was time-dependent and reversible in bone marrow and spleen. That is, Lkn-1 causes the CFU-GM, BFU-E and CFU-GEMM to be in a non-cyclic state or a low cycle state (slow cycle) within 12 hours.
The state was returned to the control level after 72 hours.

Time-dependent and reversible Lkn-1 in vivo on the proliferation of myeloid cells
The inhibitory effect at tro strongly suggested that Lkn-1 has potential clinical utility in protecting normal hematopoietic cells from cytotoxic anticancer drugs or radiation.

Example 6 Examination of Recombinant Lkn-1 as Chemokine In order to examine whether recombinant Lkn-1 causes chemotaxis, healthy human peripheral blood mononuclear cells (pheripheral blood mononuclear cells) were examined.
arc cells, PBMC) were obtained by centrifugation using a Ficoll-Hypaque gradient (1.0 77 gm / cm ³ ). Thereafter, monocytes were isolated from the PBMC obtained based on the adhesion to the plastic surface, and the isolation step was performed twice. Thereafter, the remaining cells were obtained as lymphocytes. The purity of the monocytes and lymphocytes thus obtained was determined by Diff-Quick (
Cytospin (cy) stained with Baxter Scientific, USA
tospin) by microscopic observation. As a result, the purity of monocytes and lymphocytes was 90% and 88%, respectively.

In addition, 3% Dextran T 500 (Pharmacia Fine C)
chemicals, USA) to sediment erythrocytes and Ficoll-Hyp.
The erythrocytes were obtained by centrifugation using an aque gradient. The red blood cells thus obtained were dissolved in a hypotonic solution to obtain human neutrophils. The purity of the neutrophils thus obtained was determined morphologically. As a result, the purity was 95%.

The isolated monocytes and lymphocytes were purified from 0.5% low endotoxin BSA (Sig
ma Chemical Co. , USA) and 20 mM Hepes in RPMI 1640 (Gibco, USA) at a concentration of 2 × 10 ⁶ cells / ml and 8 × 10 ⁶ cells / ml, respectively. Neutrophils were also suspended in HBSS at a concentration of 1 × 10 ⁶ cells / ml.

Next, a 48-well microchamber (Neuroprobe, USA)
The level of cell migration in was measured as follows. Buffer wells only (control) or recombinant Lkn-1, hMIP-1α (Pe
proTech. , USA), RANTES (PeproTech., USA)
, IL-8 (PeproTech., USA), or eotaxin (eota).
xin, PeproTech. , USA) and the upper wells were filled with 50 Fl of the cell suspension. Wells are fractionated into lower and upper wells by a suitable filter without polyvinylpyrrolidone. When neutrophils and lymphocytes were used, the pore diameter of the filter was 3 Fm, and when monocytes were used, the pore diameter of the filter was 5 Fm. After incubating the microchamber at 37 ° C. for 1 hour (for neutrophils), 2 hours (for monocytes) or 4 hours (for lymphocytes), the filter is removed from the chamber, washed, The cells on the filter were fixed and stained with Diff-Quick, and the number of cells was counted (see FIGS. 8A to 8C).

8A to 8C, a value obtained by dividing the number of migrated cells in the experimental group treated with the chemokine by the number of migrated cells in the control was used as an index of chemotaxis (chemo).
Tactic index). FIG. 8A shows recombinant Lkn-1 and Rkn-1.
It is a graph which shows the chemotaxis which attracts a lymphocyte by ANTES. FIG. 8B
Fig. 4 is a graph showing chemotaxis of attracting monocytes by recombinant Lkn-1 and hMIP-1α. FIG. 8C is a graph showing chemotaxis inducing neutrophils by recombinant Lkn-1 and IL-8.

As shown in FIGS. 8A to 8C, recombinant Lkn-1 was found to be a potent chemokine that attracts human peripheral blood neutrophils, monocytes and lymphocytes. Recombinant Lkn-1 exhibits chemotaxis to attract neutrophils in the same manner as IL-8 (see FIG. 8C), and chemotactic to attract monocytes in the same manner as hMIP-1α. (See FIG. 8B) and attract lymphocytes in a concentration-dependent manner up to Lkn-1 concentrations of 10 Fg / ml, despite showing lower levels of chemotaxis for lymphocytes than RANTES. It showed improved chemotaxis (see: FIG. 8A).

