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WO1997012914A1 - Nouvelle chemokine cc humaine - Google Patents

Nouvelle chemokine cc humaine Download PDF

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Publication number
WO1997012914A1
WO1997012914A1 PCT/JP1996/002851 JP9602851W WO9712914A1 WO 1997012914 A1 WO1997012914 A1 WO 1997012914A1 JP 9602851 W JP9602851 W JP 9602851W WO 9712914 A1 WO9712914 A1 WO 9712914A1
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WIPO (PCT)
Prior art keywords
human
peptide
sequence
dna
seq
Prior art date
Application number
PCT/JP1996/002851
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English (en)
Japanese (ja)
Inventor
Motoji Kitaura
Toshihiro Nakajima
Shigenori Harada
Original Assignee
Shionogi & Co., Ltd.
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Publication date
Application filed by Shionogi & Co., Ltd. filed Critical Shionogi & Co., Ltd.
Priority to AU70976/96A priority Critical patent/AU7097696A/en
Publication of WO1997012914A1 publication Critical patent/WO1997012914A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a peptide having activity against human eosinophils: a structural gene of the peptide, an expression vector having the gene, a transformant into which the expression vector is introduced, and the peptide using the transformant.
  • the present invention relates to a method of producing a monoclonal antibody against the peptide: a method of measuring the peptide using the monoclonal antibody; and a method of screening an agonist or an antagonist of the peptide.
  • Leukocytes differ in the types that exude to the site of inflammation depending on the disease. Neutrophils are mainly in acute inflammation such as normal bacterial infection, immune complex deposition and trauma, monocytes are mainly in bacillus infection, typhoid infection, and delayed type hypersensitivity, and mainly in virus infection. Lymphocytes accumulate and infiltrate, and eosinophils and basophils leach out with immediate allergy or parasite infection (Baggi ol ini, M.
  • chemokine chemokine; chemoattoactant and cytokitoacti ty.
  • the peptide is derived from human small intestine.
  • the DNA molecule of the present invention encodes the peptide described in any of the above.
  • the DNA molecule has a nucleotide sequence consisting of G at position 18 to A at position 389 of SEQ ID NO: 1 in the sequence listing.
  • the aforementioned DNA molecule has a nucleotide sequence consisting of A at position 9 9 to A at position 3 8 9 of SEQ ID NO: 1 in the sequence listing.
  • the above DNA molecule has a nucleotide sequence consisting of G at position 1 to A at position 559 of SEQ ID NO: 1 in the sequence listing.
  • the expression vector of the present invention comprises the DNA molecule described in any of the above.
  • the transformant of the present invention can be obtained by introducing the above expression vector into a host.
  • the host is an insect cell, in particular a silkworm-derived cell line.
  • the method for producing a peptide of the present invention includes the steps of culturing the above transformant to produce a peptide, and recovering and purifying the produced peptide from the culture medium.
  • the monoclonal antibody of the present invention is directed to the above peptide.
  • the method for measuring a peptide of the present invention is characterized by using the above-mentioned monoclonal antibody.
  • the method of screening for an agonist or antibody of the peptide of the present invention comprises reacting a sample presumed to contain the agonist or anthrax with a receptor specific for the peptide, and binding the same. And Z or measuring the reactivity.
  • FIGS. 1A and 1B show the results of Northern blot analysis for the expression of mRNA of the hyataxin of the present invention in each tissue of rabbit respectively.
  • FIG. 2 is a diagram showing a comparison of the amino acid sequences of the human betaxin peptide of the present invention and the previously reported CC chemokine.
  • FIG. 3 shows the human betaxin peptide of the present invention and the previously reported MCP-3 peptide.
  • Eotaxin has been cloned (Jose, PJ et al., Biochem. Biophys. Res. Commun., 205. 788-794 (1994) ''. This is a CC chemokine that does not cause neutrophils to migrate. is there.
  • chemokine receptors having migration activity of human eosinophils have been conducted on chemokine receptors having migration activity of human eosinophils, and they are specific to CC CKRK MCP-1 which is a specific receptor for MIP-1 and MCP-3 and RANTES.
  • CC CKR2B which is a specific receptor CC CKR2B which is specific for MCP-1 and MCP-3
  • CC CKR3 which is a receptor which is not specific for these chemokines has been reported (Combadiere. C Biol. Chem., 270. 16 491-16494 (1995)). Disclosure of the invention
  • the cDN ⁇ of guinea pig thataxin is first cloned, and this is used as a probe for leukocyte running from the human genome DMA library.
  • a new DNA fragment of the chemoattractant CC chemokine was cloned.
  • Northern blot analysis using the obtained DNA fragment as a probe, it was found that this novel CC chemokine is highly expressed in human small intestine.
  • a full-length cDNA of the novel CC chemokine was cloned from a human (small intestine) cDNA library and named hy- Subsequently, the human ectocine gene was expressed in insect cells, and it was found that this human ectokinin peptide had an activity to increase the intracellular calcium ion concentration of human eosinophils, and the present invention was completed. .
  • the peptide of the present invention comprises the amino acid sequence from Gly at position 24 to SEQ ID NO: 1 of SEQ ID NO: 1 in the sequence listing, or a similar sequence thereof.
  • the peptide comprises the amino acid sequence from Met at position 1 to Pro at position 97 of SEQ ID NO: 1 in the sequence listing or a similar sequence thereof.
  • the amino acid sequence from Met at position 1 to Ala at position 23 of SEQ ID NO: 1 in the sequence listing is considered to be a signal peptide.
  • the peptide has a calcium concentration in human eosinophils It may contain a modified amino acid or base.
  • PHA-P phytohemagglutinin-P
  • the purified mRNA obtained from guinea pig thymocytes can be used to synthesize single-stranded cDNA, for example, by a commercially available kit.
  • PCR reaction is carried out using the primer 1 obtained in the above (A) and a thermostable DNA polymerase.
