WO1996036708A1 - Autoantigen which can be used to detect a tendency to thrombosis - Google Patents

Autoantigen which can be used to detect a tendency to thrombosis Download PDF

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Publication number
WO1996036708A1
WO1996036708A1 PCT/DE1996/000904 DE9600904W WO9636708A1 WO 1996036708 A1 WO1996036708 A1 WO 1996036708A1 DE 9600904 W DE9600904 W DE 9600904W WO 9636708 A1 WO9636708 A1 WO 9636708A1
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WO
WIPO (PCT)
Prior art keywords
dna
autoantigen
thrombosis
tendency
expression
Prior art date
Application number
PCT/DE1996/000904
Other languages
German (de)
French (fr)
Inventor
Gerd Schmitz
Udo Schlosser
Christa BÜCHLER
Original Assignee
Progen Biotechnik Gmbh
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Filing date
Publication date
Application filed by Progen Biotechnik Gmbh filed Critical Progen Biotechnik Gmbh
Priority to EP96918581A priority Critical patent/EP0826046A1/en
Publication of WO1996036708A1 publication Critical patent/WO1996036708A1/en

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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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • 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 invention relates to an autoantigen which is suitable for determining a tendency to thrombosis, such a DNA encoding such an autoantigen and a method for producing such an autoantigen and its use.
  • Arterial and venous thrombosis is a common complication that occurs with a variety of multi-organ disorders, and especially with prolonged bedriddenness. So far there is no way to predict whether a person is prone to thrombosis or whether there is no risk of thrombosis. However, it would be very desirable to be able to determine this, since then blood thinners would not have to be administered as standard in the event of prolonged bed rest and, on the other hand, a person with a determined tendency to thrombosis could be cared for and treated much more thoroughly.
  • the present invention is therefore based on the object of providing a means with which a tendency to thrombosis can be determined.
  • the invention thus relates to an autoantigen which comprises the amino acid sequence of FIG. 2 or a functional derivative or fragment thereof.
  • the present invention is based on the knowledge of the applicant that specific autoantibodies are present in sera from persons with a tendency to thrombosis.
  • the applicant has such sera for screening a human lambda phage expression library, for example gt 1 1, Clontech # HL 1 123 b (cf. Huynh, TV et al., DNA Cloning, (1985), IRL Press Ltd., Oxford, England, Volume 1) and positive phages were obtained.
  • the inserts of these phages were subcloned and sequenced, whereby an insert, HMhk-D, was found which has the sequence indicated in FIG. 1.
  • This sequence comprises the nucleotide sequence and the amino acid sequence of an autoantigen derived therefrom. Fragments of HMhk-D were prepared by conventional PCR methods. These were incubated with the above sera, as a result of which the binding site of the autoantibodies was limited to the sequence of the autoantigen indicated in FIG. 2.
  • the above expression "functional derivative or fragment of the amino acid sequence of FIG. 2" includes any derivative or fragment of this amino acid sequence against which an autoantibody can be formed.
  • the term refers to epitopes in the amino acid sequence that can act as autoantigens.
  • the amino acid sequence of FIG. 2 can also have additions, substitutions and / or deletions of one or more amino acids, which also applies to the functional derivatives or fragments.
  • Another object of the invention is a nucleic acid coding for the above autoantigen.
  • 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.
  • the DNA of Fig. 2 was obtained from the DSM (German Collection of Microorganism and cell cultures) as pX-DIV1 under DSM 9963 on May 6, 1995.
  • a human lambda phage expression library e.g. ⁇ gt1 1 to screen Clontech # HL 1 123 b (see above) with sera from patients with a tendency to thrombosis.
  • the inserts can then be subcloned from positive phages and sequenced, as a result of which the autoantigen-coding sequences can be recognized.
  • the binding sites for the autoantibodies can be limited.
  • a DNA 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.
  • an expression vector for E. coli these are e.g. ⁇ gt1 1, pGEMEX, pUC derivatives, pGEX-2T, pET3b, pXal and pQE31.
  • yeast e.g. to call pY100 and Ycpadl
  • animal cells e.g. pKCR, pEFBOS, cDM8 and pCEV4 must be specified.
  • the baculovirus expression vector pAc SG His NT-A is particularly suitable for expression in insect cells.
  • suitable cells for expressing a DNA according to the invention which is present in an expression vector.
