WO1997012969A2 - Fmr1-related protein - Google Patents
Fmr1-related protein Download PDFInfo
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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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
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- 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
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- 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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Abstract
Description
FMR 1 -verwandtes ProteinFMR 1 -related protein
Beschreibungdescription
Die vorliegende Erfindung betrifft ein FMR 1 -verwandtes Protein, eine ein solches codierende DNA und ein Verfahren zur Herstellung eines solchen. Ferner betrifft die Erfindung die Verwendung der DNA und des Proteins sowie gegen das Protein gerichtete Antikörper.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 ist ein RNA-bindendes Protein, das z.B. mRNA von FMR1 und mRNA von fötalem Gehirn bindet. FMR1 findet sich häufig in Hoden, fötalen Ovarien und Gehirnneuronen. Defekte in FMR1 bzw. seine Defizienz finden sich bei Patienten mit mentaler Retardierung, insbesondere bei solchen des fragilen X-Syndroms. Diese Geisteskrankheit, die sich u.a. in Hyperaktivität äußert, ist vererbbar und tritt mit einer Häufigkeit von etwa 1 : 1 500 bei Männern und 1 :2500 bei Frauen auf, wobei ihr Schweregrad in der Regel bei Männern höher ist als bei Frauen.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.
Die genaue Wirkungsweise, wie Defekte in FMR1 bzw. seine Defizienz eine menta- le Retardierung, insbesondere das fragile X-Syndrom, bedingen, ist allerdings nicht bekannt. Dies wäre aber notwendig, um bei mentaler Retardierung diagnostisch und therapeutisch besser eingreifen zu können.However, the exact mode of action, such as defects in FMR1 or its deficiency, require mental retardation, especially the fragile X syndrome, is not known. However, this would be necessary in order to be able to intervene diagnostically and therapeutically better with mental retardation.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzu- stellen, mit dem mentale Retardierungen, insbesondere das fragile X-Syndrom, besser untersucht und gegebenenfalls therapiert werden kann.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.
Erfindungsgemäß wird dies durch die Gegenstände in den Patentansprüchen erreicht. Gegenstand der vorliegenden Erfindung ist somit ein FMR1 -verwandtes Protein, das die Aminosäuresequenz von Fig. 1 oder eine hiervon durch eine oder mehrere Aminosäuren unterschiedliche Aminosäuresequenz umfaßt. Ein solches Protein wird nachstehend mit "FXR2" bezeichnet.According to the invention, this is achieved by the subject matter in the claims. 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. Such a protein is hereinafter referred to as "FXR2".
Die vorliegende Erfindung beruht auf der Erkenntnis des Anmelders, daß in Tieren, besonders Säugetieren, ganz besonders dem Menschen, ein Protein existiert, das Homologien zu FMR1 , gegebenenfalls eine FMR1 Aktivität aufweist, sich aber von FMR1 auf dem DNA-Level durch Hybridisierung unter üblichen Bedingungen unterscheidet. Ein solches Protein umfaßt die Aminosäuresequenz von Fig. 1 oder eine hiervon durch eine oder mehrere Aminosäuren unterschiedliche Aminosäurese¬ quenz.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. Such a protein comprises the amino acid sequence of FIG. 1 or an amino acid sequence which differs therefrom by one or more amino acids.
Ein weiterer Gegenstand der vorliegenden Erfindung ist eine für FXR2 kodierende Nukleinsäure. Dies kann eine RNA oder eine DNA sein. Letztere kann z.B. eine genomische DNA oder eine cDNA sein. Bevorzugt ist eine DNA, die folgendes umfaßt: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:
(a) die DNA von Fig. 1 oder eine hiervon durch ein oder mehrere Basenpaare unterschiedliche DNA,(a) the DNA of FIG. 1 or a DNA different therefrom by one or more base pairs,
(b) eine mit der DNA von (a) hybridisierende DNA, oder(b) a DNA hybridizing with the DNA of (a), or
(c) eine mit der DNA von (a) oder (b) über den degenerierten genetischen Code verwandte DNA.(c) a DNA related to the DNA of (a) or (b) via the degenerate genetic code.
Der Ausdruck "hybridisierende DNA" weist auf eine DNA hin, die unter üblichen Bedingungen, insbesondere bei 20°C unter dem Schmelzpunkt der DNA, mit einer DNA von (a) hybridisiert.The term "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.