  • the resulting reaction solution is treated with a restriction enzyme to obtain a low melting point
  • Fig. 7 is a graph showing the results of IL-8 activity on human eosinophils, monocytes and neutrophils (activity to increase intracellular calcium ion concentration).
  • FIG. 7 is a graph showing the results of IL-8 activity on human eosinophils, monocytes and neutrophils (activity to increase intracellular calcium ion concentration).
  • FIG. 5 is a graph showing the results for the activity (activity to increase the intracellular calcium ion concentration) of the human etataxin peptide of the present invention and the previously reported chemokine on the CC chemokine receptor CC CKR3 expressing K562 cell line.
  • 'Chemokines' are specific attractants for leukocytes to show chemotaxis to the inflammatory response area, have some selectivity for migrating leukocytes, and have characteristic 4 system residues It refers to the family of polypeptides. These are related to amino acid sequence and biological activity.
  • the four cysteine residues of the chemokine are disulfide bonded between the first and third residues and between the second and fourth residues, respectively.
  • the chemokine which does not contain another amino acid between the first and second cysteine residues is distinguished as “C C chemokine”, and the chemokine containing one other amino acid as C X C chemokine.
  • CC chemokines are known to migrate monocytes but not neutrophils
  • CxC chemokines are known to migrate neutrophils and not monocytes.
  • the hytaxin peptide which is a novel ⁇ CC chemokine of the present invention, has an activity to increase the intracellular calcium concentration of human eosinophils.
  • the "similar sequence" to the amino acid sequence or DNA sequence is not necessarily limited to a specific sequence, and includes, in addition to this sequence, substitutions, insertions, deletions, etc. known to those skilled in the art and encoded. Refers to a sequence containing a modification to which the function or activity of the peptide is substantially the same. Alternatively, if the function or activity of the peptide is of substantially the same degree, chemical or biochemical modification, or non-natural or derivative Plasmid DNA is prepared from the resulting single-celled bacterial cells by alkaline SDS method, for example, treated with alkaline denaturation method (treated with NaOH, then neutralized with ammonia acetate and precipitated with ethanol), Prepare strand plasmid DNA.
  • nucleotide sequence of cDNA including the translated region of guinea pig etataxin can be determined, for example, by the dihydroxy method.
  • molmototetaxin cDNA shown in SEQ ID NO: 4 in the sequence listing is obtained.
  • guinea pig etataxin cDNA obtainable in the above (1)
  • appropriate forward and reverse primers are synthesized and used for the next PCR.
  • the single-stranded plasmid DMA of guinea pig etataxin obtained in the above (D) is used as a template, PCR reaction is performed using Taq polymerase, separated by agarose gel electrophoresis, and the desired size (about 330 bp)
  • the DNA band of (1) can be excised to purify the DNA fragment. This DNA fragment is labeled with 32 P or the like, which can be used as a probe.
  • infect E. coli with an appropriate human genome library for example, human lymphocyte genome library, human placenta genome library, human brain genome library
  • an appropriate human genome library for example, human lymphocyte genome library, human placenta genome library, human brain genome library
  • put plaques on the plate as a primary screen After formation, transfer to nylon membrane. After prehybridizing this membrane, it is hybridized with the probe obtained in section (A) above. After washing the membrane, the radioactivity can be detected and positive plaques giving rise to a signal can be selected.
  • PCR for the translation region of the already reported base sequence MCP-1, MCP-3, etc.
  • the plaque having the previously reported base sequence ie, the sequence which can be amplified
  • single clones can be selected by serial dilution and secondary and tertiary screening for phages that do not contain a published nucleotide sequence. That is, the phage DNA is digested with various restriction enzymes and separated by agarose gel electrophoresis, and those having the same band pattern are regarded as the same clone, and the same hybridization is repeated to make the positive band as small as possible. Let give Separation by gel electrophoresis, DNA bands of the desired size (about 150 bp) can be excised, and DNA fragments can be purified.
  • the nucleotide sequence of this inserted DNA is obtained by, for example, the dideoxy method. Partially sequenced. As a result, the DNA sequence shown in SEQ ID NO: 3 in the sequence listing is obtained.
  • cDNA is synthesized and inserted into an appropriate cloning vector.
  • the resulting plasmid can be introduced into E. coli, yeast, etc. to prepare a cDNA library.
  • primary and secondary screening can be performed by PCR. In this screening, an appropriate primer is synthesized and used based on the: fe base sequence (SEQ ID NO: ⁇ 8) of the Mormot's DNA fragment obtained in the above (C). be able to.
  • the cDNA library is first aliquoted into, for example, a 96-well plate, cultured, PCR is performed using this as a template, and separated by agarose gel electrophoresis, and the pool containing the target clone is approximately 100 bp.
  • the pool in which DNA is amplified in the primary screening may be divided into smaller pools, which may be used as templates to perform PCR and be separated by agarose gel electrophoresis. As a result, select a pool in which amplification of the target band is observed. For this, further tertiary screening can be performed by a colony hybridization method or the like.
  • cells are collected from a selected colony, suspended in an appropriate medium, spread on a plate, cultured, and transfected on a nylon membrane. After this membrane is prehybridized, it is soaked in a hybridization solution containing thaetacine DNA fragment labeled with, eg, 32 P, fluorescein etc. and hybridized. The membrane is then washed and analyzed for radioactivity. Next, as the fourth screening, select the colonies from which the signal was obtained from each plate, pick up the cells, suspend in a suitable medium, and spread it on a plate to culture the cells of single colonies. You can get it. For example, it can be incorporated into PGEM3SR dhfr, pEF-BOS, pVL1 392, pMAL-C2 etc. to provide an expression vector for expressing human formatin peptide.
  • This expression vector is introduced into, for example, bacteria, yeast, insect cells or animal cells to prepare transformants. By culturing this transformant, the human betaxin peptide of the present invention can be produced.
  • the culture of the human taxa gene thus obtained can be purified by affinity column method, ion exchange column method, gel filtration column method or the like.
