WO2000009687A1 - Nouveau gene et proteine gmp30 codee par ce gene - Google Patents

Nouveau gene et proteine gmp30 codee par ce gene Download PDF

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Publication number
WO2000009687A1
WO2000009687A1 PCT/JP1999/004351 JP9904351W WO0009687A1 WO 2000009687 A1 WO2000009687 A1 WO 2000009687A1 JP 9904351 W JP9904351 W JP 9904351W WO 0009687 A1 WO0009687 A1 WO 0009687A1
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Prior art keywords
gmp30
sequence
protein
gene
dna
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PCT/JP1999/004351
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English (en)
Japanese (ja)
Inventor
Kiyoshi Takayamna
Makoto Yoshimoto
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Taisho Pharmaceutical Co., Ltd.
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Publication date
Application filed by Taisho Pharmaceutical Co., Ltd. filed Critical Taisho Pharmaceutical Co., Ltd.
Priority to AU51965/99A priority Critical patent/AU5196599A/en
Publication of WO2000009687A1 publication Critical patent/WO2000009687A1/fr

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to a novel protein GMP30 derived from monocytes having an inflammation regulating activity, and a gene gmp30 encoding the protein.
  • Monocytes are mononuclear phagocytes derived from hematopoietic stem cells.Infections such as the incorporation of foreign microorganisms and the like into monocyte cells and melting by the action of lysosomes, which are numerous in monocytes. Plays an important role in the defense of the body.
  • monocytes have the property of migrating from the blood through cells of the vascular basement membrane toward cells that are undergoing an inflammatory reaction due to external stimuli, etc., and in the process, monocytes eventually become macrophages. It is known to differentiate. It is also known that monocytes and macrophages are activated by interferon expressed from cells undergoing an inflammatory response. In particular, since the inflammatory response caused by a foreign stimulus is controlled by the action of immune cells such as T cells and B cells, monocytes or macrophages are immune system cells (T cells, B cells, etc.) at the site of inflammation. It is supposed to have the function of controlling inflammation through some interaction with
  • An object of the present invention is to identify such a molecule and use it for the development of a medicine or the like. Disclosure of the invention
  • the present inventors aimed at identifying factors involved in the control of inflammation, and as a result of intensive studies to determine a desired protein from genes highly expressed in human monocytes,
  • the present invention has succeeded in isolating the presence of GMP30 and the gene encoding it, gmp30, and completed the present invention.
  • the present invention relates to (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1, or (b) an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1.
  • the present invention relates to a protein comprising a sequence and having an inflammation regulating function.
  • the present invention relates to (c) a gene consisting of the DNA of SEQ ID NO: 2, or (d) a protein that hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and has an inflammatory regulatory function. It concerns the gene consisting of the encoding DNA.
  • GMP 30 of the present invention is a protein having a molecular weight of 30.3 kilodaltons (k d) consisting of a total of 266 amino acid residues as shown in SEQ ID NO: 1.
  • gmp 30 of the present invention is a gene consisting of 801 base pairs (bp) as shown in SEQ ID NO: 2.
  • the gene gmp30 can be isolated from a human monocyte-derived cDNA library as a cDNA fragment containing the gene.
  • the cDNA library used by the present inventors was prepared based on mRNA extracted from healthy male peripheral blood monocytes according to a general method. Similarly, cDNA can be prepared based on mRNA of peripheral blood monocytes.
  • Human monocyte-derived mRNA was converted to type II, and cDNA synthesis was performed using a primer obtained by binding oligo dT to one end of a vector plasmid opened with an appropriate restriction enzyme as a primer, followed by the restriction enzyme MboI. Cleavage with restriction enzyme BamHI. Since this vector was prepared using dam methylase-positive E. coli as a host, the A residue of “GATC”, which is a recognition sequence of MboI, is methylated. Thus, MboI cuts only the newly synthesized cDNA portion.
  • this vector has only one BamHI cleavage site near the end opposite to the end to which the oligo dT was ligated, the enzyme cuts the vector at one position, and C DN synthesized to If the BamHI recognition sequence is present in part A, the site is also cleaved.
  • BamHI and Mbol are composed of the sequence “GATC” and generate the same cohesive end. Plasmids can be closed by cleaving with both enzymes and then reacting with DNA ligase. Using the plasmid thus prepared, Escherichia coli was transformed to construct a 3′-end cDNA library.