  • suitable cells include the E. coli strains Y1089, HB101, DH1, x1776, JM101, JM 109, BL21 and XL1 blue, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa and the insect cells Sf9.
  • DNA 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 DNA according to the invention can be expressed in the form of a fusion protein.
  • Autoantigens according to the invention are distinguished in that they recognize specific autoantibodies in people with a tendency to thrombosis. They are therefore suitable as a means of determining (diagnosing) a tendency to thrombosis. Such a determination can be made by conventional detection methods, in particular a Western blot, an ELISA, an immunoprecipitation or by immunofluorescence.
  • the autoantigens according to the invention can, if appropriate, be labeled or be used in combination with labeled antibodies directed against them.
  • Autoantigens according to the invention can also be used in a biosensor method.
  • autoantigens according to the invention can be present in a kit.
  • This can contain the autoantigens, as stated in the above form, together with conventional additives, such as buffers, carrier material and controls.
  • conventional additives such as buffers, carrier material and controls.
  • autoantigens according to the invention are also suitable for therapeutic purposes.
  • they can be used to remove autoantibodies from the circulation of patients with a tendency to thrombosis using affinity chromatography.
  • a removal of autoantibody-producing lymphocytes specific for a tendency to thrombosis by coupling the autoantigens according to the invention to cell toxins is also contemplated.
  • nucleic acids according to the invention are suitable for diagnostic purposes of all kinds.
  • Such nucleic acids can also be used for therapeutic measures.
  • the nucleic acids can be inserted into conventional expression vectors and these can be introduced into people with a tendency to thrombosis. By expression of the nucleic acids will receive autoantigens that can then intercept the autoantibodies.
  • a human lambda phage expression library, ⁇ gt1 1 Clontech # HL 1 123 b (see above) was screened with the serum of a person HM who had a tendency to thrombosis. Positive phages were obtained.
  • the inserts (EcoRI fragments) of these phages were subcloned in a vector, pUC 18 (cf. Yanisch-Perron, C. et al., Gene 33, (1985), 103) and then sequenced.
  • An insert, HMhk-D was identified which has the sequence given in FIG. 1. This comprises the nucleotide (amino acid) sequence of an autoantigen according to the invention.
  • Fragments of HMhk-D were produced by a conventional PCR method.
  • HMhk-DIV EcoRI linker was obtained a fragment, HMhk-DIV, which reacted with the serum.
  • This Fragment is shown in Fig. 2 without its two linkers. Its sequence comprises the nucleotide (amino acid) sequence of the delimited autoantibody binding site of the autoantigen encoded by HMhk-D.
  • HMhk-DIV The insert HMhk-DIV from Example 1 was inserted between the EcoRI and PstI sites of pXal (cf. 1.1 above), whereby the expression plasmid pX-DIV1 was obtained.
  • This expression plasmid was deposited with the DSM under DSM 9963 on May 6, 1995.
  • pX-DIV1 codes for a fusion protein composed of a ⁇ Gal portion (N-terminal fusion partner) and the autoantigen according to the invention from FIG. 2 (C-terminal fusion partner).
  • pX-DIV1 was used for the transformation of E. coli XL1 blue (cf. above 1 .1). The above fusion protein was obtained. It was verified as in 1.1 above.
  • the HMhk-D insert from Example 1 was inserted into the EcoRI site of the expression vector pAc SG His NT-A, B, C (see product information from PharMingen, Dianova, Raboisen), whereby the expression plasmid pAc-D was obtained.
  • pAc-D was used to transform Sf9 insect cells (see production formation above). The above fusion protein was obtained. It was detected by an antibody directed against the histidine portion or by an antibody directed against the autoantigen.
  • the expressed fusion protein from Example 2, 1 .1 or Example 2, 2. was subjected to a polyacrylamide gel electrophoresis and transferred to a PVDF membrane.
  • the membrane was saturated by conventional methods and incubated with sera from the above person HM as well as from people without a tendency to thrombosis in a dilution of 1: 100 at 25 ° C. for 1 h.
  • PBS 0.05% Tween 20
  • a commercially available POD-conjugated anti-human Ig antibody (1: 1000, DAKO) was added and also incubated for 1 h.
  • the detection reaction took place after several washing steps with the POD substrate AEC (aminoethylcarbazole, Sigma) according to the manufacturer's instructions until bands were visible.
  • the autoantigen according to the invention recognizes specific autoantibodies in the serum of a person who has a tendency to thrombosis.