  • white blood cells cause morphological change when they come in contact with chemotactic factors, and perform linear motion in the direction of higher chemotactic factor concentration. That is, when a chemotactic factor binds to a receptor on the leukocyte membrane, a local change in membrane potential of the leukocyte occurs, and an increase in intracellular calcium ion occurs. As a result, the metabolic rate increases. With the increase of the A.sub.MP concentration, a large change occurs in the intracellular location or form of microtubules and actin fibers, and is considered to be transformed into mobile leukocytes along with receptor migration and localization.
  • changes in the intracellular calcium ion concentration of leukocytes can be measured, for example, for human eosinophils as follows. To a human eosinophil suspension, add a fluorescent reagent (eg, Fur-a-2 / AM etc.) that is affected by intracellular calcium ions, and perform a brain tube. Then, the change in fluorescence when the test solution is added to the obtained eosinophil suspension can be measured using a fluorescence spectrophotometer. As a result of measurement in this manner, it was found that the human etataxin peptide of the present invention has an activity of increasing the intracellular calcium ion concentration to a rabbit eosinophil. Also, as a result of similar experiments on human monocytes and neutrophils, there was no activity to increase intracellular calcium ion concentration.
  • a fluorescent reagent eg, Fur-a-2 / AM etc.
  • the change in intracellular calcium ion concentration can be similarly measured for human CC chemokine receptor-expressing cells established by gene recombination.
  • the human etataxin of the present invention has an activity to increase intracellular ion concentration specifically to CC CKR3 expressing cells.
  • human ectaxin which is a human CC chemokine of the present invention
  • a human small intestine cDNA library is introduced into E. coli, yeast, etc., and screening is performed using an appropriate probe to perform single clone. Can be selected.
  • phage DNA can be inserted into plasmid DMA to determine the base sequence of the inserted DNA.
  • SEQ ID NO: 1 in the sequence listing a nucleotide sequence of human etataxin (SEQ ID NO: 1 in the sequence listing), which matches the sequence estimated from the human genome library, was obtained.
  • the amino acid sequence (SEQ ID NO: 1 in the sequence listing) deduced based on the nucleotide sequence of human otataxin obtained as described above contains four cysteine residues characteristic of chemokines, and It was estimated that CC chemokines do not contain another amino acid between the cysteine residues of.
  • a homology search can be performed on a database such as Genban E MBL using a program such as DNASIS (Hitachi).
  • MCP-1, MCP-2, and MCP-3 are most similar (64% to 65% homology), MIP-1 MIP -1, 1-309, and RA TES were found to have a homology of 32 to 36%. In addition, it has a homology of 57% with guinea pig etataxin.
  • the gene encoding the human etataxin peptide of the present invention may be a suitable vector, mRNA can also be detected by PCR using appropriate primers in combination after converting the mRNA to cDNA with reverse transcriptase.
  • the peptide can be confirmed by conventional immunoprecipitation or Western blotting using a human antibody specific for human tubulin obtained in the above (7).
  • Northern blot analysis of the human tubulin of the present invention revealed that the density of the mRNA band indicates that the expression is high in the small intestine and colon.
  • human ectaxin peptide can be performed, for example, as follows. For example, a fixed amount of human ectotaxin peptide labeled with a radioactive isotope, an enzyme such as peroxidase or alkaline phosphatase, or a fluorescent dye, etc., a known concentration of unlabeled human ectaxin, and serum-derived anti-human ectotaxin polypeptide. An oral antibody or a monoclonal antibody is added to cause an antigen-antibody competition reaction.
  • the labeled antigen bound to the antibody and the labeled antigen not bound to the antibody are separated by an appropriate method, and the radioactive amount of the labeled antigen bound to the antibody, enzyme Measure the activity or fluorescence intensity. Since the amount of labeled antigen bound to the antibody decreases as the amount of unlabeled antigen increases, this relationship is graphed to obtain a standard curve.
  • one of two types of monoclonal antibodies that recognize different epitopes on human ectaxin peptide is immobilized, the other is labeled by any of the methods described above, and the amount of human ectaxin bound to the immobilized antibody is labeled.
  • a method (sandwich method) of detecting and quantifying with an antibody is also possible.
  • a sample containing an unknown amount of antigen is added to the above reaction system in place of the unlabeled antigen of known degree, and the amount of radioactivity, enzyme activity or fluorescence intensity obtained after this reaction is applied to a standard curve.
  • the amount of antigen in the sample can be measured.
  • human monocytic cell line THP-1 The PCR method can be performed using cDNA or chick genomic DNA as a template. Based on the previously reported base sequences, for example, both ends of the coding region of a desired chemokine receptor can be selected as a primer.
  • the antibody against the hypotaxin peptide of the present invention may be, for example, a synthetic peptide synthesized on a conventional peptide synthesizer based on a part of the amino acid sequence of the putative hypotaxin peptide, or a vector that expresses human alphaxin.
  • a peptide obtained by purifying human etataxin peptide produced by transformed bacteria, yeast, animal cells, insect cells and the like by a conventional protein chemical method is used as an immunogen to produce a mouse, rat, hamster, An animal such as Usagi can be immunized to produce an antibody (polyclonal antibody) derived from its serum.
  • cells may be removed from the spleen or lymph node of the immunized mouse or rat and fused with myeloma cells, as described by Kohler and MUstein (Nature, 256. 495-497 (1975)) or a modification thereof.
  • a hybridoma is prepared according to the method of Ueda et al. (Pro Natl. Acad. Sci. USA, 79, 4386-439 (1982)
  • monoclonal antibodies can be produced from this hybridoma.
  • a monoclonal antibody of heat taxin peptide can be obtained by the following steps.
  • the presence of the hypotaxin mRNA and peptide of the present invention can be carried out using a conventional specific mRNA and peptide detection method.
  • mRNA can be detected by Northern blot analysis or in situ hybridization method using antisense RNA or cDNA as a probe.
