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WO2002066049A1 - Agents pour changement plasmique - Google Patents

Agents pour changement plasmique Download PDF

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Publication number
WO2002066049A1
WO2002066049A1 PCT/JP2002/001536 JP0201536W WO02066049A1 WO 2002066049 A1 WO2002066049 A1 WO 2002066049A1 JP 0201536 W JP0201536 W JP 0201536W WO 02066049 A1 WO02066049 A1 WO 02066049A1
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WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
protein
dna
Prior art date
Application number
PCT/JP2002/001536
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English (en)
Japanese (ja)
Inventor
Yukiko Hikichi
Yasushi Shintani
Hideki Matsui
Original Assignee
Takeda Chemical Industries, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Chemical Industries, Ltd. filed Critical Takeda Chemical Industries, Ltd.
Publication of WO2002066049A1 publication Critical patent/WO2002066049A1/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
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a novel cell transformation agent and the like.
  • RMS Rhabdomyosarcoma
  • RMS is one of the most common malignancies in childhood and is a sarcoma arising from immature mesenchymal cells. Since RMS is similar in shape to normal fetal skeletal muscle and expresses muscle-specific genes (J. Clin. Oncol., 13, 21 23-2139 (1995), Semin. Diagn. Pathol, 11 , 39-46 (1994)), and are thought to have arisen from immature cells that should become muscle tissue. RMSs are also classified into several subtypes due to their morphological differences. Although the survival rate of patients varies considerably due to morphological differences in sarcomas, the survival rate of children with RMS is generally 50-70. It is.
  • RMS cell the fetal rhabdomyosarcoma cell line RD.
  • RD cells LOHdoss of heterozygosity on chromosome 11 ⁇ 15 (Cancer Research, 57, 4493-4497 (1997)) and functional mutation due to base substitution of p53 (Biochem. Biophys. Res. , 17-24 (1994)) or a deletion mutation of ⁇ 16 (British J. Cancer, 79, 1032-1036 (1999)).
  • it is difficult to make the final differentiation of RD cells into skeletal muscle cells which is presumed to play an advantageous role in obtaining the proliferation necessary for tumor formation. .
  • GR-891 is a 5- iluorouracil acy c 1 onuc 1 eoside with a novel structure, which causes morphological changes without exerting cytotoxic activity on RD cells, and as a differentiation marker.
  • IP6 inosiol hexaphosphate
  • TL4 (W09 8/03648), a ligand molecule belonging to the TNF family, unexpectedly delays the growth of rhabdomyosarcoma cell line RD and increases the cytoplasmic enlargement. The fact that the accompanying remarkable morphological ability was retained was determined. Further research has led to the completion of the present invention. That is, the present invention
  • a cell transforming agent comprising a partial peptide of the protein according to (1) or a salt thereof,
  • a cell transforming agent comprising a DNA encoding the DNA according to the above (1) or the partial peptide according to the above (3);
  • the agent according to the above (5) wherein the DNA is a DNA having a base sequence represented by any one of SEQ ID NOs: 4 to 10 or SEQ ID NO: 30;
  • SEQ ID NO: 1, SEQ ID NO: 2 characterized in that an effective amount of a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 31 or a partial peptide thereof or a salt thereof is administered.
  • a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 31 with respect to mammals
  • a method of preventing (and / or) treating rhabdomyosarcoma, leiomyosarcoma, muscular dystrophy or uterine fibroids which comprises administering an effective amount of the partial peptide or a salt thereof.
  • a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 31 with respect to mammals
  • a cell transformation method comprising administering an effective amount of a DNA containing a DNA encoding a partial peptide thereof,
  • a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 31 with respect to mammals
  • a method for preventing (and / or) treating rhabdomyosarcoma, leiomyosarcoma, muscular dystrophy or uterine fibroids which comprises administering an effective amount of DNA containing DNA encoding a partial peptide thereof.
  • FIG. 1 shows the results of the analysis of the expression of the TNF receptor Yuichi family in RD cells performed in Example 1.
  • FIG. 2 shows the results of the effect of the TNF family ligand molecule (BrdU method) on the cell proliferation of RD cells performed in Example 2.
  • “-” indicates TL4
  • “Ichizono” indicates TNF «,“ ⁇ ”indicates TNF 3,“ Hin-ichi ”indicates LTo; 1/32, and _ * _ indicates TNF / 3 + L Ta1 ⁇ 2.
  • FIG. 3 shows the effect (number of living cells) of TNF family ligand molecule (50 ng / ml) on RD cell proliferation performed in Example 2.
  • the opening indicates the number of cells at the start of the culture, and the open square indicates the number of cells on the sixth day of the culture.
  • FIG. 4 is a diagram showing the results of cell cycle analysis of TL4-treated RD cells performed in Example 3.
  • (A) shows the analysis results in the control group
  • (B) shows the analysis results in the TL4 added group.
  • Individual peaks represent GO ZG phase 1, S phase, and G2ZM phase.
  • FIG. 5 shows the results of NF- ⁇ B activation by TNF family ligands of RD cells performed in Example 4.
  • - ⁇ is control
  • - ⁇ TL4
  • X is TNF
  • LT is 132 Show.
  • FIG. 6 shows the effect of a TNF family monoligand molecule on chemokine production of RD cells performed in Example 5.
  • FIG. 5 shows the results of cell immunostaining of RD cells using MY-32, an antibody recognizing skeletal muscle myosin heavy chain, upon stimulation of the TNF family monoligand performed in Example 6.
  • FIG. 8 shows the results of Western blot analysis using a skeletal muscle myosin heavy chain-recognizing antibody, MY-32, of a crude extract of RD cells at the time of TNF family ligand stimulation performed in Example 7.
  • M is the molecular weight marker
  • Lane 1 is the control
  • Lane 2 is TL 4
  • Lane 3 is TNF
  • Lane 4 is TNF 3
  • Lane 5 is TNFi3 + LTo! 1] 32
  • Lane 6 is LT.
  • ct 1/32 lane 7 shows TGF 31/33
  • lane 9 shows TPA
  • lane: ⁇ 0 shows TGF / 3 1/33 + TPA.
  • FIG. 9 shows the results of cell immunostaining of RD cells using the antibody recognizing smooth and non-muscle myosin heavy chain, F126.16D9, upon stimulation of the TNF family monoligand performed in Example 6.
  • FIG. 10 shows the results of the analysis of muscle-specific transcript expression in RD cells and the cell morphology performed in Example 7.
  • lane 1 is control
  • lane 2 is TL4
  • lane 3 is TNF o
  • lane 4 is TNF / 3
  • lane 5 is TNF3 + L ⁇ 132
  • lane 6 is LT ⁇ 1
  • 32 Lane 7 shows TGF / 31 + TGF / 33
  • lane 8 shows TPA
  • lane 9 shows TGF31 + TGF33 + TPA
  • lane 10 shows TL4 (cultured for 8 days).
  • the protein contained in the cell transforming agent of the present invention (hereinafter, sometimes referred to as the protein of the present invention) is as follows: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or Contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 31.
  • the protein of the present invention can be used, for example, in any cells of human or non-human warm-blooded animals (eg, guinea pigs, rats, mice, chickens, egrets, bushes, sheep, higgies, horses, pests, monkeys, etc.) (eg, , Spleen cells, nerve cells, glial cells, knees) 8 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells ( Eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, Osteoclasts, mammary cells, hepatocytes or stromal cells, or their precursors, stem cells or cancer cells), or any
  • Examples of the protein of the present invention include the proteins described in WO98Z0348 and WO97 / 34911.
  • examples of the protein of the present invention include ligands for the receptor protein described in J. Clin. Invest., 102, 1142-1511 (1998) and US Pat. No. 5,874,240. Active proteins (polypeptides) and the like are also included.
  • amino acid sequence substantially identical to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 examples include, for example, the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 An amino acid sequence having homology of about 40% or more, preferably 60% or more, more preferably about 80% or more, still more preferably about 90% or more, and most preferably about 95% or more. And the like.
  • the 84th to 240th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 the 82nd to 240th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2
  • it has a homology of about 90% or more.
  • amino acid sequence substantially identical to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is a constituent amino acid that is the 8th to 21st amino acid sequence represented by SEQ ID NO: 1. , No. 55-59, No. 93-102, No. 109-; L16 No., No. 118-126, No. 128-134, No. 144-149, No. 162-170 , 176th to 182nd, 184th to 189th, 193rd to 213th, 215th to 219th and amino acid sequences having the 228th to 228th amino acid sequences, etc. Is also preferred.
  • amino acid sequences are common to the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3.
  • the amino acid sequence substantially the same as SEQ ID NO: 1 or SEQ ID NO: 2 includes, as constituent amino acids, the S-21st, the 54th to the 59th, and the 54th 93 ⁇ ; L 02th, 109th to 116th, 118th to 126th, 128th to 134th, 144th to 149th, 162th to 170th, 176th to 182th
  • amino acid sequences having the amino acid sequence at positions 184-189, 193-213, 215-219 and 228-240.
  • amino acid sequences are the 6th to 20th, the 52nd to 57th, the 91st to 100th, the 107th to 114th, and the 116th to 124th positions of the amino acid sequence represented by SEQ ID NO: 2. , 126th-132nd, 142nd-147th, 162nd-170th, 176th-182nd, 184th-189th, 192st-2nd 2nd, 214th-21st It is an amino acid sequence corresponding to the 8th and 227th to 239th amino acid sequences and common to the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2.
  • amino acid sequence substantially identical to SEQ ID NO: 31 includes, as constituent amino acids, the 8th to 21st, 57th to 66th, and 73rd to 73rd amino acids of the amino acid sequence represented by SEQ ID NO: 31. 80th, 82nd to 90th, 92nd to 98th, 10th S ⁇ l 1 1st, 1st 26th to 13th 4th, 1st 40th to 16th, 1st 4th to 15th, 3rd, 15th to 17th, 17th Amino acid sequences having the 9th to 18th and 19th to 204th amino acid sequences, and the like.
  • the protein of the present invention containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 31 is as described above.
  • SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 31 having substantially the same amino acid sequence as the 7 amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 2,
  • a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 31 is preferred.
  • Examples of substantially the same activity include activities such as cell transformation.
  • the cell transformation action refers to an action that suppresses cell proliferation and causes a change in cell morphology.
  • cancer preferably, (fetal) rhabdomyosarcoma, etc.)
  • tumor preferably, (embryonic) rhabdomyosarcoma, etc.
  • tumor preferably, (embryonic) rhabdomyosarcoma, etc.
  • myocyte-like cells with cytoplasmic hypertrophy induces differentiation into multinucleated cells, etc.
  • It refers to the action of inducing a specific marker protein, for example, ⁇ -actin specific to smooth muscle or skeletal muscle, and altering the trait. Substantially the same means that their activities are qualitatively (eg, physiochemical or pharmacological).
  • the activities such as cell transformation activity are equivalent (eg, about 0.01 to 20 times, preferably about 0.2 to 5 times, more preferably about 0.5 to 2 times).
  • the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the activity such as cell transformation activity is determined by a known method or a method analogous thereto (for example, Cancer Research, 50, 3377-3382 (1990), British J. Cancer, 79, 807-813 (1999), Exp Cell Res., 204, 210-216 (1993)) and the method described in, for example, Examples described later.
  • the protein of the present invention includes (1) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 1 or 2 or more in the amino acid sequence represented by 3 or SEQ ID NO: 31 (for example, 1 to 80, preferably about 1 to 20, more preferably about 1 to 9, more preferably about 1 to 9, , 1 to 5) amino acids, (2) one or more amino acid sequences (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 31)
  • 1 to 80 preferably about 1 to 20, more preferably about 1 to 9, and still more preferably a number (eg, 1 to 5) amino acids have been added
  • the position of the deletion or substitution is not particularly limited.
  • Xaa Xaa Xaa lie Thr His Gly Leu Tyr Lys Arg Thr Xaa Arg Tyr Pro
  • a protein having the amino acid sequence [amino acid sequence represented by SEQ ID NO: 25] represented by the following formula is also preferably used.
  • one or two or more may be deleted at preferably 1 to 40, more preferably 1 to 20, still more preferably 1 to 9, and most preferably a number (1 to 5) positions.
  • the amino acid represented by Xaa may be any of a hydrophilic amino acid and a hydrophobic amino acid, and may be any of an acidic amino acid, a basic amino acid, and a neutral amino acid.
  • G 1 y, A 1 a, Va K Le u, I 1 e, Ser, Thr, Cys, Met, Glu, Asp, Lys, Arg, His, Ph e, Tyr, Trp, Pro, Asn, Gin and the like are used.
  • the third Xaa is preferably Glu or may be missing.
  • a hydrophilic amino acid is preferable, and specifically, Arg or G1n is preferable.
  • the 22nd Xaa is preferably a hydrophilic amino acid, and specifically, Thr or Arg is preferred.
  • a hydrophilic amino acid is preferable, and specifically, G1y or G1u is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Arg or G1n is preferable.
  • the 27th Xaa is preferably a hydrophilic amino acid, and specifically, Ser or Asn is preferred.
  • a hydrophilic amino acid is preferable, and specifically, G1n or Arg is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Ser or Arg is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Ser or G1y is preferable.