  • the library 1 includes a region from the poly A site at the 3 ′ end of each mRNA to the site where the base sequence of GATC first appears in the 5 ′ side thereof.
  • An appropriate number of recombinants are randomly selected from the 3′-end cDNA library, and the entire nucleotide sequence of the cDNA fragment in each recombinant is determined.
  • An organ-specific gene and a highly expressed gene can be identified based on how many cDNA fragments having the specific sequence determined in this manner are confirmed from randomly selected recombinants.
  • the total number of recombinants selected at random is suitably several hundreds to about 1,000, but if necessary, more recombinants may be processed.
  • the present inventors carried out the above method, determined all the nucleotide sequences of the cDNA fragments in the 117 recombinants, and found that the frequency of appearance as cDNA having the same sequence was 5Z11
  • the 74 cDNA fragment was selected as a candidate DNA fragment of a gene highly expressed in human monocytes.
  • the cDNA fragment contains only a part of the 3 'end of the mRNA. Therefore, the present inventors obtained a full-length cDNA based on the nucleotide sequence information of the region (hereinafter, 3 ′ fragment).
  • a human peripheral blood monocyte cDNA library commercially available from Clontech Co., Ltd. was designated as type ⁇ , and an oligonucleotide of an appropriate length having a sequence in the above 3 ′ fragment and a similar sequence having a sequence in a vector were used. Oligonucleotides of each length were synthesized, and PCR was performed using these as primers. As a result, a DNA fragment of about 1. 1 kb could be amplified. At this time, it can also be carried out by using mRNA extracted from human monocytes as type I and using a 5 'RACE kit from Clonetech or Gibco. Furthermore, it also uses the above 3 ′ fragment as a probe to clone the above human peripheral blood monocyte cDNA library by colony hybridization or Screening can also be performed by plaque hybridization.
  • the cDNA fragment amplified by the above method is incorporated into a vector pTBlue Vector (Novagen), and transfected into E. coli to cut out the fragment containing the cDNA from the clone as a circular plasmid to obtain all bases.
  • the sequence was determined, to obtain 2 clones of recombinant DNA independently, by determining the nucleotide sequence of each of c DNA fragment was confirmed sequence.
  • One protein translation region (Open Reading Flame, ORF) was found in this sequence, and this gene was named gmp30, and the protein encoded by the gene was named GMP30.
  • the gene gmp30 can be made into a recombinant gene by a general gene recombination technique using an appropriate host vector system.
  • Suitable vectors include E. coli-derived plasmids (eg, pBR322, pUC118, etc.), and Bacillus subtilis-derived plasmids (eg, pUB110, pC194, etc.) ), Yeast-derived plasmids (eg, PSH19 and others), and animal viruses such as Pacteriophage II retrovirus and vaccinia virus can be used.
  • a translation initiation codon and a translation termination codon can be added using an appropriate synthetic DNA adapter.
  • an appropriate expression promoter is connected upstream of the gene.
  • the promoter to be used may be appropriately selected depending on the host. For example, when the host is Escherichia coli, T7 promoter, 1 ac promoter, trp promoter, ⁇ PL promoter, etc., and when the host is Bacillus, S S0 promoter, etc. If the host is yeast, use 5 promoters, GAP promoter, ADH promoter, etc.If the host is animal cells, use SV40-derived promoter, retrovirus promoter, etc. it can.
  • the gene can also be expressed as a fusion protein with another protein (eg, glutathione S transferase, protein A, etc.).
  • the fused GMP30 expressed in this manner can be excised using an appropriate protease (eg, thrombin and others).
  • Hosts that can be used for expression of the gene gmp30 include Escherichia bacteria. Various strains of certain Escherichia coli, various strains of Bacillus subtilis belonging to the genus Bacillus, various strains of Saccharomvces cerevisiae as yeast, and COS-7 cells and CHO cells as animal cells can be used. As a method for transforming host cells using the above-described recombinant vector, a transformation method generally used for each host cell can be applied.
  • a DNA that hybridizes with the sequence and encodes a protein having an inflammation regulating function is also within the scope of the present invention.
  • the degree of the above-mentioned DNA mutation is within an allowable range as long as it has 90% or more homology with the DNA sequence of the gene gmp30.