  • the agonist or antagonist of the human thataxin peptide of the present invention can be screened by carrying out a sample presumed to contain an agonist or angonist of the ataxin peptide.
  • cells expressing a receptor specific for human etataxin of the present invention that is, cells expressing CC CKR3 are incubated with the above-mentioned sample under appropriate conditions.
  • the sample may contain an agonist if it has biological activity to increase intracellular calcium ion concentration. Also, if it has no biological activity but has binding activity, the sample may contain an anionist.
  • the above sample is incubated with cells expressing Receptin and human taxax to affect the biological activity and / or binding activity of human tubulin, that is, if the activity is inhibited, this sample is an agonist of hyutaxin. Or may include an evening goonist.
  • the first and second cysteines A forward-directed degenerate primer f42 (SEQ ID NO: 5 in the Sequence Listing), which encodes the amino acid sequence of the amino acid sequence of the region including (from lie to position 10 of Phe in SEQ ID NO: 2 of SEQ ID NO: 2)
  • Guinea pig (Hartley, male, 4 weeks old) The thymus of 1 animal was removed, and thymocytes were released and suspended in 20 ml of RPMI 1640 medium containing 10% FCS (fusial serum). To this was added PH AP (Sigma, NO. L9017) to a final concentration of 5 g / ml and cultured for 1 hour, followed by washing the thymocytes with Dulbecco's PBS (-) (Nissy) and quick plate. The gene fragment obtained by the reaction is integrated into an appropriate cloning vector such as pCRII, pUC19, pBluescriptll KS (+) and amplified. The amplified sequence may then be excised with an appropriate restriction enzyme and incorporated into an appropriate vector, such as pEF-B0S, pGEM3SRadhfr. PREP9, etc. to obtain an expression vector.
  • PH AP Sigma, NO. L9017
  • the expression vector of human CC chemokine receptor 1 obtained in the above (10) is treated with an appropriate restriction enzyme, phenol / clonal format extraction and ethanol precipitation are performed, and then expression is carried out by the electroporation method.
  • the expression vector is introduced into cells of, for example, human red blood cell line K562 cells.
  • CC chemokine receptors can be expressed by culturing cells transformed by electoral plating.
  • CC chemokine receptor after dissolving the DNA of the expression vector of human CC chemokine receptor, for example, in distilled water, calcium chloride is added, and then 2 XBBS solution (50 mM BE S (SIGMA). 280 mM sodium chloride (Nacalai Tesque) After adding, and 1.5 mM disodium hydrogen phosphate (Nacalai Tesque), let stand at room temperature for 25 minutes.
  • the DNA solution thus prepared is added dropwise to, for example, a petri dish in which human embryonic kidney cell line 293T cells are cultured, and cultured for 20 hours in the presence of 35: 3% carbon dioxide gas to introduce DNA into the cells. .
  • CC chemokine receptor By culturing cells into which DNA has been introduced, CC chemokine receptor can be expressed.
  • the human etataxin peptide of the present invention is labeled with 125 I using, for example, Bolton Hunter reagent or labeled with an enzyme such as alkaline phosphatase.
  • a suspension of CC chemokine receptor-expressing cells for example, human erythroleukemia cell line K562 cells or human fetal kidney cell line 293T cells, and incubate at a constant temperature Do. After washing, the amount of monoclonal antibody bound to the cells can be measured by measuring the amount of labeling.
  • the stock solution 1 was diluted 2000-fold, LB / Amp plate (10 g of Tribton in 1 L, 5 g of yeast extract, 10 g of sodium chloride, and Fif 50 pieces were spotted on an agar medium containing 50 mg of ampicillin sodium (one at a time) and cultured at 37 ° C. for 1 cell. Thirty colonies of each colony were collected from each strain, PCR was carried out using it as a template, and separated by agarose gel electrophoresis. As a result, several colonies in which amplification of the target band was observed were found in each plate (pool). Among them, one colony was selected for each boule and third screening was performed by colony hybridization.
  • a 32 P labeled etataxin DNA fragment (25 ng of the guinea pig autaxin DNA fragment shown in SEQ ID NO: 3 in the sequence table) was subjected to 32 using the multi-prime DNA labeling system (Amersham).
  • the plate was dipped in a hybridization solution containing P (labeled) and hybridized at 65 ° C.
  • the membrane was washed with 2 X SSC (0.3 M NaCK 0.03 M sodium citrate) containing 0.1% SDS, twice for 10 minutes at room temperature and once for 15 minutes at 65 °. This membrane was analyzed with a bioimage analyzer (Fuji).
  • For the fourth screening select one colony from each plate for which signal was obtained, pick up the cells, suspend in each medium and add several tens of colonies to LB / Amp plate. The cells were sown and cultured, and single colony cells were collected.
  • single-stranded cDNA was synthesized using the Ready-To- Go-T-primed First-Strand kit (Pharmacia). Using this single-stranded cDNA as a template, and using f42 and r53 as primers and Taq as a thermostable DNA polymerase, PCR reaction (1 minute at 94, 1 minute at 50, 72 minutes at 72) For 40 cycles).
  • the resulting reaction solution is treated with restriction enzyme EcoRI, separated by low melting point agarose gel electrophoresis, a DNA band of the desired size (about 150 bp) is cut out, and DNA RNA is purified using Wizard PCR Preps DNA purification system (Promega). The fragment was purified.
  • the obtained DNA fragment was inserted into pBluescript KS (+) vector treated with EcoRI and ss small intestinal alkaline phosphatase (CIAP), using a DNA ligation kit (Takara).
  • the base sequence of the inserted DNA was determined using AutoRead Sequencing Kit (Pharmacia) and ALF DNA Sequencer.
  • the resulting Morphax ertaxin c DNA sequence is shown in SEQ ID NO: 3 in the sequence listing.
  • cDNA was synthesized using Superscript Plasmid System (Gibco BRL) and inserted into cloning vector pSPORT1.
