WO1997012969A2 - Proteine apparentee a fmr1 - Google Patents

Proteine apparentee a fmr1 Download PDF

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Publication number
WO1997012969A2
WO1997012969A2 PCT/DE1996/001787 DE9601787W WO9712969A2 WO 1997012969 A2 WO1997012969 A2 WO 1997012969A2 DE 9601787 W DE9601787 W DE 9601787W WO 9712969 A2 WO9712969 A2 WO 9712969A2
Authority
WO
WIPO (PCT)
Prior art keywords
dna
protein
fxr2
immunization
fmr1
Prior art date
Application number
PCT/DE1996/001787
Other languages
German (de)
English (en)
Other versions
WO1997012969A3 (fr
Inventor
Annemarie Poustka
Johannes Coy
Original Assignee
Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts filed Critical Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority to EP96945134A priority Critical patent/EP0851923A2/fr
Priority to JP9513875A priority patent/JP2000500962A/ja
Publication of WO1997012969A2 publication Critical patent/WO1997012969A2/fr
Publication of WO1997012969A3 publication Critical patent/WO1997012969A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an FMR 1 -related protein, such a DNA coding and a method for producing such.
  • the invention further relates to the use of the DNA and the protein and antibodies directed against the protein.
  • FMR1 is an RNA binding protein that e.g. mRNA from FMR1 and mRNA from fetal brain binds. FMR1 is often found in testicles, fetal ovaries and brain neurons. Defects in FMR1 or its deficiency can be found in patients with mental retardation, especially those with fragile X syndrome. This mental illness, which among other things Expressed in hyperactivity, is heritable and occurs with a frequency of around 1: 1,500 in men and 1: 2,500 in women, although its severity is usually higher in men than in women.
  • the present invention is therefore based on the object of providing a means with which mental retardations, in particular the fragile X syndrome, can be better examined and, if appropriate, treated.
  • the present invention thus relates to an FMR1-related protein which comprises the amino acid sequence of FIG. 1 or an amino acid sequence which differs from this by one or more amino acids.
  • FXR2 Such a protein is hereinafter referred to as "FXR2".
  • the present invention is based on the knowledge of the applicant that in animals, especially mammals, very particularly humans, a protein exists which has homologies to FMR1, possibly an FMR1 activity, but which differs from FMR1 at the DNA level by hybridization under conventional methods Conditions differs.
  • a protein comprises the amino acid sequence of FIG. 1 or an amino acid sequence which differs therefrom by one or more amino acids.
  • Another object of the present invention is a nucleic acid coding for FXR2.
  • This can be an RNA or a DNA.
  • the latter can e.g. be a genomic DNA or a cDNA.
  • a DNA is preferred which comprises the following:
  • hybridizing DNA indicates a DNA which hybridizes with a DNA of (a) under customary conditions, in particular at 20 ° C. below the melting point of the DNA.
  • a DNA according to the invention is described below in the form of a cDNA. This is an example of every DNA covered by the present invention.
  • a human cDNA library for example one from a fetal brain, such as the cDNA library ⁇ -Zap, Stratagene, cat. no. 936206, or one from a fetal heart, such as the HL 3018b cDNA library, Clontech.
  • Clones of such cDNA libraries are fixed on a filter membrane and hybridized with a labeled FMR1 cDNA sample with reduced stringency, in particular at 27 ° C. below the melting point of the DNA. Positive clones are sequenced, thereby identifying those that comprise DNA encoding FXR2.
  • a cDNA according to the invention can be present in a vector or expression vector.
  • examples of such are known to the person skilled in the art.
  • these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred.
  • yeast e.g. to call pY100 and Ycpadl
  • animal cells e.g. pKCR, pEFBOS, cDM8 and pCEV4 must be specified.
  • the baculovirus expression vector pAcSGHisNT-A is particularly suitable for expression in insect cells.
  • suitable cells in order to express a cDNA according to the invention which is present in an expression vector.
  • suitable cells include the E. coli strains HB101, DH1, x1776, JM101, JM 109, BL21 and SG 1 3009, the latter being preferred, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa and the insect cells sf9.
  • a cDNA according to the invention has to be inserted into an expression vector. He is also aware that this DNA can be inserted in conjunction with a DNA coding for another protein or peptide, so that the cDNA according to the invention can be expressed in the form of a fusion protein.
  • Another object of the present invention is an antibody directed against an above protein or fusion protein.
  • Such an antibody can be produced by conventional methods. It can be polyclonal or monoclonal. For its production, it is favorable to immunize animals, in particular rabbits or chickens for a polyclonal and mice for a monoclonal antibody, with an above (fusion) protein or fragments thereof. Further "boosters" of the animals can be carried out with the same (fusion) protein or fragments thereof. The polyclonal antibody can then be obtained from the serum or egg yolk of the animals. For the monoclonal antibody, animal spleen cells are fused with myeloma cells.
  • the present invention makes it possible to better investigate mental retardations, in particular the fragile X syndrome.
  • FXR2 can be detected in body fluids of people. A relationship between FXR2 and the development and development of the above diseases can be established.