  • 35th Xaa for example, Va1 or Thr is preferable.
  • 36th Xaa for example, a hydrophobic amino acid is preferable, and specifically, Ala or Va1 is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Is preferably Arg or G1n.
  • a hydrophilic amino acid is preferable, and specifically, G1y or Ser is preferable.
  • the 41st Xaa for example, 01 or 81a is preferable.
  • the 43rd Xaa for example, a hydrophobic amino acid is preferable, and specifically, Leu or Va1 is preferable.
  • the 47th Xaa is preferably Met or may be deleted.
  • Va 1 or Thr is preferable.
  • 60th Xaa for example, a hydrophilic amino acid is preferable, and specifically, G1n or Arg is preferable.
  • Trp or G1n is preferable.
  • Trp or G1n is preferable.
  • 67th Xaa for example, an acidic amino acid is preferable, and specifically, G1u or Asp is preferable.
  • a hydrophobic amino acid is preferable, and specifically, Met or I1e is preferable.
  • Thr or A1a is preferable.
  • 71st Xaa for example, a basic amino acid is preferable, and specifically, Arg or His is preferable.
  • the 76th Xaa for example, Pro or G1y is preferable.
  • As the 77th Xaa for example, Ala or Lys is preferable.
  • the 82nd Xaa is, for example, preferably a hydrophilic amino acid, and specifically, Gin or Lys.
  • an acidic amino acid is preferable, and specifically, G1u or Asp is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Arg or G1n is preferable.
  • a hydrophilic amino acid is preferable, and specifically, G1u or G1n is preferable.
  • a hydrophobic amino acid is preferable, and specifically, Va 1 or A1a is preferable.
  • 103rd Xaa for example, Ser or A1a is preferable.
  • 106th Xaa for example, Thr or Ile is preferable.
  • the 108th Xaa is preferably, for example, Ser or Ile.
  • 117th Xaa for example, a hydrophilic amino acid is preferable, and specifically, G1n or Arg is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Ser or Thr is preferable.
  • the 135th Xaa for example, Va 1 or Thr is preferable.
  • the 136th Xaa for example, Thr or Met is preferable.
  • the 137th Xaa for example, a hydrophilic amino acid is preferable, and specifically, Lys or G1u is preferable.
  • a hydrophobic amino acid is preferable, and specifically, A1a or Pro is preferable.
  • the 143rd Xaa is, for example, preferably a hydrophobic amino acid, and specifically, I 1 e or Va 1 is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Gy or Ser is preferable.
  • 156th Xaa for example, Leu or G1n is preferable.
  • 160th Xaa for example, a hydrophilic amino acid is preferable, and specifically, Ser or Asn is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Thr or G1y is preferable.
  • the 162nd Xaa is preferably Leu or may be deleted.
  • the 163rd Xaa is preferably Pro or may be deleted.
  • the 173rd Xaa for example, Pro or Ser is suitable.
  • the 178th Xaa for example, a hydrophilic amino acid is preferable, and specifically, G1u or Lys is preferable.
  • hydrophilic amino acids are preferable, and specifically, G1n or Aig is preferable.
  • the 193rd Xaa is preferably Thr, or may be deleted.
  • a hydrophilic amino acid is preferable, and specifically, Ser or Asn is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Lys or G1u is preferable.
  • a hydrophobic amino acid is preferable, and specifically, Leu or Pro is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Asp or G1y is preferable.
  • a hydrophilic amino acid is preferable, and specifically, G1u or Asn is preferable.
  • a hydrophobic amino acid is preferable, and specifically, Leu or Pro is preferable.
  • a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 31 specifically, a protein represented by the general formula:
  • Xaa represents an arbitrary amino acid residue or a bond
  • a protein having an amino acid sequence [amino acid sequence represented by SEQ ID NO: 32] represented by the following formula is also preferably used.
  • one or two or more (for example, 1 to 40, preferably 1 to 20, more preferably 1 to 9, and most preferably a number (1 to 5)) Xaa may be deleted at the position.
  • the amino acid represented by Xaa may be any of a hydrophilic amino acid and a hydrophobic amino acid, and may be any of an acidic amino acid, a basic amino acid, and a neutral amino acid. Specifically, Gly, Ala, VaLeu, Ile, Ser, Thr, Cys, Met, Glu, Asp, Lys, Arg, His, Phe, Tyr , Tr, Pro, Asn, Gin and the like are used.
  • the third Xa a is preferably G lu or lacks. You may have lost.
  • a hydrophilic amino acid is preferable, and specifically, Ar g or G 1 ⁇ is preferable.
  • the 22nd Xaa is preferably a hydrophilic amino acid, and specifically, Thr or Arg is preferred.
  • a hydrophilic amino acid is preferable, and specifically, G1y or G1u is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Arg or G1n is preferable.
  • the 27th Xaa is preferably a hydrophilic amino acid, and specifically, Ser or Asn is preferred.
  • a hydrophilic amino acid is preferable, and specifically, G1n or Arg is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Ser or Arg is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Ser or G1y is preferable.
  • 35th Xaa for example, Va1 or Thr is preferable.
  • 36th Xaa for example, a hydrophobic amino acid is preferable, and specifically, Ala or Va1 is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Aig or G1n is preferable.
  • the 40th Xaa for example, Pro or G1y is preferable.
  • the 41st Xaa for example, Al a or Lys is preferable.
  • the 46th Xaa for example, a hydrophilic amino acid is preferable, and specifically, G1n or Lys is preferable.
  • an acidic amino acid is preferable, and specifically, G1u or Asp is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Is preferably Arg or G1n.
  • a hydrophilic amino acid is preferable, and specifically, G1u or G1n is preferable.
  • a hydrophobic amino acid is preferable, and specifically, Va1 or A1a is preferable.
  • 67th Xaa for example, Ser or A1a is preferable.
  • 70th Xaa for example, Thr or I1e is preferable.
  • 72nd Xaa for example, 361 'or 11e is preferable.
  • 81st Xaa for example, a hydrophilic amino acid is preferable, and specifically, G1n or Arg is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Ser or Thr is preferable.
  • a1 or Thr is preferable.
  • 100th Xaa for example, Thr or Met is preferable.
  • 101st Xaa for example, a hydrophilic amino acid is preferable, and specifically, Lys or G1u is preferable.
  • a hydrophobic amino acid is preferable, and specifically, A1a or Pro is preferable.
  • a hydrophobic amino acid is preferable, and specifically, I 1 e or Va 1 is preferable.
  • a hydrophilic amino acid is preferable, and specifically, G1y or Ser is preferable.
  • the 120th Xaa for example, Leu or G1n is preferable.
  • the 124th Xaa for example, a hydrophilic amino acid is preferable, and specifically, Ser or Asn is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Thr or G1y is preferable.
  • the 135th Xaa is preferably Pro or may be deleted.
  • a hydrophilic amino acid is preferable, and specifically, G1u or Lys is preferable.
  • a hydrophilic amino acid is preferable, and specifically, G1n or Arg is preferable.
  • the 155th Xaa is preferably Thr or may be deleted.
  • a hydrophilic amino acid is preferable, and specifically, Ser or Asn is preferable.
  • a hydrophilic amino acid is preferable, and specifically, Lys or G1u is preferable.
  • the 184th Xaa is, for example, preferably a hydrophobic amino acid, and specifically, Leu or Pro.
  • a hydrophilic amino acid is preferable, and specifically, Asp or G1y is preferable.
  • hydrophilic amino acid is preferable, and specifically, G1u or Asn is preferable.
  • the 191st Xaa is, for example, preferably a hydrophobic amino acid, and specifically, Leu or Pro.
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling.
  • the proteins of the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 1, usually have a carboxyl group (—COOH), carboxylate (one COO one), amide (—CONH 2 ) Or ester (—COOR).
  • the R of the ester group for example, methyl, Echiru, .eta. propyl, d_ 6 alkyl group, such as I an isopropyl or n- butyl, cyclopentyl ⁇ ', C 3 _ s cycloalkyl group such as cyclohexyl , for example, phenyl, - C 6 _ 12 Ariru groups such as naphthyl, for example, benzyl, Fei such phenylene relay C one 2 alkyl or ⁇ - naphthylmethyl such phenethyl - such as naphthyl -C ⁇ s alkyl group in addition to C 7 _ 14 Ararukiru groups, it is widely used as an ester for oral administration Bivaloyloxymethyl group and the like are used.
  • the protein of the present invention When the protein of the present invention has a carboxyl group (or carboxylate) at a position other than the C-terminus, the protein of the present invention includes a protein in which the carbonyl group is amidated or esterified.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • Amino group protecting groups ⁇ amino acid residues at the N-terminus e.g., formyl group, C DOO 6 Ashiru groups such Arukanoiru such Asechiru group
  • Dartamyl group formed by cleavage of the N-terminal side in vivo, oxidized with dalamine, substituents on the side chain of amino acid in the molecule for example, _OH, one SH, amino group , imidazole group, indole group, etc.
  • Guanijino group appropriate protecting groups (e.g., formyl group, ⁇ C Interview such as cetyl group - are protected by like 6 C i-6 Ashiru groups such as Al force Noiru) shall Or complex proteins such as so-called glycoproteins to which sugar chains are bound.
  • appropriate protecting groups e.g., formyl group, ⁇ C Interview such as cetyl group - are protected by like 6 C i-6 Ashiru groups such as Al force Noiru
  • complex proteins such as so-called glycoproteins to which sugar chains are bound.
  • examples of the protein of the present invention include a protein derived from human liver having the amino acid sequence represented by SEQ ID NO: 1, and a protein derived from a mouse embryo having the amino acid sequence represented by SEQ ID NO: 2 Preferred are a rat liver-derived protein having the amino acid sequence represented by SEQ ID NO: 3, a human liver-derived protein represented by SEQ ID NO: 31, and the like.
  • the partial peptide of the protein of the present invention may be any peptide as long as it has the same activity as that of the protein of the present invention described above, for example, an activity such as a cell transformation activity.
  • an activity such as a cell transformation activity.
  • the amino acid sequence of the protein of the present invention at least about 20 or more, preferably about 50 or more, more preferably about 70 or more, still more preferably about 100 or more, and most preferably about 200 or more.
  • Peptides having an amino acid residue are preferably used.
  • a partial peptide having at least one amino acid sequence selected from the amino acid sequences at positions 15 to 219 and 228 to 239 that is, the partial peptide having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3; 20th, 53th to 57th, 91st to 100th, 107th to 114th, 116th to 124th, 126th to 132nd, 142th to 147th, 162th to 170th, 176th to:
  • L a partial peptide having at least one amino acid sequence selected from the amino acid sequences at positions 82, 184 to 189, 192 to 212, 214 to 218 and 227 to 238), (2) No. 8-21, No. 54-59, No.
  • a partial peptide having the amino acid sequence at positions 84 to 240 of the amino acid sequence represented by SEQ ID NO: 1, and the amino acid sequence at positions 82 to 239 of the amino acid sequence represented by SEQ ID NO: 2 A partial peptide having the amino acid sequence of position 82. Amino acid sequence at positions 82 to 239 of the amino acid sequence represented by SEQ ID NO: 3.
  • a partial peptide having a sequence, a partial peptide having an amino acid sequence of the 4Sth to 204th amino acids of the amino acid sequence represented by SEQ ID NO: 31 and the like are preferably used.
  • the partial peptide of the present invention includes: (1) an amino acid sequence represented by SEQ ID NO: 1 which has an amino acid sequence that is substantially the same as the 84th to 240th amino acid sequence; A peptide having substantially the same activity as the peptide containing the 84th to 240th amino acid sequence of the amino acid sequence represented by 1; A peptide having an amino acid sequence that is substantially the same as the amino acid sequence of the 2nd to 239th amino acids, and containing the 8th to 239th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2; A peptide having substantially the same activity; 3 having an amino acid sequence substantially the same as the amino acid sequence of the 8th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 3, and SEQ ID NO: Amino acid represented by 2 A peptide having substantially the same activity as the peptide containing the 8th to 23rd amino acid sequence of the sequence, ⁇ the 48th to 2nd amino acid sequence represented by SEQ ID NO: 31 0 has
  • amino acid sequence substantially identical to the amino acid sequence at positions 84 to 240 of the amino acid sequence represented by SEQ ID NO: 1 include, for example, the amino acid sequence of SEQ ID NO: 1
  • the amino acid sequence of the 2nd to the 240th amino acid and about 40% or more, preferably 60% or more, more preferably about 80% or more, still more preferably about 90% or more, and most preferably about 95% or more is used.
  • amino acid sequences that is substantially the same as the amino acid sequence at positions 82 to 239 of the amino acid sequence represented by SEQ ID NO: 2 include, for example, the amino acid sequence of amino acid sequence represented by SEQ ID NO: 2
  • Amino acid sequences with the above homology are used Can be
  • amino acid sequence substantially the same as the amino acid sequence of the 82nd to 239th amino acids of the amino acid sequence represented by SEQ ID NO: 3 include, for example, the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 3
  • the amino acid sequence of the 2nd to 239th amino acids is about 40% or more, preferably 60% or more, more preferably about 80%. /. Above, more preferably about 90% or more, most preferably about 95% or more amino acid sequence having homology is used.