  • the obtained plasmid was introduced into E. coli (ElectroMAX DH10B, Gibco BRL) by electroporation to prepare a cDNA DNA library of 210,000 clones (4 ml).
  • Primary and secondary screening for cloning etataxin cDNA from this cDNA library was performed by PCR. In this case, based on the nucleotide sequence of the etataxin cDNA fragment of mormot obtained in the above (3) (SEQ ID NO: 3 in the sequence listing), the forward direction eoF and 103 corresponding to positions 22 to 45 are used.
  • PCR was carried out using Taq polymerase in 30 cycles of reaction of 94 "for 1 minute, 58" C for 1 minute, and 72 for 2 minutes.
  • 1 pool of cDNA library 201 (about 1000 clones) is aliquoted into a 96-well plate, and Super Broth medium (in 1 L) Then, the membrane was detected by a bioimage analyzer, and 14 positive plaques were selected.
  • the 14 phage DNAs were subjected to PCR on the translation region of MCP-1 and MCP-3 based on the previously reported nucleotide sequences, and each of them was included because it was included.
  • the remaining 12 phages were serially diluted and subjected to secondary and tertiary screening in the same manner to select 10 single clones.
  • the ten phage DNAs were cleaved with various restriction enzymes and separated by agarose gel electrophoresis. Those with the same band pattern were repeated with the same hybridization for the two clones as the same clone, and cut with a restriction enzyme to give a positive band of the smallest possible size.
  • the positive DNA fragments of these two clones were inserted into the pBlues crisp KS (+) vector, and the nucleotide sequence of the inserted DNA was determined.
  • the three exon Z 2 intron structure characteristic of the human CC chemokine gene (Baggio lini, M. et al., Ad. I ⁇ unol., 55, 97-179 (1994)) (2 introns are G and 17 of positions 174 of the base sequence of SEQ ID NO: 1 in the sequence listing, respectively). It was present between position 5 and between position 286 and position 287 C.
  • One of the two clones is a known MCP-2 and the other (clone 141) is a novel human It was CC chemokine (human etataxin)
  • the cDNA sequence of human etataxin is as shown in SEQ ID NO: 1 in the sequence listing.
  • a probe was prepared as follows. Based on the nucleotide sequence of human etataxin genome gene, the forward-directed DNA sequence (corresponding to positions 288 to 309 of SEQ ID NO: 1) and the reverse-directed DNA sequence (corresponding to SEQ ID NO. (Corresponding to positions 483 to 504 in SEQ ID NO: 1) was synthesized as a primer and used for the next PCR.
  • the phage DNA of clone 141 obtained in item (2) of Example 2 above is used as a template, and 30 cycles of 1 cycle at 94, 1 minute at 55, and 2 minutes at 72 "C using Taq polymerase.
  • the PCR reaction product was separated by agarose gel electrophoresis, the DNA band of the desired size (217 bp) was cut out, and the DNA fragment was purified using the Wizard PCR Preps DNA Purification System. Fragment 25 ng, Prime-It
  • a single-stranded plasmid DNA is prepared by treating it with neutralization and ethanol precipitation to prepare a single-stranded plasmid DNA, which is used as a template for sequencing the cDNA sequence containing the translated region of guinea pig ayu-tan, Sequenase 7-deaza-dGTP It was determined by the dideoxy method using the kit (Ver. 2.0). As a result, a guinea pig strain taxin cDNA shown in SEQ ID NO: 4 in the sequence listing was obtained.
  • the PCR reaction was repeated, separated by agarose gel electrophoresis, a DNA band of the desired size (330 bp) was cut out, and the DNA fragment was purified using Wizard PCR Preps DN'A purification system.
  • the 25 ng of the DNA fragment was 32 P-labeled using a multiprime DNA labeling system and used as a probe.
  • a human genome library 1 (human lymphocyte genome library (ADASH vector), Stratagene) 600,000 clones using phage vector 1 was infected with E. coli (NM 53 8) and used as a primary screen on LB plates (10 g of lipeptone in 1 L).
  • MCP-1, MCP-2 and MCP-3 are most similar (64-65% homology), MIP-1a, MiP-1... 309, and RANTES 32 to 36% homologous It was a rate. In addition, it was 57% equivalent to guinea pig etauxin.
  • the forward primer including the Not! Site at positions 98 to 120 and its 5 'side of SEQ ID NO: 1 and reverse to include the translation region.
  • a directional primer positions 369 to 392 of SEQ ID NO: 1 and an Xbal site added to the 5 'side thereof was synthesized. Using these primers, PCR was performed using the plasmid pYEU1 obtained in Example 4 as a template, under the same conditions as in Example 1 (1).
  • the reaction solution is treated with restriction enzymes Notl and Xbal, separated by low melting point agarose gel electrophoresis, the DNA band of the desired size (about 300 bp) is cut out, and the DNA fragment is digested using the Wizard PCR Prep DNA Purification System. Refined.
  • the resulting DNA fragment was introduced into a Baculovirus transfer vector PVL1392 (PHARMINGEN) treated with Notl and Xbal to construct pVLl.
  • the recombinant vector PVL141 and a linear AcNPV DNA having a lethal deletion were simultaneously introduced into Sf9 insect cells to obtain a recombinant baculovirus.
  • This virus was infected with Tn5B-4 insect cells (I nvnrogen) (1.2 x 10 7 cells per 150 cm 2 flask) with M. 0. I, 10 to 20, and the EX- CELL 400 serum-free medium was used.
  • the cells were cultured at 27 for 3 days in URH Biosciences (20 ml per 150 cm 2 flask).
  • the culture supernatant was recovered and filtered through a 0.2200 filter membrane.
  • the filtrate was dialyzed against 20 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl.
  • the dialyzed culture supernatant was applied to HiTrap Heparin (Pharmacia) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) and 150 mM NaCl.