Nachstehend wird eine erfindungsgemäße DNA in Form einer cDNA beschrieben. Diese steht beispielhaft für jede unter die vorliegende Erfindung fallende 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.
Zur Herstellung einer erfindungsgemäßen cDNA ist es günstig, von einer menschlichen cDNA-Bibliothek, z.B. einer von fötalem Gehirn, wie der cDNA- Bibliothek Λ-Zap, Stratagene, Kat. -Nr. 936206, oder einer von fötalem Herz, wie der cDNA-Bibliothek HL 3018b, Clontech, auszugehen. Klone solcher cDNA- Bibliotheken werden auf einer Filtermembran fixiert und mit einer markierten FMR1 cDNA-Probe bei reduzierter Stringenz, insbesondere bei 27 °C unter dem Schmelzpunkt der DNA, hybridisiert. Positive Klone werden sequenziert, wodurch jene identifiziert werden, die eine für FXR2 kodierende DNA umfassen.To produce a cDNA according to the invention, it is advantageous to use one human cDNA library, for example one from a fetal brain, such as the cDNA library Λ-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.
Eine erfindungsgemäße cDNA kann in einem Vektor bzw. Expressionsvektor vorliegen. Beispiele solcher sind dem Fachmann bekannt. Im Falle eines Expres¬ sionsvektors für E. coli sind dies z.B. pGEMEX, pUC-Derivate, pGEX-2T, pET3b und pQE-8, wobei letzterer bevorzugt ist. Für die Expression in Hefe sind z.B. pY100 und Ycpadl zu nennen, während für die Expression in tierischen Zellen z.B. pKCR, pEFBOS, cDM8 und pCEV4, anzugeben sind. Für die Expression in Insek- tenzellen eignet sich besonders der Baculovirus-ExpressionsvektorpAcSGHisNT-A.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. In the case of an expression vector for E. coli, these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred. For expression in yeast e.g. to call pY100 and Ycpadl, while for expression in 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.
Der Fachmann kennt geeignete Zellen, um eine, erfindungsgemäße, in einem Expressionsvektor vorliegende cDNA zu exprimieren. Beispiele solcher Zellen umfassen die E.coli-Stämme HB101 , DH1 , x1776, JM101 , JM 109, BL21 und SG 1 3009, wobei letzterer bevorzugt ist, den Hefe-Stamm Saccharomyces cerevisiae und die tierischen Zellen L, 3T3, FM3A, CHO, COS, Vero und HeLa sowie die Insektenzellen sf9.The person skilled in the art knows suitable cells in order to express a cDNA according to the invention which is present in an expression vector. Examples of such 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.
Der Fachmann weiß, in welcher Weise eine erfindungsgemäße cDNA in einen Expressionsvektor inseriert werden muß. Ihm ist auch bekannt, daß diese DNA in Verbindung mit einer für ein anderes Protein bzw. Peptid kodierenden DNA inse¬ riert werden kann, so daß die erfindungsgemäße cDNA in Form eines Fusions¬ proteins exprimiert werden kann.The person skilled in the art knows how 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.
Desweiteren kennt der Fachmann Bedingungen, transformierte bzw. transfizierte Zellen zu kultivieren. Auch sind ihm Verfahren bekannt, das durch die erfindungs¬ gemäße cDNA exprimierte Protein zu isolieren und zu reinigen. Ein solches Protein, das auch ein Fusionsprotein sein kann, ist somit ebenfalls Gegenstand der vor¬ liegenden Erfindung.Furthermore, the person skilled in the art knows the conditions for culturing transformed or transfected cells. He is also aware of processes for isolating and purifying the protein expressed by the cDNA according to the invention. Such a protein that can also be a fusion protein is thus also the subject of the present invention.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein gegen ein vorstehendes Protein bzw. Fusionsprotein gerichteter Antikörper. Ein solcher Antikörper kann durch übliche Verfahren hergestellt werden. Er kann polyklonal bzw. monoklonal sein. Zu seiner Herstellung ist es günstig, Tiere, insbesondere Kaninchen oder Hühner für einen polyklonalen und Mäuse für einen monoklonalen Antikörper, mit einem vorstehenden (Fusions)protein oder Fragmenten davon zu immunisieren. Weitere "Booster" der Tiere können mit dem gleichen (Fusions)protein oder Frag¬ menten davon erfolgen. Der polyklonale Antikörper kann dann aus dem Serum bzw. Eigelb der Tiere erhalten werden. Für den monoklonalen Antikörper werden Milzzellen der Tiere mit Myelomzellen fusioniert.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.