  • the degree of hybridization with the gene gmp30 can be determined by using a DIG DNA labeling kit under normal conditions (for example, DIG DNA Labeling kit, Boehringer's Mannheim Cat No. 1175033). Conditions for hybridizing in Easy Hyb solution (Boehringer Mannheim Cat No. 1603558) and washing the membrane in 50 x 0.5 XSSC solution (containing 0.1% [w / v] SDS) ( (1 XSSC is 0.15M NaC 0.015M sodium citrate), and only needs to hybridize to the gene gmp30.
  • a protein encoded by a mutant gene having high homology to the gene gmp30 as described above, which has an inflammation regulating function, is also included in the scope of the present invention.
  • the mutant has an inflammatory regulatory function. If the protein has the following, the mutant is within the scope of the present invention.
  • the side chains of the amino acids that are the constituents of proteins differ in hydrophobicity, charge, size, etc., but do not substantially affect the three-dimensional structure (also called three-dimensional structure) of the entire protein In this sense, some highly conservative relationships are known empirically and by physicochemical measurements.
  • the mutant protein is a mutation due to substitution, insertion, deletion, etc. in the amino acid sequence of the novel protein GMP30 shown in SEQ ID NO: 1, the mutation is a mutation that is highly conserved in the three-dimensional structure of the GMP30 protein. If the mutant protein has a function of regulating inflammation like GMP30, these can be said to be within the scope of the present invention.
  • the degree of mutation is within a range in which homology to the amino acid sequence shown in SEQ ID NO: 1 is 90% or more.
  • GMP30 Since GMP30 has an inflammation regulating function, it is presumed that abnormal expression of the gene gmp30 or abnormal expression of the activity of GMP30 affects the inflammation enhancement and sedation. Therefore, a substance that regulates the expression of the gene or a substance that regulates the function of GMP 30 can be expected as an anti-inflammatory agent, and the gene gmp 30 and the protein GMP 30 are used to search for such a physiologically active substance. Can be used for For example, the effect of a test substance on gene expression can be investigated by coexisting a test substance in the transcriptional expression system of the gene gmp 30 and detecting the expression level of the gene gmp 30 by an appropriate method such as PCR. it can.
  • a 3′-terminal cDNA library was prepared according to the method of Okubo et al. (Okubo et al. 1. Nature Genet., 1992> 2> pl73). From the library, 1174 recombinants were randomly selected, and the nucleotide sequence of the cDNA portion was determined. For sequencing, DNA sequencing (PRISM377, manufactured by ABI) and a reaction kit manufactured by ABI were used.
  • the expression frequency of the gene having the following sequence was 5/117.
  • the cDNA fragment containing sequence-1 was cloned by the following method. First, an oligonucleotide (Sequence No. 2) which is a reverse complementary strand to a part of Sequence No. 11 and an oligonucleotide (Sequence No. 13) having a sequence near the cDNA insertion site of lambda phage cloning vector (A gtll) were prepared. , DNA synthesizer (ABI 380B) was synthesized.
  • Human Monocyte cDNA 1 ibrary manufactured by Clontech Laboratories
  • ⁇ gt11 as a cloning vector
  • oligonucleotides of sequence 12 and 13 were used as primers.
  • the following PCR operation was performed using PCR Kit Ver.2 and PCR Thermal Cycler MP (both from Takara Shuzo).
  • PCR cycle After holding at 94 ° C for 2 minutes, incubate at 98 ° C for 20 seconds, cool to 68 ° C at a rate of 11 ° C / 2 seconds, and heat at 68 ° C for 3 minutes. The holding was further performed 30 times at 72 ° C. for 10 minutes.
  • the DNA fragment amplified in 2) was fractionated by agarose gel electrophoresis (gel concentration 1%). The gel was stained with ethidium bromide and irradiated with ultraviolet light to cut out the gel containing the target band. Extraction and purification of DNA fragments from agarose gels were performed using GENECLEAN II Kit (manufactured by Hi-101).
  • This extracted and purified DNA fragment was used as a base sequence determination vector pT7blue (Novagen). Subcloning). The ligation solution was reacted for 1.5 hours at 16 ° C with the following composition using Evening DNA Ligation Kit Ver.2 (Takara Shuzo).
  • Escherichia coli K12 strain DH5 was transformed. Transformants were treated with ampicillin (Amp) 50 g / ml, 5-Bromo-4-Chloro-3-indolyl- ⁇ -D-galactoside 40 g / m1, 1 sopropy 1-5-D-Thio- G lactopyranoside 100; plated on LB agar medium containing uM, and cultured at 37 ° C. at ⁇ ° C.
  • the nucleotide sequence was determined using DNA Sequencer 1 (PRISM377, manufactured by ABI) and using the Daiyuichi Minator method. Oligonucleotides were synthesized based on the determined base sequences, and the entire base sequences of both strands were determined by the primer-walking method. SEQ ID NO: 3 shows the entire nucleotide sequence of the same DNA of the clone. Since the base sequence contained the upstream region of sequence 12 of sequence 12 and sequence 11, it was confirmed that the desired gene gmp30 was cloned.