  • An FXR2 according to the invention can also be used to detect an autoantibody directed against this protein.
  • interactions with other proteins, in particular those associated with mental retardation can be detected with an FXR2 of the present invention, as a result of which the functions of these proteins can be elucidated and, if appropriate, therapeutic measures can be taken for or against the proteins .
  • the above detection can be carried out by conventional methods, in particular a Western blot, an ELISA, immunoprecipitation or by immunofluorescence.
  • the expression of the gene coding for FXR2 can be detected using a nucleic acid according to the invention, in particular a DNA and primers derived therefrom. This detection can be done in the usual way, especially in a Southern blot. 5
  • the present invention is suitable for taking measures for and against the presence of FXR2 in people.
  • FXR2 can be inhibited in people with an antibody according to the invention.
  • an FXR2 according to the invention in particular after coupling to a protein not regarded as foreign by the body, e.g. Transferrin, or BSA, increases the amount of FXR2 in people.
  • a nucleic acid according to the invention in particular a DNA, which is under the control of a specific tissue, e.g. Brain, heart, inducible promoter is put and after their expression leads to the provision of FXR2 in these tissues.
  • nucleic acid according to the invention in particular a DNA, can also be used to inhibit FXR2.
  • nucleic acid e.g. used as the basis for the creation of anti-sense oligonucleotides for expression inhibition of the gene coding for FXR2.
  • the present invention thus represents a major contribution to the diagnostic and therapeutic detection of mental retardation, in particular the fragile X syndrome.
  • FIG. 1 shows the base sequence and the amino acid sequence derived therefrom of a protein FXR2 according to the invention.
  • the DNA of FIG. 1 was used as a template to produce a protein FXR2 according to the invention.
  • a PCR procedure was carried out. The following was used as a primer pair: 5'-CAGAGATCTATGCTGGCAATTGGGACTCACGGTGC-3 ' and 5'-GGGAAGCTTTTAAGATTTATCCACAATCTCCTGGA-3 '.
  • the PCR approach and the PCR conditions were as follows:
  • the amplified DNA was cleaved in each case with Bgl II and Hind III and inserted into the expression vector pQE-8 (Diagen) cleaved with Bam HI and Hind III.
  • the expression plasmid pQ / FXR2 was obtained.
  • pQ / FXR2 became a trans formation by E. coli SG 13009 (see. Gottesman, S. et al., J. Bacteriol. 148, (1 981), 265-273).
  • the bacteria were cultivated in an LB medium with 100 // g / ml ampicillin and 25 ⁇ g / ml kanamycin and induced for 4 h with 60 // M isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the bacteria were lysed by adding 6 M guanidine hydrochloride, and then chromatography (Ni-NTA resin) was carried out with the lysate in the presence of 8 M urea in accordance with the manufacturer's (Diagen) instructions for the chromatography material.
  • the bound fusion protein was eluted in a pH 3.5 buffer.
  • the fusion protein was subjected to 18% SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue (cf. Thomas, JO and Kornberg, RD, J. Mol. Biol. 149 (1975), 709-733).
  • a fusion protein according to the invention can be produced in a highly pure form.
  • Example 2 Production and detection of an antibody according to the invention
  • a fusion protein according to the invention from Example 1 was subjected to 1 8% SDS-polyacrylamide gel electrophoresis. After staining the gel with 4 M sodium acetate, an approximately 8.8 kD band was cut out of the gel and incubated in phosphate-buffered saline. Pieces of gel were sedimented before the protein concentration of the supernatant was determined by SDS-polyacrylamide gel electrophoresis followed by a Coomassie blue stain. Animals were immunized with the gel-purified fusion protein as follows:
  • the rabbit's serum was tested in the immunoblot.
  • a fusion protein according to the invention from Example 1 was subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10, (1 984), 203-209 ).
  • Western blot analysis was performed as in Bock, C.-T. et al., Virus Genes 8, (1 994), 21 5-229.
  • the nitrocellulose filter was incubated for one hour at 37 ° C. with a first antibody. This antibody was rabbit serum (1: 10,000 in PBS).
  • the nitrocellulose filter was incubated with a second antibody.
  • This antibody was a goat anti-rabbit IgG (Dianova) monoclonal antibody coupled to alkaline phosphatase (1: 5000) in PBS. After 30 minutes of incubation at 37 ° C, several washing steps with PBS followed and then the alkaline phosphatase detection reaction with developer solution (36 ⁇ M 5 'bromo-4-chloro-3-indolyl phosphate, 400 / yM nitroblue tetrazolium, 100mM Tris -HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl 2 ) at room temperature until bands were visible.
  • developer solution 36 ⁇ M 5 'bromo-4-chloro-3-indolyl phosphate, 400 / yM nitroblue tetrazolium, 100mM Tris -HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl
  • Antibodies were extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention have been detected.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne une protéine apparentée à FMR1, un ADN de codage de cette protéine, un procédé de préparation de cette protéine, l'utilisation de l'ADN et de la protéine et les anticorps dirigés contre cette protéine.
PCT/DE1996/001787 1995-09-19 1996-09-19 Proteine apparentee a fmr1 WO1997012969A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP96945134A EP0851923A2 (fr) 1995-09-19 1996-09-19 Proteine apparentee a fmr1
JP9513875A JP2000500962A (ja) 1995-09-19 1996-09-19 Ｆｍｒ１関連蛋白質