  • amino acid sequence substantially identical to the 48th to 204th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31 include, for example, the amino acid represented by SEQ ID NO: 31 At least about 40%, preferably at least 60%, more preferably at least about 80? S, even more preferably at least about 90?, Preferably, an amino acid sequence having a homology of about 95% or more is used.
  • the partial peptide of the present invention includes (1) one or more amino acids in the 84th to 240th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 (for example, 1 to 80, preferably Is an amino acid sequence in which about 1 to 20 amino acids have been deleted, more preferably about 1 to 9 amino acids, and still more preferably a number (eg, 1 to 5) amino acids, and the amino acid sequence represented by SEQ ID NO: 1
  • One or two or more amino acids in the 84th to 240th amino acid sequence e.g., 1 to 80, preferably about 1 to 20, more preferably about 1 to 9, more preferably about 1 to 9) (E.g., 1 to 5) amino acid sequence (s), one or two or more amino acids in the 84th to 240th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1
  • 1 to 80 pieces preferably about 1 to 20 pieces, more preferably about 1 to 9 pieces
  • 1 or 2 or more (for example, 1 to 80, preferably about 1 to 20, more preferably 1) to the amino acid sequence of the 82nd to 239th amino acids of the amino acid sequence represented by No. 2
  • One or more e.g., 1 to 80, preferably about 1 to 20, more preferably about 1 to 9, more preferably about 1 to 5 amino acids
  • a peptide having an amino acid sequence obtained by removing the 1st to 83rd amino acids from the amino acid sequence represented by the general formula (I), or the like is preferable. Used.
  • C-terminal are usually in the carboxyl partial peptide of the present invention (one COOH), carboxylate (-C 00-), amides (an C_ ⁇ _NH 2) or an ester (one COOR) (R is as defined above Shown).
  • the partial peptide of the present invention includes, as in the protein of the present invention, those in which the amino group of the N-terminal amino acid residue is protected with a protecting group in the partial peptide described above, Glutamine residue generated by cleavage of the side in vivo, which is oxidized with pyrrole, or a substituent in which the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, or a sugar chain is bound
  • Complex peptides such as so-called glycopeptides are also included.
  • the partial peptide of the present invention includes the 84th to 240th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1, and the 82nd to 2nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2.
  • 39 Peptide having the 9th amino acid sequence, 8th to 239th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 3, 4th amino acid sequence represented by SEQ ID NO: 31 Peptides having the 8th to 204th amino acid sequence are preferred.
  • a salt with a physiologically acceptable acid eg, an inorganic acid, an organic acid
  • a base eg, an alkali metal
  • Acceptable acid addition salts are preferred.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic acid
  • the protein of the present invention or a salt thereof can be produced from a cell or tissue of a human or non-human warm animal described above by a known protein purification method. It can also be produced by culturing a transformant containing a DNA encoding the protein described above. In addition, the protein can also be produced according to the protein synthesis method described below or according thereto. Specifically, it can be produced by the method described in WO98Z034848 or WO97 / 34911.
  • the human or non-human warm-blooded animal tissues or cells are homogenized and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange. It can be purified and isolated by combining chromatography such as chromatography.
  • a commercially available resin for protein synthesis can be usually used.
  • resins include chloromethyl resin, hydroxymethyl resin, benzylhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and PAM resin.
  • an amino acid having a suitably protected amino group and side chain functional group is condensed on the resin according to the sequence of the target protein according to various known condensation methods.
  • the protein is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein, its partial peptide, or an amide thereof.
  • various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
  • DCC, N '-diisopropylcarpoimide, ⁇ -ethyl- ⁇ '-(3-dimethylaminoprolyl) carpoimide, and the like are used.
  • Activation by these involves adding the protected amino acid directly to the resin along with a racemization inhibitor additive (e.g., HOBt, HOOBt) or pre-forming the protected amino acid as a symmetrical acid anhydride or HOBt ester or HOOBt ester. It can be added to the fat after activation.
  • a solvent used for activation of the protected amino acid or condensation with the resin It can be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • the reaction temperature is appropriately selected from the range that can be used for the protein bond formation reaction, and is usually selected from the range of about 120 ° C. to 5 (TC.
  • the activated amino acid derivative is usually selected from the range of TC.
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, arylamyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, CutZ, Br-Z, and adamantyl.
  • Z Boc, arylamyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, CutZ, Br-Z, and adamantyl.
  • oxycarbonyl, trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the carboxyl group may be, for example, an alkyl ester (eg, an ester group such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cyclohexyl, cyclooctyl, 2-adamantyl), benzyl ester, 4 Leading to 12-trobenzyl ester, 4-methoxybenzyl ester, 4-methyl benzyl ester, benzhydryl ester, phenacin ester, benzyloxycarbonyl hydrazide, Yuichi shaributoxycarbonyl hydrazide, trityl hydrazide, etc. Can be protected by an alkyl ester (eg, an ester group such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cyclohe
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • Groups suitable for this esterification include, for example, lower alkynyl groups such as an acetyl group, aroyl groups such as a benzoyl group, and groups derived from carbon such as a benzyloxycarbonyl group and an ethoxycarbonyl group. Is used.
  • groups suitable for etherification include a benzyl group, a tetrahydroviranyl group, and a t-butyl group.
  • a protecting group for the phenolic hydroxyl group of tyrosine for example, Bzl, Cl 2 -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like are used.
  • imidazole protecting group for histidine for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • activated carboxyl groups in the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5_trichloromouth phenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, H0NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt).
  • active esters eg, pentachlorophenol, 2,4,5_trichloromouth phenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, H0NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt.
  • Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia are also used. .
  • the elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about 120 ° C. to 40 ° C.
  • a cation scavenger such as cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-enedithiol.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment
  • the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1,4 -In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution or dilute ammonia.
  • Another method for obtaining an amide form of a protein is to first protect the carboxy-terminal amino acid by amidating the high carboxyl group, and then extend the peptide (protein) chain to the desired length on the amino group side. Then, a protein in which only the N-terminal ⁇ -amino group protecting group of the peptide chain is removed and a protein in which only the C-terminal carboxyl group protecting group is removed are produced, and these two proteins are mixed as described above. Condensate in solvent. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained. The crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
  • an ester of a protein an ⁇ -carboxy group of the carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester of the desired protein is obtained in the same manner as the amide of a protein.
  • the partial peptide of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase.
  • a peptide synthesis method for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used.
  • the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion, and when the product has a protecting group, removing the protecting group. it can.
  • Known methods of condensation and elimination of the protecting group include, for example, the methods described in the following 1 to 5.
  • this method combines ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, liquid chromatography, and recrystallization.
  • the protein of the invention can be purified and isolated.
  • the protein obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the protein is obtained as a salt, a known method or analogous method Thus, it can be converted into a free form or another salt.
  • the DNA encoding the protein of the present invention may be any DNA containing the aforementioned DNA encoding the protein of the present invention. Further, it may be any of genomic DNA, genomic DNA library, the above-mentioned cell / tissue-derived cDNA, the above-mentioned cell / tissue-derived cDNA library, and synthetic DNA.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can also be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using an mRNA fraction prepared from the above-mentioned cell tissue.
  • DNAs containing a nucleotide sequence encoding the protein of the present invention described in WO 98/03648 or WO 97/34911 are also included in the present invention.
  • examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 1 of the present invention include: (1) DNA having the base sequence represented by SEQ ID NO: 4, (2) SEQ ID NO: It hybridizes under high stringent conditions to DNA having the nucleotide sequence represented by 4 and has the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 1 (eg, cell transformation activity).
  • DNA encoding a protein can be used.
  • Examples of the DNA which can hybridize with the DNA having the nucleotide sequence represented by SEQ ID NO: 4 under highly stringent conditions include, for example, 'a nucleotide sequence represented by SEQ ID NO: 4 and at least about 40%, preferably about 60% Or more, more preferably 80% or more, even more preferably about 90% 'or more, most preferably about 95%' or more. DNA containing a base sequence having the same is used.
  • Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 2 of the present invention include: (1) DNA having the nucleotide sequence represented by SEQ ID NO: 7, and (2) nucleotide sequence represented by SEQ ID NO: 7. DNA that hybridizes to DNA having the same under high stringent conditions and encodes a protein having the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 2 is used.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 7 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 7 and at least about 40%, preferably about 60% or more. More preferably, a DNA containing a nucleotide sequence having a homology of 80% or more, more preferably about 90% or more, and most preferably about 95% or more is used.
  • Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 3 of the present invention include: (1) DNA having the base sequence represented by SEQ ID NO: 10, and (2) having DNA having the base sequence represented by SEQ ID NO: 10.
  • DNA that hybridizes to DNA under high stringent conditions and encodes a protein having the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 3 is used.
  • Examples of a DNA that can hybridize with the base sequence represented by SEQ ID NO: 10 under high stringent conditions include, for example, the base sequence represented by SEQ ID NO: 10 and at least about 40%, preferably at least about 60%, More preferably, a DNA containing a nucleotide sequence having a homology of 80 'or more, more preferably about 90% or more, and most preferably about 95% or more is used.
  • Examples of the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 31 of the present invention include: (1) DNA having the base sequence represented by SEQ ID NO: 30; For example, DNA that hybridizes to DNA having a sequence under high stringency conditions and encodes a protein having the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 2 is used.
  • Examples of the DNA which can hybridize with the base sequence represented by SEQ ID NO: 30 under high stringent conditions include, for example, about 40% or more, preferably about 40% or more of the base sequence represented by SEQ ID NO: 30.
  • DNA containing a base sequence having a homology of 60% or more, more preferably 80% or more, further preferably about 90% or more, and most preferably about 95% or more is used.
  • Hybridization can be performed by a known method or a method analogous thereto, for example, a method described in Molecular Cloning 2nd (J. Sambrook e. Al., Cold Spring Harbor Lab. Press, 1989). It can be done according to the following. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
  • Highly stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 70 ° C.
  • the conditions at ⁇ 65 are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
  • DNA having the base sequence represented by SEQ ID NO: 4 and the like are used as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 and the like.
  • DNA containing the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 1 of the present invention include, for example, the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6
  • DNA having the following is used.
  • DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 2 DNA having the base sequence represented by SEQ ID NO: 7 and the like are used.
  • Examples of the DNA containing the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 2 of the present invention include, for example, the nucleotide sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9 And the like having DNA are used.
  • DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 3 a DNA having the base sequence represented by SEQ ID NO: 10 or the like is used.
  • DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 31 a DNA having the base sequence represented by SEQ ID NO: 30 or the like can be used.
  • the DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. Any of the above-described cell-tissue-derived cDNA, the above-described cell-tissue-derived cDNA library, and synthetic DNA may be used.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be performed directly by RT-PCR using an mRNA fraction prepared from the cells and tissues described above.
  • a DNA having at least one nucleotide sequence is used selected from the 682-7 1 seventh (or the 682-72 0 th) of the base sequence.
  • Examples of the DNA encoding the partial peptide having at least one amino acid sequence selected from the amino acid sequences at positions 214 to 218 and 227 to 238 (or positions 223 to 239) include, for example, SEQ ID NO: : 16th to 60th, 157th to 171st (or 154th to 171st), 271st to 300th, 319th to 342th, and threeth of the base sequence represented by 10 346-372, 376-396, 424-441, 484-510, 526-546, 550-567, 574-636, 640-654, and 640-654 DNA having at least one base sequence
  • Examples of DNA encoding a partial peptide having the amino acid sequence at positions 84 to 240 of the amino acid sequence represented by SEQ ID NO: 1 include: (1) the 250th amino acid sequence of the base sequence represented by SEQ ID NO: 4; A DNA having the nucleotide sequence of the nucleotide sequence from the 720th to the 720th nucleotide, and (2) a DNA having a nucleotide sequence of the 250th to the 720th nucleotide of the nucleotide sequence represented by SEQ ID NO: 4 which is hybridized under high stringent conditions.
  • DNA encoding a partial peptide having the same activity (eg, cell transformation activity) as the partial peptide having the amino acid sequence at positions 84 to 240 of the amino acid sequence represented by No. 1 can also be used.
  • Examples of DNA that can hybridize with the 250th to 720th nucleotide sequences of the nucleotide sequence represented by SEQ ID NO: 4 under high stringency conditions include, for example, the 250th nucleotide of the nucleotide sequence represented by SEQ ID NO: 4.
  • DNA containing a base sequence is used.
  • Examples of the DNA encoding the partial peptide having the amino acid sequence of the 82nd to 239th amino acids of the amino acid sequence represented by SEQ ID NO: 2 include: (1) the 244th to 717th nucleotides of the base sequence represented by SEQ ID NO: 7 A DNA having the nucleotide sequence of SEQ ID NO: 2, which hybridizes to a DNA having the nucleotide sequence of positions 244 to 717 of the nucleotide sequence represented by SEQ ID NO: 7, and the 82nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2 A DNA encoding a partial peptide having the same activity as the partial peptide having the amino acid sequence at positions 239 to 239 (eg, cell-transforming effect) can also be used.