Example 7 Calcium efflux by recombinant Lkn-1 (calcium flu)
x) analysisExample 7-1 : Analysis of calcium efflux in lymphocytes, monocytes and neutrophils Recombinant Lkn-1 binds to receptors and activates lymphocytes, monocytes and neutrophils
To determine whether it would induce calcium efflux. Other agoni to the receptor
The competition between the strike and Lkn-1 was also examined. Using MSIII Fluorometer (Photon Technology International, USA)
[Ca in isolated peripheral blood leukocyte subsets (lymphocytes, monocytes and neutrophils) ²⁺ ] To determine receptor activation. That is, cells
And 2FM's fura-2AM (Sigma Chemical Co., US)
A) is reacted at 37 ° C. for 45 minutes, washed twice and containing 0.05% BSA
1 × 10 in HBSS (pH 7.4)⁷Resuspended to a concentration of cells / ml. 2 ml of the cell suspension was added to a stirred water jacketed cuvette (wa).
ter-jacketed cuvette) plus 340 nm at 37 ° C and
It was activated continuously at 380 nm. The emitted fluorescence was determined by the 25 nM agonist (
Recombinant Lkn-1, hMIP-1α, RANTES, IL-8 or eotaxy
Before and after the addition of (n) (see FIGS. 9A to 9F).

In FIGS. 9A to 9F, the relative fluorescence is shown by the relative ratio of the fluorescence activated at 340 nm and 380 nm. FIG. 9A shows RANTES and recombinant Lkn-
Figure 5 shows the relative fluorescence measured in lymphocytes stimulated by one series of additions. FIG. 9B shows the relative fluorescence measured in lymphocytes stimulated by a series of additions of recombinant Lkn-1 and RANTES. FIG. 9C shows hMIP-1α.
And the relative fluorescence measured in monocytes stimulated by a series of additions of recombinant Lkn-1. FIG. 9D shows the relative fluorescence measured on monocytes stimulated by a series of additions of recombinant Lkn-1 and hMIP-1α. FIG.
Figure 5 shows the relative fluorescence measured in neutrophils stimulated by a series of additions of L-8 and recombinant Lkn-1. FIG. 9F also shows the relative fluorescence measured in neutrophils stimulated by a series of additions of recombinant Lkn-1 and IL-8. As shown in FIGS. 9A to 9F, it was revealed that recombinant Lkn-1 induces calcium efflux in lymphocytes, monocytes and neutrophils.

On the other hand, G protein-coupled receptor (G protein-coupled r)
When receptors are continuously exposed within a short period of time to a ligand having the same binding site as the receptor signal, the receptor becomes desensitized.
). Such a phenomenon appeared when the cells expressing the receptor with recombinant Lkn-1 were stimulated.

As shown in FIGS. 9A to 9F, when stimulated later with RANTES and hMIP-1α, recombinant Lkn-1 was found to completely desensitize lymphocytes and monocytes. (See: FIGS. 9B and 9D). When subsequently stimulated with recombinant Lkn-1, RANTES or hMIP-1α did not completely desensitize lymphocytes and monocytes (see FIGS. 9A and 9C). This indicates that recombinant Lkn-1
Shares a receptor with RANTES and hMIP-1α, RANTES and hMI
This indicates that calcium efflux is further induced by P-1α. In addition, even though recombinant Lkn-1 induced calcium efflux in neutrophils, neutrophils were not desensitized by recombinant Lkn-1 during further stimulation with IL-8 (see: FIG. 9F). )
. In addition, IL-8 did not desensitize neutrophils during further stimulation with recombinant Lkn-1 (see: FIG. 9E). Therefore, this means that recombinant Lkn-1
Despite being a potent factor in inducing calcium efflux in neutrophils like 8, it clearly shows that it does not share a receptor with IL-8.