Die vorliegende Erfindung ermöglicht es, mentale Retardierungen, insbesondere das fragile X-Syndrom, besser zu untersuchen. Mit einem erfindungsgemäßen Antikör¬ per kann FXR2 in Körperflüssigkeiten von Personen nachgewiesen werden. Es kann eine Beziehung von FXR2 zur Entstehung und Ausbildung vorstehender Erkrankungen hergestellt werden. Ferner kann mit einem erfindungsgemäßen FXR2 ein gegen dieses Protein gerichteter Autoantikörper nachgewiesen werden. Des¬ weiteren können mit einem FXR2 der vorliegenden Erfindung Wechselwirkungen mit anderen Proteinen, insbesondere solchen, die mit mentalen Retardierungen in Bezug gebracht werden, detektiert werden, wodurch die Funktionen dieser Protei- ne aufgeklärt und gegebenenfalls therapeutische Maßnahmen für oder gegen die Proteine ergriffen werden können. Vorstehende Nachweise können durch übliche Verfahren, insbesondere einen Western Blot, einen ELISA, eine Immunpräzipitation oder durch Immunfluoreszenz, erfolgen. Desweiteren kann mit einer erfindungs¬ gemäßen Nukleinsäure, insbesondere einer DNA und hiervon abgeleiteten Primern, die Expression des für FXR2 kodierenden Gens nachgewiesen werden. Dieser Nachweis kann in üblicher Weise, insbesondere in einem Southern Blot, erfolgen. 5The present invention makes it possible to better investigate mental retardations, in particular the fragile X syndrome. With an antibody according to the invention, 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. Furthermore, 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. Furthermore, 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
Darüberhinaus eignet sich die vorliegende Erfindung, Maßnahmen für und gegen das Vorliegen von FXR2 in Personen zu ergreifen. Mit einem erfindungsgemäßen Antikörper kann FXR2 in Personen inhibiert werden. Andererseits kann mit einem erfindungsgemäßen FXR2, insbesondere nach Kopplung an ein vom Körper nicht als fremd angesehenes Protein, z.B. Transferrin oder BSA, die Menge von FXR2 in Personen erhöht werden. Entsprechendes kann auch mit einer erfindungsgemäßen Nukleinsäure, insbesondere einer DNA, erreicht werden, die unter die Kontrolle eines in bestimmten Geweben, z.B. Gehirn, Herz, induzierbaren Promotors gestellt wird und nach ihrer Expression zur Bereitstellung von FXR2 in diesen Geweben führt. Darüberhinaus kann eine erfindungsgemäße Nukleinsäure, insbesondere eine DNA, auch zur Inhibierung von FXR2 genutzt werden. Hierzu wird die Nukleinsäu¬ re, z.B. als Basis für die Erstellung von Anti-Sinn-Oligonukleotiden zur Expressions- Inhibierung des für FXR2 kodierenden Gens verwendet.Furthermore, 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. On the other hand, with 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. The same can also be achieved with 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. In addition, a nucleic acid according to the invention, in particular a DNA, can also be used to inhibit FXR2. For this the nucleic acid, e.g. used as the basis for the creation of anti-sense oligonucleotides for expression inhibition of the gene coding for FXR2.
Die vorliegende Erfindung stellt somit einen großen Beitrag zur diagnostischen und therapeutischen Erfassung von mentalen Retardierungen, insbesondere des fragilen X-Syndroms, dar.The present invention thus represents a major contribution to the diagnostic and therapeutic detection of mental retardation, in particular the fragile X syndrome.
Kurze Beschreibung der Zeichnung:Brief description of the drawing:
Fig. 1 zeigt die Basensequenz und die davon abgeleitete Aminosäurese¬ quenz eines erfindungsgemäßen Proteins FXR2.1 shows the base sequence and the amino acid sequence derived therefrom of a protein FXR2 according to the invention.