  • DNA that can be hybridized under stringent conditions include, for example, the nucleotide sequence from the 24th to the 71st nucleotide of the nucleotide sequence represented by SEQ ID NO: 7 and about 40% or more.
  • DNA containing a base sequence having a homology of about 60% ′ or more, more preferably 80 ′ or more, further preferably about 90% or more, and most preferably about 95% or more is used.
  • Examples of the DNA encoding the partial peptide having the 8th to 23rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 3 include: (1) the 24th amino acid sequence represented by SEQ ID NO: 10; A DNA having the 4th to 717th nucleotide sequence, 2 hybridizing to a DNA having the 24th to 717th nucleotide sequence of the nucleotide sequence represented by SEQ ID NO: 10, DNA encoding a partial peptide having an activity equivalent to that of the partial peptide having the 8th to 23rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 3 (eg, cell transformation activity) Used by all.
  • Examples of the DNA encoding the partial peptide having the 48th to 204th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31 include, for example, 1) the base sequence represented by SEQ ID NO: 30 DNA having the nucleotide sequence from the 14th to the 61st to the 12th of the nucleotide sequence, 2 into the DNA having the nucleotide sequence from the 14th to the 61st to the 12th of the nucleotide sequence represented by SEQ ID NO: 30 It hybridizes under high stringent conditions and has the same activity as the partial peptide having the 48th to 204th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31 (eg, cell transformation activity) DNA encoding a partial peptide having the following is also used.
  • Examples of the DNA that can hybridize under the stringent conditions with the 24th to 717th base sequences of the base sequence represented by SEQ ID NO: 10 include, for example, SEQ ID NO: 10 Approximately 40% or more, preferably about 60% or more, more preferably 80% or more, and still more preferably about 90% or more of the 24th to 717th base sequence of the base sequence represented by DNA containing a nucleotide sequence having a homology of 3 ⁇ 4 or more, most preferably about 95 3 ⁇ 4 or more is used.
  • the DNA encoding the partial peptide having the 84th to 240th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 is the DNA encoding the 250th nucleotide of the base sequence represented by SEQ ID NO: 4.
  • DNA having a base sequence from the 720th position to the 720th position is used.
  • the DNA encoding the partial peptide having the 82nd to 239th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2 includes the 244th to 717th nucleotide of the base sequence represented by SEQ ID NO: 7.
  • DNA having a base sequence of The DNA encoding the partial peptide having the 82nd to 239th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 3 includes the 244th to 717th nucleotide of the base sequence represented by SEQ ID NO: 10.
  • DNA having the second base sequence is used.
  • the DNA coding for the partial peptide having the 48th to 204th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 31 is a DNA encoding the 142nd to 142nd amino acid sequence of the base sequence represented by SEQ ID NO: 30
  • DNA having the nucleotide sequence of 6 1 2 is used.
  • the synthetic DNA primer having a partial nucleotide sequence of the DNA encoding the protein of the present invention is used for the cloning by the PCR method.
  • the nucleotide sequence of DNA can be converted using known kits such as Mutan TM -super Express Km (Takara Shuzo Co., Ltd.) and Mutan TM -K (Takara Shuzo Co., Ltd.). It can be carried out according to a known method such as the Gapped du 1 ex method or the Kimke 1 method or a method analogous thereto.
  • D encoding the cloned protein of the present invention or a partial peptide thereof NA can be used as it is depending on the purpose, or digested with a restriction enzyme or added with a linker, if desired.
  • the DNA may have ATG as a translation initiation codon on its 5 'end, and may have TAA, TGA or TAG as a translation termination codon on its 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • An expression vector for a DNA encoding the protein of the present invention or a partial peptide thereof is, for example, (a) cutting out a DNA fragment of interest from the DNA encoding the protein of the present invention, and It can be produced by ligating downstream of a promoter in a suitable expression vector.
  • the vector examples include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), In addition to yeast-derived plasmids (eg, pSH19, pSHI5), bacteriophages such as phage, animal viruses such as retrovirus, vaccinia virus, paculovirus, etc., pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. are used.
  • Escherichia coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis eg, pUB110, pTP5, pC194
  • yeast-derived plasmids eg, pSH19,
  • the promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression.
  • SRa promoter SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter and the like can be mentioned.
  • CMV promoter cytomegalovirus promoter
  • SRa promoter SV40 promoter
  • LTR promoter LTR promoter
  • HSV-TK promoter cytomegalovirus promoter
  • the host is Escherichia, trp promoter, lac promoter, recA promoter, ⁇ PL promoter, 1 pp promoter, etc.
  • yeast such as penP promoter, AOX1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferred.
  • the expression vector may include, in addition to the above, an enhancer, a splicing signal, a polyA addition signal, a selection marker, and an SV40 replication origin (hereinafter, sometimes abbreviated as SV40o1-i), if desired. What they contain can be used.
  • the selection marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhf 1-) gene, an ampicillin resistance gene (hereinafter sometimes abbreviated as Ampr), a neomycin resistance gene (hereinafter abbreviated as Ampr). , Neo, or G418 resistance).
  • the dh fr gene confers resistance to methotrexate (MTX), and Neo confers G418 resistance.
  • MTX methotrexate
  • Neo confers G418 resistance.
  • the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the protein. If the host is a bacterium belonging to the genus Escherichia, PhoA signal sequence, OmpA • signal sequence, etc. If the host is a bacterium belonging to the genus Bacillus, the amylase signal sequence, subtilisin signal sequence, etc. In some cases, MFa signal sequence, SUC2 signal sequence, etc. When the host is an animal cell, for example, insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule, signal sequence, etc. it can.
  • a transformant can be produced by introducing a vector containing the DNA encoding the protein of the present invention thus constructed into cells.
  • the host for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia examples include, for example, Escherichia coli K12, DH1 [Procedures of the National Academy of Sciences, Obs. Natl. Acad. Sci. US A), 60, 160 (1968)], JM103 [Nucleic Acids Research, (Nucleic Acids Research), 9, 309 (1981)], JA221 [Journal of Molecular Biology], Volume 120, 5 17 (1 978)], HB 10 1 [Journal of Molecular Biology] Rekiura Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc. are used.
  • Bacillus spp. include, for example, Bacillus subtilis MI 11 [Gene, 24, 255 (1983)], 207-21 [Journal ⁇ Biochemistry, 95 , 87 (1984)].
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22R, NA87-11A, DKD-5D, 20B12, Schizosaccharomyces bomb (Sctiizosaccharomyces pombe) NCYC 1913, NCYC 2036, Pichia pastoris Pichia pastoris) KM71 or the like is used.
  • insect cells for example, when the virus is Ac NPV, a cell line derived from a larva of Spodoptera (Spodoptera irugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, High Five derived from egg of Trichoplusia ni TM cells, cells derived from amestra brass icae, cells derived from Estigmena acrea, and the like are used.
  • Sf cells include Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, JL et al., In Vitro, 13, 213-217, (1977) ) Is used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster Yuichi cell CHO (hereinafter abbreviated as CHO (dh fr-) cell). ), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, 293 cells, C127 cells, BALB 3T3 cells, Sp-2 cells, etc. are used. Among these, CH ⁇ cells, CHO (dhfI- ”) cells, 293 cells and the like are preferable.
  • Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
  • To transform animal cells for example, see Cell Engineering Separate Volume 8 New Cell Engineering Experiment Protocol. 263—267 (1995) (published by Shujunsha), Virology, 52, 456 (1 973).
  • Examples of the method for introducing the expression vector into cells include the calcium phosphate method [Graham, FL and van der Eb, AJ Virology 52, 456-467 (1973)], the electroporation method [Nueniann, E. et. al. Embo Journal (EMBO J.) 1, 841-845 (1982)].
  • a method for stably expressing the protein of the present invention using animal cells there is a method of selecting, by clone selection, cells in which the expression vector introduced into the animal cells is integrated into the chromosome. .
  • a transformant can be selected using the selection marker as an indicator.
  • a stable animal cell line having a high expression ability of the protein of the present invention can be obtained by repeatedly performing clone selection on the animal cells obtained using the selection marker.
  • the dhfr gene is used as a selection marker, the DNA encoding the protein of the present invention is amplified in the cell together with the dhfr gene by culturing the MTX concentration gradually and selecting a resistant strain. By doing so, it is possible to obtain an animal cell strain with a higher expression.
  • the above transformant is cultured under conditions in which DNA encoding the protein of the present invention or a partial peptide thereof can be expressed, and the protein of the present invention or a partial peptide thereof is produced and accumulated, whereby the present invention is obtained.
  • a protein, a partial peptide thereof, or a salt thereof can be produced.
  • a liquid medium is suitable as the medium used for the culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like.
  • the inorganic or organic substance and the inorganic substance include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • M9 medium containing glucose and casamino acid As a medium for culturing the genus Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experimen in 'Molecular' Genetics (Journal of Experiments in Mo1e eCullar Genetics), 43 1-43 3, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3 / 3-indolylacrylic acid can be added to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually carried out at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
  • the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours. If necessary, aeration and stirring may be added. '
  • the culture medium When culturing a transformant in which the host is yeast, the culture medium may be, for example, Burkholder's minimal medium such as CBostian, KL, or the like. Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and 0.5% casamino acids SD medium [Bitter, GA et al., Proc. Natl. Acad. Sci. USA, Proc. Natl. Acad. Sci. USA] 1, 5330 (1 984)]. The pH of the medium is preferably adjusted to about 5-8. The cultivation is usually performed at about 20 to 35 ° C for about 24 to 72 hours, and aeration and agitation are added as necessary.
  • the culture medium When culturing a transformant whose host is an insect cell, the culture medium was Grace's Insect Medium (Grace, TC, Nature, 195, 788 (1962)). And the like to which additives such as the above are appropriately added are used.
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • the medium When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20 ml of fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association at ion, Vol. 199, 519 (1967)] ], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], and the like.
  • the pH is about 6-8.
  • Culture is usually performed at about 30 ° C (about 40 ° C for about 15 to 72 hours, and aeration and agitation are added as necessary.
  • DMEM medium containing dialysed fetal serum containing almost no thymidine.
  • the protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells or cells are collected by a known method after culture, and suspended in an appropriate buffer. After the cells or cells are disrupted by lysozyme and / or freeze-thawing, a method of obtaining a crude extract of the protein of the present invention by centrifugation or filtration may be suitably used.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM .
  • the protein is secreted into the culture solution, after the culture is completed, the bacterial cells or cells are separated from the supernatant by a known method, and the supernatant is collected.
  • Purification of the protein of the present invention contained in the culture supernatant or the extract thus obtained can be carried out by appropriately combining known separation and purification methods.
  • These known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • differences in molecular weights methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity mouth chromatography, hydrophobic methods such as reversed-phase high-performance liquid chromatography, etc.
  • a method using a difference in gender, a method using a difference in isoelectric point such as isoelectric focusing, and the like are used.
  • the thus obtained protein of the present invention when obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto. It can be converted to a free form or another salt by an analogous method.
  • the protein of the present invention produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, dalicosidase and the like are used.
  • the presence of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody or the like.
  • An antibody against the protein of the present invention, its partial peptide or a salt thereof is an antibody capable of recognizing the protein of the present invention, its partial peptide or a salt thereof (hereinafter, sometimes abbreviated as the protein of the present invention). If so, it may be either a polyclonal antibody or a monoclonal antibody.
  • An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can be produced using the protein of the present invention as an antigen according to a known antibody or antiserum production method.
  • the protein of the present invention is administered to a human or a non-human warm-blooded animal at a site capable of producing an antibody by administration to itself or together with a carrier or a diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. Administration can usually be performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • human or non-human warm-blooded animals include monkeys
  • mice Rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens are used, and mice and rats are preferably used.
  • a non-human warm-blooded animal immunized with the antigen for example, an individual with an antibody titer was selected from a mouse, and the spleen or lymph node was collected 2 to 5 days after the final immunization.
  • an individual with an antibody titer was selected from a mouse, and the spleen or lymph node was collected 2 to 5 days after the final immunization.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting a labeled protein or the like described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) -Sendai virus and the like, and preferably PEG is used.
  • myeloma cells for example, myeloma cells derived from non-human warm-blooded animals such as NS-1, P3U1, SP 2/0, and AP-1 are used, and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the concentration of PEG (preferably PEG 1000 to PEG 6000) is about 10 to 80%.
  • PEG preferably PEG 1000 to PEG 6000
  • Various methods can be used for screening monoclonal antibody-produced lipids.
  • the hybridoma culture supernatant is applied directly or onto a solid phase (eg, microplate) on which a protein antigen is adsorbed together with a carrier. Then, add an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is mouse) or protein A labeled with a radioactive substance or enzyme, and bind to the solid phase.
  • a solid phase eg, microplate
  • an anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is mouse
  • protein A labeled with a radioactive substance or enzyme bind to the solid phase.
  • a method to detect the monoclonal antibody that has been bound the hybridoma culture supernatant is added to the solid phase to which the anti-immunoglobulin antibody or protein A is adsorbed, and a protein labeled with a radioactive substance, an enzyme, or the like is added, and the solid phase is bound to the solid phase.