Example 7-2 : Analysis of calcium efflux in cell lines expressing CC chemokine receptor In Example 7-1, recombinant Lkn-1 was RANTES and hMIP-1α
Has been shown to activate receptors that can be activated by CC or C
Cell lines expressing the XC chemokine receptor were tested for whether recombinant Lkn-1 activated. Instead of using isolated leukocyte subsets, recombinant CCR1,
HOS expressing CCR2B, CCR3, CCR4, CCR5 or CXCR4
Cell lines (see: NIH AIDS Research and Related Reagents Program: AIDS Reseach and Reference Reagent Program)
of NIH, USA), except for using Example 7-1. hMIP-1α binds to both CCR1 and CCR5, and RANTES is known to bind to CCR1, CCR3 and CCR5.

FIG. 10 (A) shows the relative fluorescence measured in a CCR1-expressing HOS cell line stimulated by the continuous addition of various reagents, and FIG. 10 (B) shows the relative fluorescence stimulated by the continuous addition of various reagents. FIG. 4 is a graph showing relative fluorescence measured in HOS cell lines expressing CCR3.

As can be seen from FIGS. 10 (A) and 10 (B), it was revealed that recombinant Lkn-1 strongly induced calcium efflux in HOS cell lines expressing CCR1 or CCR3. However, no calcium efflux by recombinant Lkn-1 was observed in HOS cell lines expressing other receptors.

Further, as can be seen from FIG. 10 (A), CCR1-HOS cells were first transformed with recombinant Lk
When stimulated by n-1 it is not stimulated by hMIP-1α or RANTES, but when CCR1-HOS cells are stimulated first by hMIP-1α or RANTES, stimulation by recombinant Lkn-1 is affected. Did not.

FIG. 10 (A) shows that recombinant Lkn-1 is more C-expressed than hMIP-1α or RANTES.
It shows that it is a potent agonist for CR1,
This is clear from FIG. 10 (C) in which the calcium response depending on the concentration of -1 and hMIP-1α was investigated.

Furthermore, as can be seen from FIG. 10 (B), when CCR3-HOS cells were previously stimulated with eotaxin, almost completely desensitized during further stimulation with eotaxin or recombinant Lkn-1. CCR3-HOS cells were preceded by recombinant Lkn
-1 stimulated almost completely during further RANTES stimulation; however, the CCR-HOS cells were preceded by recombinant Lkn-1 or RA.
When stimulated by NTES, stimulation by additional eotaxin is unaffected. Furthermore, as a result of examining the concentration-dependent calcium response of eotaxin, recombinant Lkn-1 and RANTES (see FIG. 10 (D)), recombinant Lkn-1 is less potent than eotaxin, but more reactive with CCR3 than RANTES. It was confirmed to be a potent agonist.

As described and demonstrated in detail above, the present invention provides a cDNA encoding a novel Lkn-1 protein belonging to C6β-chemokine, and the amino acid sequence deduced therefrom. The open reading frame of Lkn-1 cDNA encodes 113 amino acids including a 21 amino acid signal peptide, of which the mature protein consisting of 92 amino acids has a molecular weight of 10,16.
It is estimated to be 2 Daltons. The recombinant Lkn-1 expressed and purified in Escherichia coli or insect cells using the Lkn-1 cDNA significantly suppresses colony formation and cell proliferation, attracts neutrophils, monocytes and lymphocytes, and performs chemical reaction. Causing chemotaxis,
Binds to CCR1 and CCR3 receptors. Therefore, the recombinant Lkn-1 protein can be used as a drug that has the potential to produce antibodies, treat HIV-1 infection, protect bone marrow stem cells during chemotherapy or radiation therapy, and suppress leukemia. it can.

[0059]

[Sequence list]

[Brief description of the drawings]

The objects and configurations of the present invention described above will become more apparent from the accompanying drawings and description below.

FIG. 1 (A) shows the nucleotide sequence of a 202 bp DNA fragment (SEQ ID NO: 1) and the amino acid sequence deduced therefrom (SEQ ID NO: 2). FIG. 1 (B) shows the amino acid sequence of Lkn-1 (SEQ ID NO: 2) and 87 of mMRP-2.
3 shows a comparison with the amino acid sequences of Nos. 110 to 110 (SEQ ID NO: 3).