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19534763.3 1995-09-19
DE1995134763 DE19534763C1 (de) 1995-09-19 1995-09-19 FMR1-verwandtes Protein

Publications (2)

Publication Number Publication Date
WO1997012969A2 true WO1997012969A2 (fr) 1997-04-10
WO1997012969A3 WO1997012969A3 (fr) 1997-07-17

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ID=7772586

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE1996/001787 WO1997012969A2 (fr) 1995-09-19 1996-09-19 Proteine apparentee a fmr1

Country Status (4)

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EP (1) EP0851923A2 (fr)
JP (1) JP2000500962A (fr)
DE (1) DE19534763C1 (fr)
WO (1) WO1997012969A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20000072201A (ko) * 2000-08-17 2000-12-05 김희태 여러 종류의 디엔에이 프로브들을 사용한 취약-엑스증후군의 진단 방법

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038467A1 (fr) * 1995-05-31 1996-12-05 The Trustees Of The University Of Pennsylvania Proteines liees a l'x fragile, leurs compositions et procedes de production et d'utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038467A1 (fr) * 1995-05-31 1996-12-05 The Trustees Of The University Of Pennsylvania Proteines liees a l'x fragile, leurs compositions et procedes de production et d'utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMBO JOURNAL, Bd. 14, Nr. 11, 1.Juni 1995, EYNSHAM, OXFORD GB, Seiten 2401-2408, XP000673943 SIOMI, M.C. ET AL.: "FRX1, an autosomal homolog of the fragile X mental retardation gene" *
EMBO JOURNAL, Bd. 14, Nr. 21, 1.November 1995, EYNSHAM, OXFORD GB, Seiten 5358-5366, XP000673942 ZHANG, Y. ET AL.: "The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2" *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20000072201A (ko) * 2000-08-17 2000-12-05 김희태 여러 종류의 디엔에이 프로브들을 사용한 취약-엑스증후군의 진단 방법

Also Published As

Publication number Publication date
DE19534763C1 (de) 1997-05-15
JP2000500962A (ja) 2000-02-02
EP0851923A2 (fr) 1998-07-08
WO1997012969A3 (fr) 1997-07-17

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