Die vorliegende Erfindung wird durch die nachstehenden Beispiele erläutert.The present invention is illustrated by the following examples.
Beispiel 1 : Herstellung und Reinigung eines erfindungsgemaßen Proteins FXR2Example 1: Production and purification of an FXR2 protein according to the invention
Zur Herstellung eines erfindungsgemäßen Proteins FXR2 wurde die DNA von Fig. 1 als Template verwendet. Es wurde ein PCR- Verfahren durchgeführt. Als Primer- Paar wurde verwendet: 5'-CAGAGATCTATGCTGGCAATTGGGACTCACGGTGC-3' und 5'-GGGAAGCTTTTAAGATTTATCCACAATCTCCTGGA-3'. Der PCR-Ansatz bzw. die PCR-Bedingungen waren jeweils wie folgt: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:
PCR-AnsatzPCR approach
Template DNA (Fig. 1 ) 1/y| = 1 ngTemplate DNA (Fig. 1) 1 / y | = 1 ng
Pfu-Polymerase 10x-Puffer 10//I = 1 xPfu polymerase 10x buffer 10 // I = 1 x
DMSO ^ 0μ\ = 10 %
Oligonukleotide, je 1 ,5μl 3μ\ = je 150 ngOligonucleotides, 1, 5μl 3μ \ = 150 ng each
H2O-bidest ad 99/vlH 2 O bidest ad 99 / vl
PCR-BedingungenPCR conditions
- 92°C - 5 min - Zugabe von 1//I Pfu-Polymerase (Stratagene) = 2,5 Einheiten- 92 ° C - 5 min - addition of 1 // I Pfu polymerase (Stratagene) = 2.5 units
- Zugabe von Paraffin- Add paraffin
PCRPCR
92°C 1 min 58°C 1 min 1 Zyklus 72°C 10 min 92°C 1 min 58°C 1 min 39 Zyklen 72°C 2 min 72°C 10 min 1 Zyklus92 ° C 1 min 58 ° C 1 min 1 cycle 72 ° C 10 min 92 ° C 1 min 58 ° C 1 min 39 cycles 72 ° C 2 min 72 ° C 10 min 1 cycle
Die amplifizierte DNA wurde jeweils mit Bgl II und Hind lll gespalten und in den mit Bam Hl und Hind lll gespaltenen Expressionsvektor pQE-8 (Diagen) inseriert. Es wurde das Expressionsplasmid pQ/FXR2 erhalten. Ein solches kodiert für ein Fusionsprotein aus 6 Histidin-Resten (N-Terminuspartner) und einem erfindungs¬ gemäßen Protein FXR2 von Fig. 1 (C-Terminuspartner). pQ/FXR2 wurde zur Trans- formation von E.coli SG 13009(vgl. Gottesman, S. et al., J. Bacteriol. 148, ( 1 981 ), 265-273) verwendet. Die Bakterien wurden in einem LB-Medium mit 100//g/ml Ampicillin und 25μg/ml Kanamycin kultiviert und 4 h mit 60//M Iso- propyl-ß-D-Thiogalactopyranosid (IPTG) induziert. Durch Zugabe von 6 M Guani- dinhydrochlorid wurde eine Lyse der Bakterien erreicht, anschließend wurde mit dem Lysat eine Chromatographie (Ni-NTA-Resin) in Gegenwart von 8 M Harnstoff entsprechend der Angaben des Herstellers (Diagen) des Chromatographie-Materials durchgeführt. Das gebundene Fusionsprotein wurde in einem Puffer mit pH 3,5 eluiert. Nach seiner Neutralisierung wurde das Fusionsprotein einer 18 % SDS- Polyacrylamid-Gelelektrophorese unterworfen und mit Coomassie-Blau angefärbt (vgl. Thomas, J.O. und Kornberg, R.D., J.Mol. Biol. 149 (1975), 709-733).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. Such a code for a fusion protein from 6 histidine residues (N-terminus partner) and a protein FXR2 according to the invention from FIG. 1 (C-terminus partner). 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 μg / ml kanamycin and induced for 4 h with 60 // M isopropyl-β-D-thiogalactopyranoside (IPTG). 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. After its neutralization, 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).