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

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

FIG. 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), m
FIG. 9 shows a comparison with the amino acid sequences of MIP-1α (SEQ ID NO: 11) and hMIP-1β (SEQ ID NO: 12).

FIG. 5 (A) is a graph showing the effect of Lkn-1 on myeloid cell colony formation in bone marrow. FIG. 5 (B) is a graph showing the effect of Lkn-1 on myeloid cell colony formation in the spleen. FIG. 5 (C) is a graph showing the effect of Lkn-1 on the proliferation rate of myeloid cells in bone marrow. FIG. 5 (D) is a graph showing the effect of Lkn-1 on the proliferation rate of myeloid cells in the spleen.

FIG. 6 (A) is a graph showing the dose-dependent inhibitory effect of Lkn-1 on the proliferation rate of myeloid cells in bone marrow. FIG. 6 (B) is a graph showing the dose-dependent inhibitory effect of Lkn-1 on the proliferation rate of myeloid cells in the spleen.

FIG. 7 (A) is a graph showing the time-dependent inhibitory effect of Lkn-1 on the growth rate of CFU-GM in bone marrow. FIG. 7 (B) is a graph showing the time-dependent inhibitory effect of Lkn-1 on the proliferation rate of BFU-E in bone marrow. FIG. 7 (C) is a graph showing the time-dependent inhibitory effect of Lkn-1 on the growth rate of CFU-GEMM in bone marrow. FIG. 7 (D) is a graph showing the time-dependent inhibitory effect of Lkn-1 on the growth rate of CFU-GM in the spleen. FIG. 7 (E) is a graph showing the time-dependent inhibitory effect of Lkn-1 on the proliferation rate of BFU-E in the spleen. FIG. 7 (F) is a graph showing the time-dependent inhibitory effect of Lkn-1 on the growth rate of CFU-GEMM in the spleen.

FIG. 8 (A) shows recombinant Lkn-1 and RANTES (regulated-on activation, normal T cell expressed and s).
Figure 4 is a graph showing chemotaxis to attract lymphocytes by ecreted). FIG. 8 (B) is a graph showing the chemotaxis of attracting monocytes by recombinant Lkn-1 and hMIP-1α. FIG. 8 (C) is a graph showing chemotaxis inducing neutrophils by recombinant Lkn-1 and IL-8.

FIG. 9 (A) is a graph showing relative fluorescence measured in lymphocytes stimulated by continuous addition of RANTES and recombinant Lkn-1. FIG. 9 (B) is a graph showing relative fluorescence measured in lymphocytes stimulated by continuous addition of recombinant Lkn-1 and RANTES. FIG. 9 (C) is a graph showing relative fluorescence measured on monocytes stimulated by continuous addition of hMIP-1α and recombinant Lkn-1. FIG. 9 (D) is a graph showing relative fluorescence measured on monocytes stimulated by continuous addition of recombinant Lkn-1 and hMIP-1α. FIG. 9 (E) is a graph showing relative fluorescence measured in neutrophils stimulated by continuous addition of IL-8 and recombinant Lkn-1. FIG. 9 (F) is a graph showing relative fluorescence measured in neutrophils stimulated by continuous addition of recombinant Lkn-1 and IL-8.

FIG. 10 (A) shows CCR1-expressing H stimulated by sequential addition of various reagents.
Figure 4 is a graph showing relative fluorescence measured in an OS cell line. FIG. 10 (B) shows CCR3-expressing H stimulated by successive addition of various reagents.
Figure 4 is a graph showing relative fluorescence measured in an OS cell line. FIG. 10 (C) is a graph showing the magnitude of the calcium response peak depending on the concentrations of Lkn-1 and hMIP-1α. FIG. 10 (D) is a graph showing the magnitude of the calcium response peak depending on the concentrations of Lkn-1, eotaxin and RANTES.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 5/00 B (72) Inventor Chang, So-the-second Second Korea 463-050 Gyeonggi-do , Seongnam, Theohyun-Dong, Hyundai Apartments 112-902 (72) Inventor Park, Do-Hong 137-062 Seoul, Theochok, Bambaye-2-dong, 2754-3 (72) Inventor Beek, Seung 449-900 Gyeonggi-do, Yangguin, Kiheang-Iup, Singal-li, 422-16 (72) Inventor Lee, Eung-Kyong Republic of Korea 134-022 Seoul, Kangdong-ku, Cheongho 2-dong 320- 8 F term (reference) 4B024 AA01 BA21 CA03 CA04 CA09 CA12 DA02 DA06 EA04 GA14 4B065 AA26X AA90X AA94Y AB01 BA03 BD14 CA24 CA44 4C084 AA02 AA06 AA07 BA01 BA21 BA23 NA06 ZB261 ZB262 ZB271 ZB272 ZC801 ZC802 4H045 AA10 AA20 AA30 BA10 CA42 DA01 EA28 FA