  • elution was performed with 20 Tr-HC1 buffer (pH 7.5) and 600 mM NaCl.
  • the salt concentration of the eluted fraction is 20 mM Tris-HCl buffer
  • the label was 32 P-labeled using a random primer labeling kit (Stratagene) and used as a probe.
  • Hybridization is carried out using a commercially available membrane (Human Multiple Tissue Northern Blot, Clonetech) to which various human tissues mRNA have been separated by electrophoresis and transferred, and the probe obtained in the above (1).
  • the membrane was washed in 2 X SSC containing 0.1% SDS, twice for 10 minutes at room temperature, and once for 15 minutes at 55, and then dipped in 0.5 X SSC containing 0.1% SDS to 55 I did it once for 15 minutes. This membrane was detected by a bioimage analyzer. The results are shown in A and B of FIG. Arrows in the figure indicate the position of the band of mRNA of human being. As apparent from the band density, strong signals were obtained in the order of small intestine> colon heart.
  • Example 3 Human small intestine cDNA library 1 (human small intestine cDNA library 1 (AMaxl Bek 1), Clontech)]
  • the probe obtained in item (1) of Example 3 above was introduced into E. coli (K802) 0, 000 clones.
  • the first to third screenings were performed in the same manner as in (2) of Example 2 above to select two single clones.
  • Phage (AMaxl) DNA was inserted into plasmid (pYEUra3) DNA for these two clones, and the nucleotide sequence of the insertion DNA was determined in the same manner as in Example 1 (3).
  • the amino acid sequence deduced based on the nucleotide sequence of human etataxin contains four cysteine residues characteristic of chemokines and another amino acid between the first and second cysteine residues. It is presumed that there is no CC chemokine.
  • the amino acid sequence was compared to the sequences of known human CC chemokines and morphoetaxins (a DNASIS (Hitachi) solution using Genbank as a database) Therefore, it has been found that the human tubulin protein has an activity to specifically increase the intracellular calcium ion concentration on human eosinophils.
  • CC CKR1 and CC CKR2B (Combadiere, C. et al., Supra) which are two kinds of human CC chemokine receptor 1 reported previously
  • the cDNA of human monocytic cell line THP-1 was used below.
  • the PCR reaction was performed as follows. The primers shown in SEQ ID NOS: 7 and 8 of the Sequence Listing are used for amplification of CC CKR1 and primers shown in SEQ ID NOS: 9 and 10 of the Sequence Listing, using 500 ng of cDNA of THP-1 cell as template. One 500 ng of each was used for amplification of CC CKR2B.
  • Taq polymerase (Takara Shuzo) was used as an enzyme for the reaction. The reaction was cycled for 3 minutes at 94 minutes, followed by 35 cycles of 1 minute at 94, 2 minutes at 55, 3 minutes at 72, and a reaction for 3 minutes at 72.
  • the gene fragments of CC CKR1 and CC CKR2B obtained by this reaction were each incorporated into the TA cloning site of pCRII (Invitrogen).
  • PCR was performed as follows in order to add Xbal-digested sequences to both ends of the CC CKR1 and CC CKR2B gene fragments.
  • the primers shown in SEQ ID NOS: 11 and 12 in the sequence listing are for amplification of CC CKR1.
  • the primer shown in was used for amplification of CC CKR2B.
  • ExTaq (Takara Shuzo Co., Ltd.) was used as an enzyme for carrying out the reaction. The reaction was cycled at 94 for 2 minutes, followed by 10 cycles of 94 for 30 seconds, 60: 1 minute, 72 * C for 2 minutes, and 72 minutes for 5 minutes. went.
  • CC C KR1 and CC CKR2B were digested with Xbal (Takara Shuzo), respectively, and then the Xbal site of pEF-BOS (Mizushima, S. et al., Nucleic Acids Res., 18, 5322 (1990)) Incorporated into the The plasmids thus obtained were named as pEBCC CKR1 and pEBCC CKR2B, respectively.
  • the concentration of the peptide was determined using the BCA kit (Pierce) with ushi serum albumin as a standard substance. From 100 ml of culture supernatant, 54 ⁇ g of purified human rat taxin peptide was obtained. The amount of contaminating endotoxin was determined by using Limulus amebocyte lysate (QCL-1000, Bio Whitaker), which was less than 2 pg / zg.
  • the N-terminal amino acid sequence of the purified human tubulin peptide was determined using an amino acid sequencer (Shimadzu) and found to be Gly Pro Ala Xaa Val Pro Thr This amino acid sequence was predicted from the nucleotide sequence.
  • the signal peptide (Ala at position 1 to SEQ ID NO: 1 to Ala at position 3 of SEQ ID NO: 1) was cleaved to coincide with the N-terminal amino acid sequence of the mature secretory peptide consisting of 74 amino acids.
  • neutrophils or monocytes isolated from rabbit blood After washing human eosinophils, neutrophils or monocytes isolated from rabbit blood according to a conventional method with Dulbecco's PBS (-), 125 mM of HACM buffer (in 20 mM Hepes. PH 7.4) Suspended in NaCl 3 x 10 6 in 1 ml of 5 mM KCL Im MgCl 2 , 1 mM CaCl 2 , 0.5 m glucose, and 0.025% BSA), and added fura-2 / ⁇ (Molecular Prote) 0.3 mtiol / 10 6 was added and incubated at 37 for 30 minutes.
  • eosinophils, neutrophils or monocytes were suspended in the same buffer at 5 ⁇ 10 6 Zml.
  • the change in the degree of fluorescence when each of the obtained eosinophils, neutrophils, or monocyte suspension 500 ⁇ 1 is added with chemokine, is measured using a fluorescence spectrophotometer (LS50B, PERK1N EL MER).
  • the excitation wavelength was 340 nm and 380 nm
  • the fluorescence wavelength was 510 nm
  • the response was 0.02 seconds.
  • the results are shown in FIG. 3 as the ratio of the fluorescence intensities of 340 nm and 380 ⁇ .