Es zeigte sich, daß ein erfindungsgemäßes Fusionsprotein in hochreiner Form hergestellt werden kann.It was found that a fusion protein according to the invention can be produced in a highly pure form.
Beispiel 2: Herstellung und Nachweis eines erfindungsgemäßen AntikörpersExample 2: Production and detection of an antibody according to the invention
Ein erfindungsgemäßes Fusionsprotein von Beispiel 1 wurde einer 1 8 % SDS- Polyacrylamid-Gelelektrophorese unterzogen. Nach Anfärbung des Gels mit 4 M Natriumacetat wurde eine ca. 8.8 kD Bande aus dem Gel herausgeschnitten und in Phosphat gepufferter Kochsalzlösung inkubiert. Gel-Stücke wurden sedimentiert, bevor die Proteinkonzentration des Überstandes durch eine SDS-Polyacrylamid- Gelelektrophorese, der eine Coomassie-Blau-Färbung folgte, bestimmt wurde. Mit dem Gel-gereinigten Fusionsprotein wurden Tiere wie folgt immunisiert: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:
Immunisierungsprotokoll für polyklonale Antikörper im KaninchenImmunization protocol for polyclonal antibodies in rabbits
Pro Immunisierung wurden 35μg Gel-gereinigtes Fusionsprotein in 0,7 ml PBS und 0,7 ml komplettem bzw. inkomplettem Freund's Adjuvans eingesetzt. Tag O: 1 . Immunisierung (komplettes Freund's Adjuvans) Tag 14 2. Immunisierung (inkomplettes Freund's Adjuvans; icFA) Tag 28 3. Immunisierung (icFA) Tag 56 4. Immunisierung (icFA) Tag 80 Ausbluten35 μg of gel-purified fusion protein in 0.7 ml of PBS and 0.7 ml of complete or incomplete Freund's adjuvant were used for each immunization. Day O: 1st Immunization (Freund's Complete Adjuvant) Day 14 2nd immunization (incomplete Freund's adjuvant; icFA) Day 28 3rd immunization (icFA) Day 56 4th immunization (icFA) Day 80 Bleeding
Das Serum des Kaninchens wurde im Immunoblot getestet. Hierzu wurde ein erfindungsgemäßes Fusionsprotein von Beispiel 1 einer SDS-Polyacrylamid-Gelelek- trophorese unterzogen und auf ein Nitrocellulosefilter übertragen (vgl. Khyse- Andersen, J., J. Biochem. Biophys. Meth. 10, ( 1 984), 203-209). Die Western Blot-Analyse wurde wie in Bock, C.-T. et al., Virus Genes 8, (1 994), 21 5-229, beschrieben, durchgeführt. Hierzu wurde das Nitrocellulosefilter eine Stunde bei 37°C mit einem ersten Antikörper inkubiert. Dieser Antikörper war das Serum des Kaninchens ( 1 : 10000 in PBS). Nach mehreren Waschschritten mit PBS wurde das Nitrocellulosefilter mit einem zweiten Antikörper inkubiert. Dieser Antikörper war ein mit alkalischer Phosphatase gekoppelter monoklonaler Ziege Anti-Kaninchen- IgG-Antikörper (Dianova) ( 1 :5000) in PBS. Nach 30-minütiger Inkubation bei 37 °C folgten mehrere Waschschritte mit PBS und anschließend die alkalische Phosphata- se-Nachweisreaktion mit Entwicklerlösung (36μM 5' Bromo-4-chloro-3-indolylphos- phat, 400/yM Nitroblau-tetrazolium, 100mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCI2) bei Raumtemperatur, bis Banden sichtbar waren.The rabbit's serum was tested in the immunoblot. For this purpose, 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. For this purpose, 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). After several washes with 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 (36μM 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.
Es zeigte sich, daß erfindungsgemäße, polyklonale Antikörper hergestellt werden können.It was found that polyclonal antibodies according to the invention can be produced.
Immunisierungsprotokoll für polyklonale Antikörper im HuhnImmunization protocol for chicken polyclonal antibodies
Pro Immunisierung wurden 40μg Gel-gereinigtes Fusionsprotein in 0,8 ml PBS und 0,8 ml komplettem bzw. inkomplettem Freund's Adjuvans eingesetzt.For each immunization, 40 μg gel-purified fusion protein in 0.8 ml PBS and 0.8 ml complete or incomplete Freund's adjuvant were used.