  • a method for detecting a monoclonal antibody is used.
  • the selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma. For example: 220.%, preferably 10-20.
  • RPMI 1640 medium containing fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma cultivation (SFM-10) 1, Nissui Pharmaceutical Co., Ltd.) can be used.
  • the cultivation temperature is usually from 20 to 40 ° (:, preferably, about 37 ° C.
  • the culturing time is usually from 5 days to 3 weeks, preferably from 1 week to 2 weeks.
  • the antibody titer of the culture supernatant of the hybridoma can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)).
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody.
  • the polyclonal antibody of the present invention can be obtained by any known or equivalent method.
  • a complex of an immunizing antigen (protein antigen) and a carrier protein is formed, and a non-human warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody, and the immunized animal contains the polyclonal antibody of the present invention. It can be produced by collecting the product and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of carrier and hapten are determined by the hapten immunized by cross-linking with the carrier.
  • Any antibody may be cross-linked at any ratio as long as the antibody can be efficiently produced.
  • perforated serum albumin, psilogloproline, hemocyanin, etc. are used in a weight ratio to hapten 1 relative to hapten 1.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • daltaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
  • the condensation product is administered to a non-human warm-blooded animal at a site where antibody production is possible, together with a carrier or diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody production ability during administration. The administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 times. It is a thing.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a non-human warm-blooded animal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above.
  • Antibody separation and purification can be performed according to the same immunoglobulin separation and purification method as the monoclonal antibody separation and purification described above.
  • Antisense DNA having a base sequence substantially complementary to DNA or mRNA encoding the protein or partial peptide of the present invention includes DNA or mRNA encoding the protein or partial peptide of the present invention.
  • the nucleotide sequence substantially complementary to the DNA or mRNA is, for example, about 40% of the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA or mRNA (that is, the complementary strand of the DNA or mRNA). % Or more, preferably about 60% or more, more preferably about 80 or more, even more preferably about 90 ° 'or more. In particular, about 40% of the total nucleotide sequence of the complementary strand of the DNA or mRNA of the present invention is complementary to the complementary strand of the nucleotide sequence encoding the N-terminal portion of the protein of the present invention (for example, the nucleotide sequence near the start codon).
  • Antisense DNA having a homology of at least 90% or more, more preferably about 90% or more is suitable. These antisense DNAs can be produced using a known DNA synthesizer or the like.
  • the protein of the present invention, its partial peptide, or a salt thereof has, for example, an action such as a cell shape conversion action. Therefore, the protein of the present invention, its partial peptide, or a salt thereof can be used for various uses based on the above-mentioned action.
  • a protein of the present invention a partial peptide thereof, or a salt thereof (hereinafter, sometimes abbreviated as the protein of the present invention), a DNA encoding the protein of the present invention, etc. )
  • antibodies against the protein and the like of the present invention sometimes abbreviated as the antibody of the present invention
  • antisense DNA sometimes abbreviated as the antibody of the present invention
  • the cell transformation activity refers to an activity that suppresses cell proliferation and induces a change in cell morphology.
  • cancer 'tumor preferably, (fetal) rhabdomyosarcoma, etc.
  • Suppresses cell proliferation and induces cancer / tumor preferably (fetal) rhabdomyosarcoma etc.
  • the protein of the present invention is reduced or deficient in the living body, there may be patients who do not sufficiently or (A) administering the DNA of the present invention to the patient, expressing the protein of the present invention in vivo, (inserting) the DNA of the present invention into (oral) cells, After the expression of the protein, the cells are transplanted into a patient, and (8) the role of the protein of the present invention in the patient is sufficiently or normally maintained by administering the protein of the present invention to the patient. Can be demonstrated.
  • the protein of the present invention suppresses cancer / tumor (preferably, (fetal) rhabdomyosarcoma, etc.) cell proliferation, and produces cancer, tumor (preferably, (fetal) rhabdomyosarcoma, etc.) cells.
  • cancer / tumor preferably, (fetal) rhabdomyosarcoma, etc.
  • tumor preferably, (fetal) rhabdomyosarcoma, etc.
  • the protein of the present invention and the DNA of the present invention include, for example, (fetal) rhabdomyosarcoma (focal rhabdomyosarcoma, rhabdomyosarcoma primary liver sarcoma, etc.), leiomyosarcoma, muscular dystrophy ( It is useful as a medicine for treating and preventing diseases such as myotonic dystrophy) or uterine fibroids.
  • the DNA of the present invention When the DNA of the present invention is used as the above-mentioned therapeutic or prophylactic agent, the DNA is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. After that, it can be administered to a human or non-human warm-blooded animal according to a conventional method.
  • the DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered by a force gun such as a gene gun or a hydrogel catheter.
  • the protein of the present invention when used as the above-mentioned therapeutic or prophylactic agent, it should be purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. Is preferred.
  • the protein of the present invention is used as the above-mentioned pharmaceutical, for example, it is orally administered as tablets, capsules, elixirs, microcapsules and the like, if necessary, or water or other pharmaceuticals. It can be used parenterally in the form of injections, such as sterile solutions with liquids that are acceptable or suspensions.
  • the DNA of the present invention is used, the DNA is administered alone or after insertion into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector, followed by administration according to a conventional method. can do.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
  • Agents for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 TM , HCO-50, etc.) You may use together with.
  • oily liquid for example, sesame oil, soybean oil, and the like are used, and may be used in combination with benzyl benzoate, benzyl alcohol, or the like as a solubilizing agent.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • Preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, antioxidants and the like.
  • the prepared injection is usually filled in a suitable ampoule.
  • the vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used parenterally.
  • the preparations obtained in this way are safe and low toxic, and thus can be used, for example, in mammals (e.g., Monkeys).
  • the dosage of the protein of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like.
  • the protein of the present invention when the protein of the present invention is orally administered as a (fetal) rhabdomyosarcoma therapeutic agent, it is generally In adults (as 6 O kg), the protein may be administered daily at a dose of about 0.1 mg to: L 0 O mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg. I do.
  • the single dose of the protein varies depending on the administration target, target disease, and the like.
  • the protein of the present invention may be used as a therapeutic agent for (fetal) rhabdomyosarcoma by injection.
  • the dose When administered to an adult (with a body weight of 6 O kg) in the form of about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 2 mg of the protein per day, It is convenient to administer by injecting about 10 mg into the affected area. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the protein of the present invention and the DNA of the present invention are used for the activation of NF- / cB by each TNF family ligand molecule (eg, TNFa ', TNF / 3, LTo; 1/32, etc.) in RD cells, It has a chemokine expression-inducing or production-promoting effect and an RD cell differentiation-inducing effect.
  • TNF family ligand molecule eg, TNFa ', TNF / 3, LTo; 1/32, etc.
  • the DNA of the present invention can be used as a probe in mammals (eg, humans, rats, mice, guinea pigs, egrets, sheep, sheep, bush, horseshoe, horses, cats, dogs, monkeys, etc.). Abnormalities (DNA abnormalities) in the DNA encoding the protein of the present invention can be detected. Therefore, the DNA of the present invention is useful as an agent for genetic diagnosis of various diseases related to the protein of the present invention.
  • mammals eg, humans, rats, mice, guinea pigs, egrets, sheep, sheep, bush, horseshoe, horses, cats, dogs, monkeys, etc.
  • a (fetal) rhabdomyosarcoma (focal horizontal) Can be diagnosed as diseases such as rhabdomyosarcoma, rhabdomyosarcoma primary liver sarcoma), leiomyosarcoma, muscular dystrophy (myotonic dystrophy) or uterine fibroids.
  • the above-described genetic diagnosis using the DNA of the present invention includes, for example, the well-known Northern Hybridization and the PCR-SSCP method (Genomics, Vol. 5, pp.
  • the antibody of the present invention can specifically recognize the protein of the present invention
  • quantification of the protein of the present invention in a test solution in particular, quantification by a sandwich immunoassay, allows the protein of the present invention to be analyzed. It can be used for diagnosis of various diseases and the like based on the cell transformation action of quality.
  • one of the antibodies is an antibody that recognizes the N-terminal of the protein of the present invention, and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention. Is desirable.
  • a monoclonal antibody against the protein of the present invention (hereinafter simply referred to as a monoclonal antibody)
  • the protein of the present invention can be quantified using an antibody (also referred to as an internal antibody), and detection by tissue staining or the like can also be performed.
  • an antibody also referred to as an internal antibody
  • the antibody molecule itself may be used, or the F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and the antibody, antigen, or antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution is measured. Any measurement method may be used as long as the amount is detected by chemical or physical means and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competition method, an immunometric method and a sandwich method are preferably used, but from the viewpoint of sensitivity and specificity, it is particularly preferable to use the San Germanti method described later.
  • a labeling agent used in a measuring method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • Radioisotopes if example embodiment, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C]
  • the enzyme large preferably stable and specific activity
  • fluorescent substances for example, fluorescamine, fluorescein isothiosianate, etc.
  • the luminescent substance for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used, respectively.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used.
  • the carrier for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.
  • a test solution is reacted with an insolubilized monoclonal antibody (primary reaction), and further reacted with a labeled monoclonal antibody (secondary reaction), and then the activity of the labeling agent on the insolubilized carrier is measured.
  • the primary and secondary reactions can be performed in reverse order Alternatively, they may be performed simultaneously or at different times.
  • the labeling agent and the method of insolubilization can be the same as those described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and two or more kinds of antibodies are used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds.
  • the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephelometry method.
  • a competition method an antigen in a test solution and a labeled antigen are applied to the antibody.
  • the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (BZ'F separation), and the amount of labeled B or F is measured. Quantify the amount of antigen in the test solution.
  • a soluble antibody is used as the antibody
  • BZF separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody against the antibody a solid phase antibody is used as the first antibody
  • An immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.
  • the amount of insoluble sediment resulting from an antigen-antibody reaction in a gel or in a solution is measured. Even if the amount of antigen in the test solution is very small and only a small amount of sediment can be obtained, use a laser-nef mouth meter that uses laser scattering Is preferably used.
  • the protein measurement system of the present invention may be constructed by adding ordinary technical considerations to those skilled in the art to the ordinary conditions and procedures in each method. For details of these general technical means, reference can be made to reviews and written documents.
  • the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • rhabdomyosarcoma focal rhabdomyosarcoma, rhabdomyosarcoma primary liver sarcoma, etc.
  • leiomyosarcoma It can be diagnosed as a disease such as muscular dystrophy (myotonic dystrophy) or uterine fibroids, or is likely to be affected in the future.
  • Antisense DNA that binds complementarily to the DNA of the present invention and can suppress the expression of the DNA is the protein of the present invention or the DNA of the present invention in vivo.
  • the above-mentioned antisense DNA is used as the above-mentioned therapeutic / prophylactic agent, it can be used in the same manner as the aforementioned therapeutic / prophylactic agent for various diseases containing the DNA of the present invention.
  • the antisense DNA when used, the antisense DNA may be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. It can be administered according to conventional means.
  • the antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and then administered using a gene gun or a catheter such as a hydrogel catheter.
  • the antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below.
  • the L-form is indicated unless otherwise specified.
  • DNA Deoxyribonucleic acid
  • RNA Liponucleic acid
  • d ATP Deoxyadenosine triphosphate
  • dTTP Deoxythymidine triphosphate
  • Th r thread ' Piano.
  • FIG. 1 shows the amino acid sequence of human-derived protein of the present invention.
  • FIG. 1 shows an amino acid sequence of a rat-derived protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding a human-derived protein having the amino acid sequence represented by SEQ ID NO: 1 of the present invention.
  • nucleotide sequence of a DNA containing a cDNA encoding a human-derived protein having the amino acid sequence represented by SEQ ID NO: 1 of the present invention inserted into plasmid pTB1939 is shown.
  • Fig. 3 shows the nucleotide sequence of DNA containing a cDNA encoding a human-derived protein having the amino acid sequence represented by SEQ ID NO: 1 of the present invention, inserted into plasmid pTB1940.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding a mouse-derived protein having the amino acid sequence represented by SEQ ID NO: 2 of the present invention.
  • FIG. 7 shows the nucleotide sequence of DNA containing cDNA encoding a mouse-derived protein having the amino acid sequence represented by SEQ ID NO: 2 of the present invention, which is inserted into plasmid pTB1958.
  • FIG. 1 shows the nucleotide sequence of genomic DNA encoding a mouse-derived protein having the amino acid sequence represented by SEQ ID NO: 2 of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding a rat-derived protein having the amino acid sequence represented by SEQ ID NO: 3 of the present invention.
  • FIG. 1 shows the nucleotide sequence of a primer used for cloning DNA encoding the human-derived protein of the present invention.
  • the base sequence of the primer used for cloning the DNA encoding the human-derived protein of the present invention is shown.
  • FIG. 1 shows the nucleotide sequence of an oligonucleotide used to clone DNA encoding the mouse-derived protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of an oligonucleotide used for cloning DNA encoding the mouse-derived protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of a synthetic oligonucleotide used for the analysis of the nucleotide sequence near the start codon of the DNA encoding the mouse-derived protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of a synthetic oligonucleotide used for analysis of the nucleotide sequence near the initiation codon of DNA encoding mouse-derived protein of the present invention.