  • Human embryonic kidney cell line 293T cells (DuBridge, RB et al., Mol. Cell Biol., 7, 379-38 7 (1987)) 5 x 10 6 in 10 ml of 10% FCS in D-MEM (GIBCO)
  • the cells were suspended in a cell culture of diameter lOcra and cultured in a petri dish (Iwaki Glass Co., Ltd.) at 37 ° C for 10% in the presence of 10% carbon dioxide gas.
  • the DNA solution thus prepared was added dropwise to a Petri dish for culturing 293T cells, and cultured in the presence of 35%, 3% carbon dioxide gas for 20 hours to introduce DNA into the cells.
  • the transfected cells were washed twice with 3 ml of PBS (+), and 10 ml of D-MEM (GIBCO) containing 10% FCS was added, and the mixture was incubated at 37 for 1 day in the presence of 5% carbon dioxide gas.
  • the cells were cultured and used for intracellular calcium concentration measurement.
  • Example 9 for CC CKR1 and CC CKR2B, the intracellular calcium mobilization activity was increased by the addition of the specific ligands ⁇ -1 ⁇ and miCP-1, respectively. The expression of receipt was confirmed.
  • CC CKR3 is considered to be similarly expressed, as similar transformation and culture were performed using the same cell line as CC CKR1 and CC CKR2B.
  • the expression of CC CKR3 mRNA was confirmed by Northern blot analysis.
  • the reaction was cycled for 3 minutes at 94: 1 and then for 40 cycles under the conditions of 94 for 1 minute, 55 for 1 minute, 72 for 2 minutes, and 72 for 5 minutes.
  • the resulting CC CKR3 gene fragment was digested with Xbal (Takara Shuzo) and then incorporated into pBluescriptll KS (+) at the Xbal position.
  • This plasmid was named pBS CKR3.
  • a CC CKR3 DNA fragment obtained by digesting pBS CKR3 with Xbal was incorporated into the Xbal site of pEF-BOS.
  • the plasmid thus obtained was named pEBCC CKR3.
  • Electrobolition Chamber 1 (BIO-RAD) and allow it to stand on ice for 10 minutes, then set the chamber to Gene-Pulser, 0.25 kV, Electroporation was performed under the conditions of 500 / iF. After pulsing the chamber 1, it was immediately left on ice for 10 minutes. The cells subjected to electoral plating were suspended at 5 ⁇ 10 6 cells / ml in R FCS 640 medium containing 10% FCS (Nichiken), and cultured for 2 days in the presence of 37% ⁇ 5% carbon dioxide gas.
  • the cells were suspended in RPMI 1640 medium containing 10% FCS (Nichiken) in medium supplemented with 800 / ig / ml GEN ETICIN (GIBC0), and aliquoted into 96-well plates at 1 ⁇ 10 5 cells / well.
  • 37 Continue culture in the presence of 5% carbon dioxide, and cells resistant to GENETICIN In pyogenic dermatitis, it infiltrates into a lesion, and it is considered that the infiltration involves a chemokine such as the human glutathione peptide of the present invention.
  • MBP major basic protein
  • ECP eosinophil cation protein
  • human rat taxin peptides according to the present invention have specific activity to eosinophils, they can be used as a therapeutic agent for parasite infection or allergic disease involving eosinophils or in search thereof.
  • activity of chemokines is changed by sequentially deleting the amino acids from the N-terminal amino acid residue to the cysteine residue. Therefore, it is expected that the partial peptide of human tubulin of the present invention will be an inhibitor of human tubulin peptide.
  • Such peptides can be used as inhibitors of human auxin peptides, that is, as therapeutic agents for allergic diseases.
  • antibodies against human ectoxin gene and human ectaxin provided by the present invention are useful for analyzing the mutational state of hytaotaxin gene and the expression state of its mRNA and peptide, Therefore, it is also useful for the diagnosis of allergic diseases.
  • a hereditary disease caused by abnormalities in the human etataxin gene by introducing the human taxaine gene provided by the present invention directly or in an appropriate vector and introducing it into cultured cells ex vivo and then administering it into the body. It is useful for developing gene therapy for various types of cancer and diseases caused by parasites.
  • an antagonist or anthonist which also can be used as an inhibitor of the human tau eustin peptide, That is, it can be used as a therapeutic agent for parasitic infection, cancer and allergic diseases.
  • the solution was added and incubated for 30 minutes at 37T :. After washing with HACM buffer, each CC chemokine receptor-expressing cell was suspended in the same buffer at 5 ⁇ 10 6 Zinl.
  • the human etataxin peptide showed an increase in the ratio of fluorescence intensity only to CC CKR3 expressing cells, and further, desensitization in which the ratio of fluorescence intensity did not change by the continuous addition was also observed.
  • an increase in the ratio of fluorescence intensity was not observed for CC CKR1 and CC CKR2B, which are other CC chemokine receptors.
  • the increase in the ratio of fluorescence intensity was recognized in MIP-1 alpha and M CP-1 peptides which are positive controls for each recipient.
  • the human X: taxin peptide of the present invention has an activity to specifically increase intracellular calcium ion concentration against CC CKR3 expressing 293T cells. From these results, it is considered that the hytaxaxin of the present invention is a specific ligand of CC CKR3 whose specific ligand is still unknown.