  • the cDNA contains an ORF encoding a protein consisting of 266 residues (GMP30) (SEQ ID NO: 3). Since a stop codon appeared in the same reading frame in the upstream region of the methionine residue which is the start codon of the protein, the amino acid sequence of the protein encoded by the cDNA fragment is the only one shown in SEQ ID NO: 3. It was confirmed that there was.
  • Example 2 Expression of the gene gmp 30 in animal cells
  • oligonucleotide having a sequence upstream of the initiation codon of the protein of SEQ ID NO: 3 (SEQ ID NO: 4) and an oligonucleotide (Sequence-5) having a sequence of a part of the reverse complementary strand downstream of the stop codon were synthesized using a DNA synthesizer (380B manufactured by ABI).
  • the PCR cycle consists of holding at 94 ° C for 2 minutes, reacting at 98 ° C for 20 seconds, cooling to 68 ° C — cooling at 1 ° C for 2 seconds, and 3 minutes at 68 ° C. The holding was further performed 30 times at 72 ° C. for 10 minutes.
  • a DNA fragment (about 1.5 kb) was specifically amplified by the above method.
  • the amplified DNA fragment (1.5 kb) is fractionated by agarose gel electrophoresis at a gel concentration of 1%, and the gel is stained with ethidium bromide and irradiated with ultraviolet light to contain the target band. The gel was cut out. Extraction and purification of DNA fragments from agarose gel were performed using GENECLEAN II Kit (Bio101).
  • the extracted and purified DNA fragment was subcloned into an animal cell expression vector pTARGET (promega).
  • the ligation solution was reacted for 1.5 hours at 16 ° C with the following composition using Evening DNA Ligation Kit Ver.2 (Takara Shuzo).
  • Escherichia coli K12 strain DH5 was transformed. Transformants were treated with ampicillin (Amp) 50 g / m 15-Bromo-4-Chloro-3-indolyl- ⁇ -D-galactoside 40 gZm1, Isopropyl-3-D-Thio-Galactopyranoside 100 / iM was plated on an LB agar medium containing and cultured overnight at 37 ° C. The colonies appearing on the above plate were inoculated into 10 ml of LB liquid medium containing 50 g / m1 Amp, cultured at 37 ° C overnight, and the cells were collected by centrifugation.
  • the recombinant DNA was purified using Plasmid Miniprep Kit (manufactured by Qiagen) to obtain pTARGET-gmp30.
  • This recombinant plasmid has a CMV promoter upstream of the inserted fragment, and gmp30 can be expressed by introducing the recombinant plasmid into an appropriate animal cell.
  • a collagen petri dish with a diameter of 60 mm was added to a collagen Petri dish, and HamF- containing 10% fetal bovine serum (Dainippon Pharmaceutical), 50 units of Zml ⁇ nicillin, and 50 gZm of 1 streptomycin. (Gibco, hereinafter referred to as the growth medium) 12 medium was used to culture at 3 7 ° C, 5% C_ ⁇ 2 presence.
  • a LIPOFECTAMINE reagent manufactured by Gibco
  • pTARGET-gmp30 was overlaid on the cells, cultured for 6 hours, then replaced with a growth medium, and cultured for 48 hours. After dispersing the cells with trypsin, the cell suspension was dispensed into a 60 mm-diameter plastic petri dish and cultured for another 24 hours.
  • the medium was replaced with a growth medium containing G418 reagent (manufactured by Gibco, 500 gZmL final concentration), and the growth medium was replaced every three days and cultured for 2 weeks.
  • G418 reagent manufactured by Gibco, 500 gZmL final concentration
  • the colonies of the cells became visible to the naked eye, three colonies were isolated using a stainless steel cup.
  • pTARGET vector Promega alone was introduced into CHO k1 cells in the same manner as above, and a stable transformant was isolated. Released.
  • the reaction product was fractionated by 1% agarose gel electrophoresis, stained with ethidium die, and irradiated with ultraviolet light to check whether the target band was amplified.
  • the band of interest was amplified only in CHO k1 cells into which pTARGET-gmp30 had been introduced, and no amplification could be confirmed in CHO k1 cells into which the control vector had been introduced.
  • monocytes isolated from healthy males were used in a 10% non-mobilized ⁇ fetal serum (Dainippon Pharmaceutical Co., Ltd.) in RM 1640 medium containing 50 units ZmL penicillin and 50 g / mL streptomycin. To achieve a final concentration of 1000 units The cells were further cultured for 24 hours after the addition of the fermenter (IFN- ⁇ ). Monocytes were prepared in a medium without IFN-I as a control.
  • the PCR cycle consists of holding at 94 ° C for 2 minutes, reacting at 98 ° C for 20 seconds, cooling to 68 ° C at a rate of 1 ° C / 2 seconds, holding at 68X for 3 minutes, The holding at 10 ° C. for 10 minutes was repeated 30 times.
  • the reaction product was fractionated by 1% agarose gel electrophoresis, stained with ethidium amide, and irradiated with ultraviolet light to check whether the target band was amplified.