  • FIG. 1 shows a nucleotide sequence of an adapter binding to both ends of a mouse chromosome DNA fragment used for analysis of a nucleotide sequence near a start codon of DNA encoding a mouse-derived protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of a synthetic oligonucleotide used for analysis of the nucleotide sequence near the start codon of the DNA encoding the mouse-derived protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of a synthetic oligonucleotide used for analysis of the nucleotide sequence near the start codon of the DNA encoding the mouse-derived protein of the present invention.
  • [SEQ ID NO: 21] 1 shows the nucleotide sequence of a primer used for cloning DNA encoding the extracellular region of the human-derived protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of a primer used for cloning DNA encoding the extracellular region of the human-derived protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of a primer used for cloning DNA encoding the rat-derived protein of the present invention.
  • FIG. 1 shows the nucleotide sequence of a primer used for cloning the DNA encoding the rat-derived protein of the present invention.
  • Example 13 shows the nucleotide sequence of a primer used in Example 7 described later.
  • Example 13 shows the nucleotide sequence of a primer used in Example 7 described later.
  • Example 13 shows the nucleotide sequence of a primer used in Example 7 described later.
  • Example 13 shows the nucleotide sequence of a primer used in Example 7 described later.
  • Example 13 shows the nucleotide sequence of a primer used in Example 7 described later.
  • Example 13 shows the nucleotide sequence of a primer used in Example 7 described later.
  • the transformants Escherichia coli DH10 ⁇ TB1939 and Escherichia coli DH10B'pTB1940 obtained in Reference Example 1 described below were obtained on July 17, 1996, respectively.
  • IFO Institute for Fermentation
  • the transformant Escherichia coli DH5 ⁇ / ⁇ 1958 obtained in Reference Example 2 described below was obtained from January 30, 1997, 1-1 1-1 Higashi, Tsukuba-shi, Ibaraki Prefecture 1 305-8566) Deposited by the National Institute of Advanced Industrial Science and Technology (AIST), Patented Depositary for Biotechnology (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NI BH)) as FERM BP-5805 and 1 997 Accession No. IF ⁇ 1 to the Fermentation Research Institute (IF ⁇ ) from January 31, 2012 Deposited as 6054.
  • AIST National Institute of Advanced Industrial Science and Technology
  • NI BH National Institute of Advanced Industrial Science and Technology
  • the transformant Escherichia coli DH5 / pTB2011 obtained in Reference Example 3 described below has been used since July 8, 1997, 1-1 1-1 Tsukuba-Higashi, Ibaraki Pref. 305—8566) Deposited at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Depositary Depositary Center (formerly National Institute of Advanced Industrial Science and Technology (NI BH)) under the deposit number FERM BP—60 12 and 1 9 Deposited with the Fermentation Research Institute (IF ⁇ ) as the deposit number IFO16109 from July 7, 1997.
  • AIST National Institute of Advanced Industrial Science and Technology
  • NI BH National Institute of Advanced Industrial Science and Technology
  • the cloning of the cDNA was carried out using a gene trapper (GENETRAPPER TM ) cDNA positive selection system (Gibco BRL).
  • E. coli DH1 2 S strain super one script TM human liver c DNA library (GIBCO copy R. El Co.), 100 g / ml ampicillin 'phosphate-containing Terrific Broth (1 2g / 1 Bacto- tryptone ( Difco), 24 g / 1 Bacto-yeast extract (Difco), 2.3 g / 1 monopotassium phosphate, 12.5 g / l dipotassium phosphate, 0.4% glycerol) at 30 ° C for 16 hours.
  • a plasmid cDNA library was prepared using Chiazien Plasmid Kit (Qiagen IIh).
  • the purified plasmid cDNA library was digested with Genell and Exo III (both from Gibcopy Inc.) to create a single-stranded cDNA library, while a synthetic oligonucleotide (SEQ ID NO: : 1) was used for screening of cDNA library.
  • the probe was labeled by biotinylating the 3 ′ end using TdT and Pyotin-141-dCTP (Gibcopy AR).
  • the single-stranded cDNA library was treated at 95 ° C for 1 minute, quenched in ice, and a biotinylated probe was added, followed by hybridization at 37 ° C for 1 hour at room temperature. After hybridization, a gene trapper cDNA positive selection system 'streptavidin beads (Gibcobiell) was added, and the mixture was left at room temperature for 30 minutes with stirring every 2 minutes. Thereafter, the cells were placed in a gene trapper cDNA positive selection system / magnet rack (Gibcovi, Inc.) and left for 2 minutes. The supernatant was discarded, and the magnetic beads were washed with Gene Trapper-cDNA positive selection system. Washing with this wash buffer was performed three times. Then magne JP02 / 01536
  • the supernatant was discarded, the Gene Trapper-cDNA positive selection system and elution buffer were added, and the mixture was allowed to stand at room temperature for 5 minutes. After placing in a magnetic rack for 5 minutes, the DNA solution of the supernatant was recovered.
  • a synthetic oligonucleotide (SEQ ID NO: 11) was added as a primer to the obtained DNA solution and treated at 95 ° C for 1 minute.
  • Gene trapper cDNA positive selection system 'Repair enzyme was added and left at 70 ° C for 15 minutes to synthesize double-stranded DNA.
  • the synthesized double-stranded DNA was introduced into Escherichia coli DH10B using an electoral poration device (Bio-Rad).
  • clone # 9 and clone # 33 contained the same DNA fragment, and had 1491 nucleotide sequences represented by SEQ ID NO: 5 including poly (A) + chain .
  • Clone # 81 had 1353 base sequences represented by SEQ ID NO: 6 including poly (A) + chain and poly (A) + additional signal (AATAA).
  • the cDNA fragments of these three clones contained the same gene and encoded a TL4 protein consisting of 240 amino acids represented by SEQ ID NO: 1.
  • Kyte-Doolittle analysis suggested that the hydrophobic region from valine 35 (Va 1) to tributophan 63 (T rp) is a transmembrane region of this protein.
  • This protein had the highest homology with human photoxin 3, but showed 33 homology at the amino acid level. Although 31% homology was found at the amino acid level with human Fas ligand, phylogenetic tree analysis by the J. Hein method (based on the PAM250 residue weight table) showed that human F Higher homology with the as ligand was seen.
  • Plasmid PTB1939 containing clone # 9 and plasmid pTB1940 containing clone # 81 among the DNAs encoding the protein of the present invention were introduced into Escherichia coli DH10B for transformation.
  • Body: 'Escherichia coli DH10B pTB1939 and Escherichia coli DH10B./pTB1940 were obtained.
  • Reference Example 2 Cloning of cDNA Encoding Mouse-Derived TL4 Protein Cloning of cDNA was performed by PCR. Superscript TM mouse 8.5 E.
  • coli DH12S strain from a 5 day embryo-derived cDNA library (Gibcopy AR) was transformed into Super Broth (32 g / 1 B acto-tryptone (32 g / 1 B Difco), 20 g / 1 Bacto-yeast extract (Difco), 0.2 g / 1 NaCl).
  • a plasmid cDNA library was prepared using Qiagen Plasmid Kit (Qiagen) and used as type I.
  • the obtained amplified fragment was inserted into pT7Blue T-vector (Novagen) using DNA ligation kit version 2 (Takara Shuzo Co., Ltd.) and introduced into E. coli DH5.
  • Plasmid DNA was extracted from the resulting transformant and reacted using the Dye Terminator-Cycle Sequence FS Ready Reaction Kit (PerkinElmer) and using the 373A DNA sequencer (PerkinElmer). The nucleotide sequence of the cDNA fragment was determined.
  • the obtained clone has a 795 nucleotide sequence represented by SEQ ID NO: 8 including the 717 nucleotide sequence represented by SEQ ID NO: 7, and the 239 amino acids represented by SEQ ID NO: 2 Encoding a mouse-derived TL4 protein.
  • This mouse-derived TL4 protein and the human-derived TL4 protein having the amino acid sequence represented by SEQ ID NO: 1 obtained in Reference Example 1 have 78% homology at the amino acid level.
  • the DNA encoding it had 77% homology at the base level.
  • the resulting plasmid pTB1958 carrying DNA encoding TL4 protein derived from mouse was introduced into Escherichia coli DH5, and a transformant: Escherichia coli DH5 ⁇ ⁇ 1958 was transformed. Obtained.
  • mouse genomic DNA used was digested with ScaI restriction enzyme in advance, and the 5 'and 3' ends were labeled with Primer API (Clontech) and Primer AP 2
  • the first PCR reaction was performed using this mouse genomic DNA solution, TaKaRa LA PCR kit version 2 (Takara Shuzo Co., Ltd.), AP1, and synthetic oligonucleotide GSP1, and a thermal cycler (GeneAmpR PCR System 2400, PerkinElma) At 94 ° C for 2 seconds, 72. C, 7 cycles of 3 minutes, 94. C, 2 seconds, 68 ° C, 3 minutes 37 cycles, 68 ° C, 4 minutes, 4 ° C. 4Synthetic oligonucleotide GSP1: (SEQ ID NO: 19) Next, this reaction solution was diluted 50-fold with sterile water and used for the second PCR reaction.
  • the second PCR reaction was performed using the first PCR reaction solution, TaKaRa LA PCR kit version 2 (Takara Shuzo Co., Ltd.), the primer AP2, and the synthetic oligonucleotide GSP2. 2400, PerkinElmer), 5 cycles of 94 ° C, 2 seconds, 72 ° C, 3 minutes, 25 cycles of 94 ° C, 2 seconds, 68 ° C, 3 minutes, 68 ° C, 4 minutes, The test was performed under the condition of standing at 4 ° C.
  • the amplified fragment of about 1.1 kbp obtained from the genomic DNA solution digested with Seal was transferred to pT7 Blue T1 Vector (Novagen) using DNA Ligation Kit Version Jon 2 (Takara Shuzo Co., Ltd.).
  • the resulting strain was inserted into E. coli DH5 strain to obtain a transformed strain.
  • Plasmid DNA was extracted from the resulting transformant and reacted using the Dita Minercycle FS Ready Reaction Kit (PerkinElmer) and transferred to the 373A DNASequencer (PerkinElmer). A part of the base sequence of the amplified fragment was determined.
  • the obtained clone has a nucleotide sequence encoding the No. 13 Asp from the No. 1 Met (start codon) of the mouse-derived protein having the amino acid sequence represented by SEQ ID NO: 2 (SEQ ID NO: 7).
  • the synthetic oligonucleotide (SEQ ID NO: 1) used for the cloning of the cDNA encoding the mouse-derived protein of the present invention. : 14) was confirmed to be a part of the actual DNA sequence encoding the mouse-derived protein of the present invention.
  • a mouse TL4 protein cDNA labeled with a lambda FI XRII library (Strata Gene Co.) incorporating 129 SVJ mouse chromosome DNAS au3AI partially digested fragment was used.
  • the lobes were isolated by the plaque hybridization method. First, 1 one 1 Q X 10 pfu (plaaue- forming unit) / phage solution diluted to ml, 0.2% maltose, 1 OmM Mg S0 4 30 ° with the added LB medium C- evening cultured E.
  • the resulting phage particles were transferred onto a membrane, and the membrane was phage-coated on a Petman 3MM paper filter (Whatman International) impregnated with a denaturing solution (1.5 M NaCl, 0.5 M NaOH). After placing for 7 minutes with the marked side up, neutralize The solution (1.5 M NaC K 0.5 M Tris-HCl (pH 7.2), lmM EDTA) was allowed to stand for 3 minutes on a filter paper impregnated with the phage facing up for 3 minutes.
  • a denaturing solution 1.5 M NaCl, 0.5 M NaOH
  • the membrane was washed with 2 XSSC solution (0.3 M NaCl, 0.03 M sodium citrate) After the membrane was air-dried, the phage adhered to the filter paper impregnated with 0.4 M Na ⁇ H. Place for 20 minutes face-up, wash with 5XSSC solution (0.75M NaC1, 75mM sodium citrate) and pack in hybridization pack.ECL gene detection system (Amersham) The prehybridization buffer was added at 5 ⁇ and prehybridization was performed at 42 ° C for 1 hour.
  • the DNA fragment obtained by amplifying the open reading frame (720 bp) of the mouse-derived TL4 protein cDNA by PCR After adding the same amount of the labeling reagent of the CL gene detection system and dal aldehyde, incubate at 37 ° C for 5 minutes and label, add this to the prehybridization pack 10/1 at a time, and add it at 42 ° C. Incubated with C for 1 hour. Thereafter, the membrane was removed from the pack, and washed with a primary washing buffer (6 M urea, 4 g / l SDS, 25 ml / l 20 XSSC) pre-incubated at 42 ° C for 20 minutes.
  • a primary washing buffer (6 M urea, 4 g / l SDS, 25 ml / l 20 XSSC) pre-incubated at 42 ° C for 20 minutes.
  • the plate was washed with the secondary washing buffer (2XS SC) for 5 minutes at room temperature.