  • Eosinophils flowing into the blood may cause parasite infection, allergic diseases, especially asthma and 55 60 6 ⁇ 70
  • GTATCCTCAT TATCAGTCCA AAGGGCATGG GTTTTATTAT ATATATATAT ATTTTTTTTT 529
  • GATCCCCTGT CACGTGTGGG CAATGTTCCC CCTCTCCTCT CTTCCCTCCT GGAATCTTGT 709
  • Sequence type nucleic acid
  • AGG AAG ATA CCC CTT CAG CGA CGA CTA GAG AGC TAC AGG AGA ATC ACC AGT 260 Arg Lys He Pro Leu Gin Arg Leu Glu Ser Ser Arg Arg Arg He Thr Ser
  • GAC AAA ATG ATA TGT GCG
  • GAC CCC AAG AAG TGG GTT CAG GAT GCC 297 Asp Lys Met lie Cys Ala Asp Pro Lys Lys Trs Trp Val Gin Asp Ala
  • Sequence type nucleic acid
  • Organism name Guinea pig
  • Sequence type nucleic acid
  • Type E nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • a peptide comprising the amino acid sequence from Gly 24 to Pro 97 of SEQ ID NO: 1 in the sequence listing or a similar sequence thereof.
  • the peptide according to claim 1 comprising the amino acid sequence from Met in position 1 to SEQ ID NO: 97 in SEQ ID NO: 1 in the sequence listing, or a similar sequence thereof.
  • DNA molecule according to claim 5 having a nucleotide sequence consisting of G at position 168 to A at position 389 of SEQ ID NO: 1 in the sequence listing.
  • DNA molecule according to claim 5 having a base sequence consisting of A at position 99 to A at position 389 of SEQ ID NO: 1 in the sequence listing.
  • DNA molecule according to claim 5 which has a nucleotide sequence consisting of G at position 1 to S at position 859 of SEQ ID NO: 1 in the sequence listing.
  • An expression vector comprising the DNA molecule of any of claims 5-8.
  • Topology Linear
  • Sequence type nucleic acid

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Abstract

Un peptide qui est l'homologue humain de l'éotaxine du cobaye, ou une nouvelle chemokine, en particulier une chémokine CC humaine présentant une activité sur les éosinocytes humains; le gène structurel du peptide; un vecteur d'expression possédant le gène; un transformant dans lequel a été introduit le vecteur d'expression; un procédé de production du peptide par utilisation du transformant; un anticorps monoclonal dirigé contre le peptide; un procédé de dosage du peptide par utilisation de l'anticorps monoclonal; et un procédé de recherche d'un agoniste ou d'un antagoniste du peptide.
PCT/JP1996/002851 1995-10-05 1996-10-01 Nouvelle chemokine cc humaine WO1997012914A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998050434A1 (fr) * 1997-05-07 1998-11-12 Schering Corporation Methodes relatives a un appauvrissement des eosinophiles et compositions afferentes
US6204024B1 (en) 1997-09-12 2001-03-20 Akzo Nobel N.V. CCR5 RNA transcription based amplification assay
WO2001066754A1 (fr) * 2000-03-03 2001-09-13 Cambridge Antibody Technology Limited Anticorps humains contre l'eotaxine et utilisation associee
US6946546B2 (en) 2000-03-06 2005-09-20 Cambridge Antibody Technology Limited Human antibodies against eotaxin
US7005509B1 (en) 1995-02-17 2006-02-28 Incyte Corporation Chemokine PANEC-1 polynucleotides and compositions and methods related thereto
US7265201B1 (en) 1995-06-23 2007-09-04 Millennium Pharmaceuticals, Inc. Human chemotactic cytokine

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. BIOPHYS. RES. COMMUN., Vol. 205, 1994, P.J. JOSE et al., "Eotaxin: Cloning of an Eosinophil Chemoattractant ...", p. 788-794. *
BIOCHEM. BIOPHYS. RES. COMMUN., Vol. 225, Aug. 1996, J. BARTELS et al., "Human Dermal Fibroblasts Express Eotaxin ...", p. 1045-1051. *
J. BIOL. CHEM., Vol. 271, No. 13, March 1996, M. KITAURA et al., "Molecular Cloning of Human Eotaxin, ...CC Chemokine Receptor 3", p. 7725-7730. *
J. CLIN. INVEST., Vol. 97, No. 3, Feb. 1996, P.D. PONATH et al., "Cloning of the Human Eosinophil Chemoattractant, Eotaxin", p. 604-612. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7005509B1 (en) 1995-02-17 2006-02-28 Incyte Corporation Chemokine PANEC-1 polynucleotides and compositions and methods related thereto
US7858326B2 (en) 1995-06-23 2010-12-28 Millennium Pharmaceuticals, Inc. Method of diagnosis of inflammatory disease using eotaxin antibodies
US7265201B1 (en) 1995-06-23 2007-09-04 Millennium Pharmaceuticals, Inc. Human chemotactic cytokine
US7285620B2 (en) 1995-06-23 2007-10-23 Millennium Pharmaceuticals, Inc. Human chemotactic cytokine
US7423129B2 (en) 1995-06-23 2008-09-09 Millennium Pharmaceuticals, Inc. Antibodies to human eotaxin
WO1998050434A1 (fr) * 1997-05-07 1998-11-12 Schering Corporation Methodes relatives a un appauvrissement des eosinophiles et compositions afferentes
US6204024B1 (en) 1997-09-12 2001-03-20 Akzo Nobel N.V. CCR5 RNA transcription based amplification assay
WO2001066754A1 (fr) * 2000-03-03 2001-09-13 Cambridge Antibody Technology Limited Anticorps humains contre l'eotaxine et utilisation associee
US7323311B2 (en) 2000-03-03 2008-01-29 Cambridge Antibody Technology Limited Methods of obtaining a specific binding member that binds eotaxin
US8067564B2 (en) 2000-03-03 2011-11-29 Medimmune Limited Methods of obtaining a specific binding member that binds eotaxin
US8715961B2 (en) 2000-03-03 2014-05-06 Medimmune Limited Methods of obtaining a specific binding member that binds eotaxin
US9284589B2 (en) 2000-03-03 2016-03-15 Medimmune Limited Methods of obtaining a specific binding member that binds eotaxin
US10577413B2 (en) 2000-03-03 2020-03-03 Medimmune Limited Human antibodies against eotaxin and their use
US6946546B2 (en) 2000-03-06 2005-09-20 Cambridge Antibody Technology Limited Human antibodies against eotaxin

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