Abstract

L'invention se rapporte à une nouvelle protéine GMP30 dérivée d'un monocyte humain et dotée d'une activité régulatrice vis à vis des inflammations ainsi qu'à un gène gmp30 codant pour ladite protéine. On fabrique ce gène gmp30 codant pour la nouvelle protéine GMP30 dotée d'une activité régulatrice vis à vis des inflammations en clonant une banque d'ADNc provenant d'un monocyte humain. En raison de l'activité régulatrice dont elle est dotée vis à vis des inflammations, cette protéine peut servir à la fabrication d'anti-inflammatoires.
PCT/JP1999/004351 1998-08-12 1999-08-11 Nouveau gene et proteine gmp30 codee par ce gene WO2000009687A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU51965/99A AU5196599A (en) 1998-08-12 1999-08-11 Novel gene and protein gmp30 encoded thereby

Applications Claiming Priority (2)

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JP10/227734 1998-08-12
JP10227734A JP2000050879A (ja) 1998-08-12 1998-08-12 新規遺伝子とそれにコードされる蛋白質

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US09/776,931 Division US6509918B1 (en) 1996-12-05 2000-12-19 Electrostatic recording apparatus and image density control method thereof

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001065259A1 (fr) * 2000-03-02 2001-09-07 Genox Research, Inc. Methode d'examen de maladies allergiques

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020130A2 (fr) * 1996-11-01 1998-05-14 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
WO1998025959A2 (fr) * 1996-12-11 1998-06-18 Chiron Corporation Proteines humaines secretees
WO1998033913A1 (fr) * 1997-01-31 1998-08-06 Incyte Pharmaceuticals, Inc. Proteine intrinseque humaine
WO1998042738A1 (fr) * 1997-03-21 1998-10-01 Human Genome Sciences, Inc. 87 proteines humaines secretees

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020130A2 (fr) * 1996-11-01 1998-05-14 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
WO1998025959A2 (fr) * 1996-12-11 1998-06-18 Chiron Corporation Proteines humaines secretees
WO1998033913A1 (fr) * 1997-01-31 1998-08-06 Incyte Pharmaceuticals, Inc. Proteine intrinseque humaine
WO1998042738A1 (fr) * 1997-03-21 1998-10-01 Human Genome Sciences, Inc. 87 proteines humaines secretees

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PITTOIS K. ET AL.: "cDNA sequence analysis, chromosomal assignment and expression pattern of the gene coding for integral membrane protein 2B", GENE,, vol. 217, no. 1-2, 14 September 1998 (1998-09-14), pages 141 - 149, XP002925252 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001065259A1 (fr) * 2000-03-02 2001-09-07 Genox Research, Inc. Methode d'examen de maladies allergiques

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AU5196599A (en) 2000-03-06

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