Claims (25)

[Claims] 1. A human C6β having a base sequence shown below (SEQ ID NO: 6)
-CDNA for the chemokine Lkn-1 (leukotactin-1): 2. A human C having the amino acid sequence shown below (SEQ ID NO: 5).
6β-chemokine Lkn-1 (leukotactin-1): 3. A cDNA of human C6β-chemokine Lkn-1 in which the nucleotide sequence of Nos. 1 to 63 has been removed from the cDNA of human C6β-chemokine Lkn-1 (leukotactin-1). 4. An expression vector comprising the cDNA according to claim 1. 5. An expression vector PVL1392 comprising the cDNA of claim 1.
-Lkn-1. 6. A method for producing recombinant leukotactin-1, comprising the steps of transforming a host cell with the expression vector according to claim 4, culturing the transformant, and obtaining recombinant Lkn-1 therefrom. 7. The method according to claim 6, wherein the host cell is Escherichia coli or an insect cell. 8. A recombinant leukotactin-1 produced by a method comprising the steps of transforming a host cell with the expression vector according to claim 4, culturing the transformant, and obtaining recombinant Lkn-1 from the culture. . 9. The recombinant leukotactin-1 according to claim 8, which suppresses colony formation. 10. The recombinant leukotactin-1 according to claim 8, which strongly attracts human peripheral blood neutrophils, monocytes and lymphocytes and causes chemotaxis. 11. The recombinant leukotactin-1 according to claim 8, which binds to CCR1 and CCR3 receptors. 12. RANTES (regulated activated normal T cell expression sequence) and h
The MIP-1α (human macrophage inflammatory protein-1α) shares a receptor, but does not share a receptor with IL-8 (interleukin-8).
2. The recombinant leukotactin-1 according to 1. An expression vector comprising the cDNA according to claim 3. 14. An expression vector pET21a containing the cDNA according to claim 3.
-Lkn-1. 15. An Escherichia coli transformed with the expression vector according to claim 14.
XL-1 Blue) hMRP-2 (ATCC 98166). 16. A host cell is transformed with the expression vector according to claim 13,
A method for producing recombinant leukotactin-1, comprising a step of culturing a transformant and obtaining recombinant Lkn-1 therefrom. 17. The method according to claim 16, wherein the host cell is Escherichia coli or an insect cell. 18. A host cell is transformed with the expression vector according to claim 13,
Recombinant leukotactin-1 produced by a method comprising culturing a transformant and obtaining recombinant Lkn-1 therefrom. 19. The recombinant leukotactin-1 according to claim 18, which suppresses colony formation. 20. The recombinant leukotactin-1 according to claim 18, which strongly attracts human peripheral blood neutrophils, monocytes and lymphocytes and causes chemotaxis. 21. The recombinant leukotactin-1 according to claim 18, which binds to CCR1 and CCR3 receptors. 22. The recombinant leukotactin-1 of claim 21, wherein the recombinant leukotactin-1 shares a receptor with RANTES and hMIP-1α, but does not share a receptor with IL-8. 23. A method for protecting myeloid cells from a cytotoxic anticancer agent or radiation, comprising the step of administering human C6β-chemokine Lkn-1 (leukotactin-1) to a subject. 24. The method according to claim 23, wherein the human C6β-chemokine Lkn-1 (leukotactin-1) is the recombinant leukotactin-1 according to claim 8. 25. The method according to claim 23, wherein the human C6β-chemokine Lkn-1 (leukotactin-1) is the recombinant leukotactin-1 according to claim 18.