Tag O. 1 . Immunisierung (komplettes Freund's Adjuvans)Day O.1 Immunization (Freund's Complete Adjuvant)
Tag 28: 2. Immunisierung (inkomplettes Freund's Adjuvans; icFA) Tag 50: 3. Immunisierung (icFA)Day 28: 2nd immunization (incomplete Freund's adjuvant; icFA) Day 50: 3rd immunization (icFA)
Aus Eigelb wurden Antikörper extrahiert und im Western Blot getestet. Es wurden erfindungsgemäße, polyklonale Antikörper nachgewiesen.Antibodies were extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention have been detected.
Immunisierungsprotokoll für monoklonale Antikörper der MausImmunization protocol for mouse monoclonal antibodies
Pro Immunisierung wurden 1 2μg Gel-gereinigtes Fusionsprotein in 0,25 ml PBS und 0,25 ml komplettem bzw. inkomplettem Freund's Adjuvans eingesetzt; bei der 4. Immunisierung war das Fusionsprotein in 0,5 ml (ohne Adjuvans) gelöst.1 2 μg of gel-purified fusion protein in 0.25 ml of PBS and 0.25 ml of complete or incomplete Freund's adjuvant were used per immunization; at the 4th immunization the fusion protein was dissolved in 0.5 ml (without adjuvant).
Tag O. 1 . Immunisierung (komplettes Freund's Adjuvans) Tag 28 2. Immunisierung (inkomplettes Freund's Adjuvans; icFA) Tag 56 3. Immunisierung (icFA) Tag 84 4. Immunisierung (PBS) Tag 87 FusionDay O.1 Immunization (Freund's complete adjuvant) Day 28 2. Immunization (Freund's incomplete adjuvant; icFA) Day 56 3. Immunization (icFA) Day 84 4. Immunization (PBS) Day 87 Fusion
Überstände von Hybridomen wurden im Western Blot getestet. Erfindungsgemäße, monoklonale Antikörper wurden nachgewiesen. Supernatants from hybridomas were tested in a Western blot. Monoclonal antibodies according to the invention have been detected.
Claims
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9513875A JP2000500962A (en) | 1995-09-19 | 1996-09-19 | FMR1-related protein |
| EP96945134A EP0851923A2 (en) | 1995-09-19 | 1996-09-19 | Fmr1-related protein |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19534763.3 | 1995-09-19 | ||
| DE1995134763 DE19534763C1 (en) | 1995-09-19 | 1995-09-19 | FMR1-related protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1997012969A2 true WO1997012969A2 (en) | 1997-04-10 |
| WO1997012969A3 WO1997012969A3 (en) | 1997-07-17 |
Family
ID=7772586
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/DE1996/001787 Ceased WO1997012969A2 (en) | 1995-09-19 | 1996-09-19 | Fmr1-related protein |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0851923A2 (en) |
| JP (1) | JP2000500962A (en) |
| DE (1) | DE19534763C1 (en) |
| WO (1) | WO1997012969A2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20000072201A (en) * | 2000-08-17 | 2000-12-05 | 김희태 | A method for diagnosing Fragile-X syndrome using DNA probes |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5876949A (en) * | 1995-05-31 | 1999-03-02 | The Trustees Of The University Of Pennsylvania | Antibodies specific for fragile X related proteins and method of using the same |
-
1995
- 1995-09-19 DE DE1995134763 patent/DE19534763C1/en not_active Expired - Fee Related
-
1996
- 1996-09-19 WO PCT/DE1996/001787 patent/WO1997012969A2/en not_active Ceased
- 1996-09-19 EP EP96945134A patent/EP0851923A2/en not_active Withdrawn
- 1996-09-19 JP JP9513875A patent/JP2000500962A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20000072201A (en) * | 2000-08-17 | 2000-12-05 | 김희태 | A method for diagnosing Fragile-X syndrome using DNA probes |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0851923A2 (en) | 1998-07-08 |
| WO1997012969A3 (en) | 1997-07-17 |
| DE19534763C1 (en) | 1997-05-15 |
| JP2000500962A (en) | 2000-02-02 |
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