  • the membrane was immersed in the detection reagent of the ECL gene detection system for 1 minute, and the membrane was overlaid on an X-ray film and exposed. One hour later, the membrane was taken out and developed, and a positive clone was selected.
  • the clones selected here were subjected to secondary screening in the same manner as above, and finally five candidate clones (# 2, 3, 4, 5, 5, 6) could be obtained. From the results of the PCR reaction, it was found that among these five candidate clones, clones containing the entire region of the gene encoding the mouse-derived TL4 protein were clones # 1 and # 6. Was.
  • the cloned vector pUC19 was digested with the restriction enzyme XbaI, and then subjected to electrophoresis using a 1.0% agarose gel to cut out a DNA fragment corresponding to 2.71 ⁇ . After recovering and purifying using (Qiagen), dephosphorylation of the terminal was carried out using ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ (Takara Shuzo). The DNA fragment from # 6 clone prepared above was ligated to this CI CI-treated pUC19 using DNA Ligation Kit Ver.2 (Takara Shuzo), and introduced into E. coli DH5a.
  • the plasmid DNA into which the desired DNA fragment was inserted was selected and isolated.
  • various synthetic oligo DNAs were 1536
  • Termine overnight cycle sequence A sequence reaction using FS Ready Reaction Kit (Perkin Elmer) was performed using the GeneAmpR PCR System 2400 according to the conditions in the attached document, and the sample was subjected to DNA sequencer 373A (Pakkin Elmer). One company). The obtained nucleotide sequence was confirmed using a gene analysis software Laser Gene (Lasergene, DNASTAR). The results showed that the chromosomal gene encoding the mouse-derived TL4 protein consisted of four exons.
  • the plasmid containing the XbaI DNA fragment derived from the # 6 clone containing the coding region of the mouse-derived TL4 protein obtained as described above was named pTB2011, and introduced into Escherichia coli DH5.
  • the obtained transformant was Escherichia coli DH5 ⁇ TB2011.
  • Reference Example 4 Expression and Western Blot Analysis of Extracellular Region of Human TL4 Protein Using Pichia Yeast as Host
  • PPICZ ⁇ (Invitrogen) was used as a vector for expressing the extracellular region of the human-derived TL4 protein of the present invention in yeast Pichia pastoris.
  • a gene encoding a secretion signal and one factor of the secretory signal of the budding yeast Saccharomyces cerevisiae, which is functional even in Pichia yeast, downstream of the promoter of the alcohol oxidase gene (AOX1) of the yeast, followed by multi-cloning Includes a cloning site, allowing the recombinant protein to be secreted into the medium.
  • AOX1 alcohol oxidase gene
  • a DNA fragment encoding the extracellular region of the human-derived TL4 protein of the present invention is prepared by a PCR method, and the following two primers are synthesized by a DNA synthesizer (01igol000M, Beckman). .
  • This primer has an XbaI recognition sequence, a termination codon (TGA) on its 3 'side, and a sequence complementary to 15 bases encoding the C-terminal 5 amino acids of the extracellular region of human-derived TL4 protein. )
  • the obtained primers were used as 5 O moK 100 ng of plasmid pTB1939 obtained in Reference Example 1, dATP, dCTP, dGTP, and dTTP, respectively, l Ornnol, and 2.5 units of native Pfu DNA polymerase (Stratagene).
  • Solution and 5/1 native Pfu buffer (Stratagene) were prepared at 94 ° C for 1 minute using a thermal cycler (GeneAmpR PCR System 2400, PerkinElmer). Subsequently, PCR was performed under the following conditions: 98 ° C, 20 seconds ⁇ 55, 30 seconds ⁇ 68 ° C, 2 minutes, 1 cycle: 30 cycles, and finally 72 ° C, 5 minutes.
  • the PCR product was recovered from the reaction mixture, digested with EcoRI and XbaI, digested with EcoRI and XbaI and ligated to linearized pPICZ ⁇ A to obtain a circularized plasmid. .
  • the plasmid DNA was again cut at the SacI unique cleavage site at the AOX1 locus, linearized, and then introduced into Pichia pastoris KM71 strain by electroporation.
  • the expression of the recombinant protein was performed in the following procedure. First, a colony of a platinum loop transformant for human-derived TL4 protein expression was transferred to a BMGY medium (1% yeast extract, 2% peptone, 10 OmM potassium phosphate (pH 6.0), 1.34.3 ⁇ 4'yea st nitrogen base with ammonium sulfate without amino acids (Difco (Di fco) Inc.), 4X 10- 5% Piochin was inoculated 1% glycerol) 25 ml, and cultured 3 0 ° C, 20 hours.
  • BMGY medium 1% yeast extract, 2% peptone, 10 OmM potassium phosphate (pH 6.0), 1.34.3 ⁇ 4'yea st nitrogen base with ammonium sulfate without amino acids (Difco (Di fco) Inc.
  • 4X 10- 5% Piochin was inoculated 1% glycerol) 25 ml, and cultured 3 0
  • Western plotting using the main culture supernatant was performed as follows. First, a peptide containing a part of the amino acid sequence of the extracellular region of the human-derived TL4 protein (the 166th to 180th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1) is synthesized. A heron antiserum recognizing was prepared according to a known method. Next, the above culture supernatant 51 is mixed with the sample processing solution (0.25MT l'is-HC1, 2% SDS, 30% glyceroK10 ⁇ -merca toethanol, 0.01% bromophenol blue, pH6.8) 5/1. After treating at 95 ° C for 5 minutes, use SDS-polyacrylamide gel electrophoresis (10-20% gradient gel).
  • the membrane was washed twice with TTBS, and then alkaline phosphatase (AP) -labeled goat anti-pea IgG diluted 1: 3000 with TTBS containing 1.0-0 'gelatin. The mixture was reacted with the antibody at room temperature for 1 hour. The membrane was washed twice with TTBS, and once more with TBS, and then detected using an AP coloring kit (Bio-Rad).
  • AP alkaline phosphatase
  • E. coli DH 1 2 S strain of super script TM rat liver c DNA library (Gibb copy Earl Erusha), 100 Terrific Broth containing ampicillin (12 g / l Bacto-tryptone (Difco) '24 g / 1 Bacto-yeast extract (Difco), 2.3 g / 1 monopotassium phosphate, 12.5 g / 1 diphosphate
  • the cells were harvested, and the DNA was prepared using a chiadienplasmid kit (Qiagen) to prepare a plasmid cDNA library.
  • PCR was carried out in a reaction system using Type II, the following two synthetic oligonucleotides as primer DNA, and TaKaRa LA Taq (Takara Shuzo Co., Ltd.) as DNA polymerase.
  • the DNA fragment was recovered using a DNA ligation kit version 2 (Takara Shuzo Co., Ltd.) into the T-cloth site of pT7Blue T-vector (Novagen) to determine its nucleotide sequence. 'Connected. After introducing the Raige one Chillon liquid into E. coli DH 5 alpha strain, select 2 black ⁇ "down from the co mouth knee groups ampicillin resistant transformant has emerged on ampicillin-containing L beta agar medium, each Plasmid DNA was prepared from 0201536
  • each plasmid DNA was converted into type II, and two types of commercially available primer DNA (PRM-007, PRM-008) (Toyobo Co., Ltd.) and a DNA synthesizer (OligolOOOM, Beckman) Oligo DNA was used as primer DNA, and Thermo-Sequenase TM dye terminator cycle sequencing pre-mix kit (Amersham's) was used. After performing on a System 2400, the sample was analyzed using a DNA sequencer 373A (PerkinElmer).
  • the resulting nucleotide sequence was analyzed with gene analysis software laser Gene (La S ergene, De Nuesuta (DNASTAR) Ltd.).
  • the T clone site was represented by SEQ ID NO: 10, which encodes a rat-derived TL4 protein consisting of 239 amino acids represented by SEQ ID NO: 3. It contained a DNA fragment with a base sequence of 784 base pairs, including an open reading frame consisting of seven base sequences.
  • the rat-derived TL4 protein and the human-derived TL4 protein having the amino acid sequence represented by SEQ ID NO: 1 obtained in Reference Example 1 have 75% homology at the amino acid level, The DNA encoding them had 74% homology at the base level.
  • the rat-derived TL4 protein and the mouse-derived TL4 protein having the amino acid sequence represented by SEQ ID NO: 2 obtained in Reference Example 2 have 96% homology at the amino acid level. And the DNAs encoding them had 94% homology at the base level.
  • the resulting plasmid PTB 2012 carrying DNA encoding the TL4 protein derived from rat was introduced into Escherichia coli DH5 ⁇ to obtain a transformant: Escherichia coli DH5 ⁇ / ⁇ TB2012.
  • Reference Example 6 Production of soluble human TL4 using insect cell expression system
  • the PCR reaction was performed using DNA Thermal Cycler 9600 at 94 ° C for 1 minute, and then using ExTaq DNA polymerase for 10 seconds at 98 ° C, 5 seconds at 55 ° C, and 1 minute at 72 ° C. The cycle was repeated 25 times.
  • the amplified fragment thus obtained was treated with restriction enzymes EcoRI and Xbal.
  • the pCM-FLAG plasmid is also treated with the restriction enzymes EcoRI and XbaI, and DNA encoding the signal sequence of prebub trypsin and FLAG protein added as a tag for the purpose of easy purification and detection, respectively. Fragments were obtained.
  • the amplified DNA fragment of soluble TL4 that had been treated with a restriction enzyme was ligated to the 3 ′ end of the DNA fragment encoding the preprotrypsin-FLAG protein.
  • the obtained DNA fragment encoding the preprotrypsin-FLAG protein-soluble human TL4 protein was treated with the restriction enzymes SacI and XbaI, and the insect cells were also treated with the restriction enzymes SacI and XbaI. It was inserted into pFAST Bacl (GI BCO BRL Lifetech).
  • the resulting TL4 expression plasmid pFAST Bacl / shTL4 was expected to be secreted into the cell culture supernatant using prebub trypsin in insect cells and produced as a FLAG-tagged fusion protein in insect cells. .
  • Bac-to-Bac Baculovirus Expression Systems (GI BCO BRL Lifetech) was used, and the experimental method was in accordance with the attached protocol. That is, the recombinant plasmid pFAST Bacl / shTL4 into which the obtained DNA encoding the human TL4 protein was inserted was introduced into the attached Escherichia coli DH10Bac to obtain a transformed bacterium. Collected. The obtained recombinant bacmid was transduced into Sf9 insect cells using the attached self-ectin reagent to obtain a recombinant paculovirus.
  • GI BCO BRL Lifetech Bac-to-Bac Baculovirus Expression Systems
  • PCR was performed using two primers, Primer 1 (SEQ ID NO: 28) and Primer 1 (SEQ ID NO: 29).
  • the composition of the reaction solution used in the reaction was 33.5 ng of the above cDNA as type III, 1/50 amount of Advantage 2 Polymerase Mix (CLONTECH), Primer 1 (SEQ ID NO: 28) and Primer 1 (SEQ ID NO: 2). : 29), 20/1 dNTPs 2.5 mM each, and 1/10 of the buffer attached to the enzyme were added to make a total volume of 501.
  • the reaction product after the PCR reaction has two products of 723 bases and 615 bases.
  • the reaction product of 615 base pairs is recovered from the gel and purified according to the method of QIAquick Gel Extraction Kit (QIAGEN).
  • the purified product was subcloned into a plasmid vector pCR2.1-T0P0 vector according to the procedure of a TA cloning kit (Invitrogen).
  • Synthetic oligonucleotides (5, -GTAGAATTCGGCCAACCCAGCAGCACATCTTAC-3 '(5, -GTAGAATTCGGCCAACCCATC)
  • a PCR reaction was performed using primers of SEQ ID NO: 33)) and a synthetic oligonucleotide (5'-AAATCTAGATATTGCTGGGTTTGAGGTGAGTCC-3 '(SEQ ID NO: 34)) to which a restriction enzyme site of Xbal was added at the 3' end, and From the alanine at residue 90 corresponding to the extracellular region of TL4, an amplified DNA fragment of soluble TL4 encoding valine at residue 239 was obtained.
  • the PCR reaction was performed using a DNA Thermal Cycler 9600 at 94 ° C for 1 minute, and then using ExTa qDNA polymerase at 98 ° C for 10 seconds and 6 (TC). A cycle of 1.5 minutes at 72 ° C. for 5 seconds was repeated 25 times.
  • the amplified fragment thus obtained was treated with restriction enzymes EcoRI and XbaI.
  • the pCMV-FLAG plasmid is treated with the same restriction enzymes EcoRL and baI, and the DNA fragment encoding the FLAG protein added for the purpose of facilitating purification and detection as a signal sequence for tryptic pre-mouthlet and a tag.
  • TL4 expression plasmid pFAST Bacl / smTL4
  • pFAST Bacl / smTL4 is secreted into the cell culture supernatant in insect cells using preprotolibsin, and is produced as a fusion protein with FLAG group added.
  • Bac-to-Bac Baculovirus Expression Systems (GI BCO BRL Lifetech) was used, and the experimental method was described in the attached protocol. That is, the obtained recombinant plasmid pFAST Bacl / smTL4 into which the DNA encoding the mouse TL4 protein was inserted was introduced into the attached Escherichia coli DH10Bac to obtain a transformed bacterium. Collected. The obtained recombinant bacmid was transformed into Sf9 insect cells using the attached self-ectin reagent to obtain a recombinant baculovirus.
  • GI BCO BRL Lifetech Bac-to-Bac Baculovirus Expression Systems
  • Example 1 Expression analysis of one molecule of the TNF receptor family in RD cells
  • RD cells were purchased from ATCC (ATCC No. CCL-136). The cells were cultured in DMEM (GIBCOBRL) containing 10% fetal bovine serum (FBS) and sodium pyruvate at a final concentration of ImM.
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • tRNA Total RNA
  • RD cells confluent in a T75 flask are collected using trypsin-EDTA, washed with PBS (-), suspended in 600 1 RLT buffer (containing 1% 3 mercaptoethanol), and After homogenization using G $ 21, homogenization was performed again using a QIAshredder column (QIAGEN).
  • QIAshredder column QIAGEN
  • the reaction composition for this reaction was 25 tRNA, 5.71 attached buffer, and Dnasel added to make a total volume of 56.71.
  • the purified RNA was subjected to a reverse transcription (RT) reaction according to the protocol of the TaqMan Gold RT-PCR Kit (PE Applied Biosys / ⁇ ).
  • the reaction composition in this reaction was lg tRNA, 5 lOxTadMan RT Buffer.
  • the expression level of each receptor in RD cells was determined by the quantitative PCR method (TadMan method) using real-time monitoring.
  • the TaqMan method is based on the principle of detecting and quantifying a specific PCR strand amplified by PCR with the SDS7700 in real time based on the fluorescence intensity of a fluorescent probe called Tad Man probe.
  • TaQMan probes and primers for each receptor were designed and synthesized using Primer Express (PE Applied Software). The sequences are described below.
  • TNF receptor I TNF receptor I
  • TNF receptor II (TNFRI I)
  • reaction composition in the TaQMan PCR reaction use the previously prepared cMA as type II, and add 2x TaMan Universal PCR Master Mix (PE Applied Biosystems) 12.5 K 200 nM TaaMan probe and TaQMan primer so that each becomes ⁇ . Liquid volume.
  • the PCR reaction was performed at 5 (TC2 min, 95 ° C / 10 min, 95 ° C
  • the PCR was performed 40 times, and at the same time as the reaction was completed, quantitative automated PCR analysis was performed.
  • TR2 has the lowest expression of 40 copies / ng total RNA, and if this expression is 1, TNFRI is 215 times, LT / 3R is 125 times, and TNFRII is 45 times expressed.1 .
  • Example 2 Effect of TNF family ligand molecule on cell proliferation and cell morphology of RD cells
  • TL4 The effect of TL4 on cell proliferation of RD cells was performed using a kit of Cell Proliferation ELISA JrdU (color imetric) (Roche). That is, RD cells (2500 cells / well) and TL4 (hTL4, lot99c, insect cells as host), purified as secreted protein by FLAG column: (protein consisting of amino acid sequence represented by SEQ ID NO: 31) , TNFct, TNF / 3, and LT ⁇ 1 / 32 (all from R & D) were added to 96-well plates at different concentrations to obtain a total IOOI volume, and cultured for 4 days.
  • FLAG column protein consisting of amino acid sequence represented by SEQ ID NO: 31
  • TNFct TNF / 3
  • LT ⁇ 1 / 32 all from R & D
  • TL4 As a result, 4 and 6 days after the addition of TL4 (hTW, lotc), the amount of BrdU incorporation was clearly suppressed and the total number of cells was reduced to about one-third compared to the non-supplemented group. Compared with the cell number, it was found that the increase was about 25-fold in the TL4 (liTL4, lotc) -added group. However, microscopic observation revealed that the addition of TL4 (hTL4, lotc) clearly reduced the cell density. It had many long-growing cells, and its morphology had changed significantly.
  • RD cells were seeded on a 12-well plate at 8 ⁇ 10 4 cells / well, cultured overnight in a head medium containing 10% FBS, and then the medium was replaced.
  • FuGENE6 Transfection Reagent (Roche) 98.5 1 and 0 ⁇ -MEMI medium (GIBC0BRL) 1.5 ⁇ 1 were mixed, and a cis element was added upstream of the reporter gene (SEAP).
  • SEAP reporter gene
  • 0.5 / xg of the vector-plasmid solution into which was incorporated was mixed and left at room temperature for 15 minutes.
  • the RD cells were dropped on the plated plate / well, stirred well, and allowed to stand overnight in the incubation to perform transfection. Thereafter, the supernatant was removed, washed twice with DMEM medium (without serum), and replaced with the same culture medium.
  • TL4 hTL4, lotc
  • TNFa, TNFj8, ⁇ ⁇ ⁇ 2, TNF / 3 + LT ⁇ 1 / 32 protein solution were prepared using DMEM medium (without serum). Reagents were added to each well to a final concentration of 50 ng / nil. At 3, 4, 5, 6, and 7 hours after the addition of the reagent, 40 liters of the supernatant were collected and immediately stored at -20 ° C.
  • SEAP activity was measured using the Great EscAPe Chemiluminescence Detection Kit (CLONTECH). The method followed the kit protocol, and detection was performed by chemiluminescence assay.
  • TL4 hTL4, lotc
  • each ligand molecule of TNF family activate NF-KB.
  • Activation of NF- ⁇ B was detected from 3 hours to at least 7 hours after the addition of the reagent, and its intensity was strongest in TNFR-mediated signals such as TNF and TNF / 3, followed by ⁇ : 1
  • the signal from / 32 via the / 3 receptor), this was about 1/3 of TNFR.
  • the signal from TL4 (hTL4, lotc) via the TR2 and LT
  • Example 5 Effect of TNF Family Monoligand Molecules on Chemokine Production of RD Cells
  • RD cells were treated with 5X10 and TL4 (hT lotc) or other TNF family monoligands (TN Fa, TNF i3, LTal 32, TNF / 3 and LTo; 132)
  • the protein solution was placed in a 12-well plate at 10 or 50 ng / ml, respectively (total 1 ml reaction system), and cultured at 37 ° C.
  • 200 l of the supernatant was collected, and immediately stored at -20 ° C.
  • the amount of IL-8 and RANTES in the supernatant was measured using an ELISA system.
  • the method followed the protocol of Quantikine human IL-8 Colorimetric Sandwich ELISA and Quantikine human RANTES Colorimetric Sandwich ELISA (both from R & D).
  • TL4 hTL4, lotc
  • TL4 (hTL4, lotc) and LTal32 produced about 2000 pg / nil.
  • the simultaneous addition of TNF, TNF / 3 or TNF 3 and LTo; 1 j32 resulted in about 7000 pg / ml.
  • RANTES was slightly higher with the simultaneous addition of TL4 (hTL4, lotc) or TNF / 3 and LTcd / 32, and all cytokines, unlike IL-8, increased their production in a culture time-dependent manner .
  • TL4 hTL4, lotc
  • cytokines unlike IL-8
  • Fig. 6 Fig. 6
  • Anti-Mouse IgG (H + L) AP Conjugate attached to ProtoBlotll APS system was diluted 5000-fold with TBST, and a secondary antibody reaction was performed at room temperature for 1 hour. After washing twice in TBST and once in TBS, the membrane was immersed in Westren Blue Stabilized Substrate for Alkaline Phosphatase attached to the system, and a staining reaction was performed.
  • TNF family ligand molecule TNF family ligand molecule
  • RD cells 6 ⁇ 10 3 I wells
  • TL4 hTL4, lotc
  • TGF / 31 and TGF 33 protein solutions each at a final concentration of 50 ng Lab-Tek Chamber Slide (Swell) (NUNC) was added to each well to make a total volume of 0.4 ml and cultured for 6 days.
  • NUNC Lab-Tek Chamber Slide
  • the expression of muscle-specific genes in RD cells was confirmed by RT-PCR.
  • the primer information for each gene is shown below.
  • forward primer 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3 '(SEQ ID NO: 49 reverse primer: 5, -CTAGAAGCATTTGCGGTGGACGATGGA GGG-3' (SEQ ID NO: 50)
  • forward primer 5'-CCGTGGGCGTGTAAGGTGTG-3 '(SEQ ID NO: 51) reverse primer 5, -ACGATGGAGGTGAGGGAGTGC-3 '(SEQ ID NO: 52)
  • reaction composition in the RT-PCR reaction one-fifth of the cDNA prepared in Example 1 was already used as type I, Advantage 25 Polymerase Mix (CL0NTECH), one-fifth, foreprimer and reverse primer. 1 M each, 200 M dNTPs, and a buffer attached to the enzyme were added to make a total, and the PCR reaction was performed under the following conditions depending on the primer. ⁇ -actin repeats a cycle of 94 ° C for 3 minutes, 94 ° C for 15 seconds, 60 ° C for 15 seconds, and 68 for 45 seconds 35 times, and finally an extension reaction of 68 ° C for 5 minutes Was.
  • 3-actin repeats a cycle of 94 ° C for 15 minutes, 94 ° C for 15 seconds, 60 ° C for 15 seconds, 68 ° C for 45 seconds 18 times, and finally an extension reaction of 68 ° C for 5 minutes Was done.
  • Myogenin repeats a cycle of 94 ° C for 3 minutes, 94 ° C for 15 seconds, 61.9 ° C for 15 seconds, 68 ° C for 45 seconds 28 times, and finally performs an extension reaction at 68 ° C for 5 minutes Was.
  • Id-1 repeats a cycle of 94 ° C for 15 minutes, 94 ° C for 15 seconds, 62.5 ° C for 15 seconds, 68 ° C for 45 seconds 25 times, and finally an extension reaction of 68 ° C for 5 minutes was done. After completion of the PCR reaction, 3/1 was analyzed by 1.5% agarose gel electrophoresis.
  • TNFa As mentioned above, TNF / 3 does not affect BrdU uptake or morphological changes in RD cells, but smooth muscle-specific a-actin was hardly expressed as in untreated RD cells.
  • TGF / 3 which has an inhibitory effect on proliferation of RD cells
  • TPA which has the ability to induce differentiation
  • the protein of the present invention is used for cell transformation.
  • (fetal) rhabdomyosarcoma (focal rhabdomyosarcoma, primary rhabdomyosarcoma, etc.), leiomyosarcoma, muscular dystrophy (myotonic dystrophy) or uterus
  • fibroids etc.
  • (embryonic) rhabdomyosarcoma (focal rhabdomyosarcoma, rhabdomyosarcoma primary liver sarcoma, etc.)
  • leiomyosarcoma muscular dystrophy (Myotonic dystrophy) or uterine fibroids.

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Abstract

L'invention concerne des médicaments possédant un effet d'induction de différentiation sur une tumeur, notamment des agents de changement plasmique contenant des protéines, ou des sels de ces protéines, dont la séquence en acides aminés est la même ou sensiblement la même que celles représentées par les SEQ ID N°1, SEQ ID N°2, SEQ ID N°3 ou SEQ ID N°31. Ces agents sont utiles pour prévenir ou traiter le rhabdosarcome, le liomyosarcome, la dystrophie musculaire et le myome de l'utérus.
PCT/JP2002/001536 2001-02-23 2002-02-21 Agents pour changement plasmique WO2002066049A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1364653A4 (fr) * 2001-02-23 2005-01-26 Takeda Pharmaceutical Inhibiteurs de caspase 3

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034911A1 (fr) * 1996-03-22 1997-09-25 Human Genome Sciences, Inc. Molecule ii inductrice d'apoptose
WO1998003648A1 (fr) * 1996-07-19 1998-01-29 Takeda Chemical Industries, Ltd. Proteine similaire au ligand fas, son procede de production et d'utilisation
WO1999035262A2 (fr) * 1998-01-07 1999-07-15 Human Genome Sciences, Inc. Molecule ii induisant l'apoptose
WO1999042584A1 (fr) * 1998-02-20 1999-08-26 Human Genome Sciences, Inc. Molecule ii induisant l'apoptose et procedes d'utilisation
WO2000053223A1 (fr) * 1999-03-11 2000-09-14 Human Genome Sciences, Inc. Molecule ii induisant l'apoptose et techniques d'utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034911A1 (fr) * 1996-03-22 1997-09-25 Human Genome Sciences, Inc. Molecule ii inductrice d'apoptose
WO1998003648A1 (fr) * 1996-07-19 1998-01-29 Takeda Chemical Industries, Ltd. Proteine similaire au ligand fas, son procede de production et d'utilisation
WO1999035262A2 (fr) * 1998-01-07 1999-07-15 Human Genome Sciences, Inc. Molecule ii induisant l'apoptose
WO1999042584A1 (fr) * 1998-02-20 1999-08-26 Human Genome Sciences, Inc. Molecule ii induisant l'apoptose et procedes d'utilisation
WO2000053223A1 (fr) * 1999-03-11 2000-09-14 Human Genome Sciences, Inc. Molecule ii induisant l'apoptose et techniques d'utilisation

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1364653A4 (fr) * 2001-02-23 2005-01-26 Takeda Pharmaceutical Inhibiteurs de caspase 3

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