Abstract

The present invention concerns: an autoantigen which can be used to detect a tendency to thrombosis; a DNA which codes for such an autoantigen; and a method of producing such an autoantigen and the use thereof.

Description

Autoantigen, geeignet zur Feststellung einer Thromboseneigung Autoantigen, suitable for determining a tendency to thrombosis
Die Erfindung betrifft ein Autoantigen, das sich zur Feststellung einer Thrombose¬ neigung eignet, eine ein solches Autoantigen kodierende DNA und ein Verfahren zur Herstellung eines solchen Autoantigens und dessen Verwendung.The invention relates to an autoantigen which is suitable for determining a tendency to thrombosis, such a DNA encoding such an autoantigen and a method for producing such an autoantigen and its use.
Arterielle und venöse Thrombose ist eine häufige Komplikation, die bei einer Vielzahl von Multiorganerkrankungen und insbesondere bei längerer Bettlägrigkeit auftritt. Bisher gibt es noch keine Möglichkeit, vorherzusagen, ob ein Mensch zu einer Thrombose neigt oder ob keine Thrombosegefahr besteht. Es wäre aber sehr wünschenswert, dies feststellen zu können, da dann nicht standardmäßig bei längerer Bettlägrigkeit Blutverdünnungsmittel verabreicht werden müßten und andererseits eine Person mit festgestellter Thromboseneigung sehr viel eingehender betreut und therapiert werden könnte.Arterial and venous thrombosis is a common complication that occurs with a variety of multi-organ disorders, and especially with prolonged bedriddenness. So far there is no way to predict whether a person is prone to thrombosis or whether there is no risk of thrombosis. However, it would be very desirable to be able to determine this, since then blood thinners would not have to be administered as standard in the event of prolonged bed rest and, on the other hand, a person with a determined tendency to thrombosis could be cared for and treated much more thoroughly.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzu¬ stellen, mit dem eine Thromboseneigung festgestellt werden kann.The present invention is therefore based on the object of providing a means with which a tendency to thrombosis can be determined.
Erfindungsgemäß wird dies durch die Bereitstellung der Gegenstände in den Patentansprüchen erreicht.According to the invention, this is achieved by the provision of the subject matter in the claims.
Gegenstand der Erfindung ist somit ein Autoantigen, das die Aminosäuresequenz von Fig. 2 oder ein funktionelles Derivat oder Fragment davon umfaßt.The invention thus relates to an autoantigen which comprises the amino acid sequence of FIG. 2 or a functional derivative or fragment thereof.
Die vorliegende Erfindung beruht auf der Erkenntnis des Anmelders, daß in Seren von Personen mit einer Thromboseneigung spezifische Autoantikörper vorliegen. Der Anmelder hat solche Seren zum Screenen einer humanen Lambda-Phagen- Expressionsbibliothek, z.B. gt 1 1 , Clontech # HL 1 123 b (vgl. Huynh, T.V. et al., DNA Cloning, (1985), IRL Press Ltd., Oxford, England, Band 1 ) verwendet und positive Phagen erhalten. Die Inserts dieser Phagen wurden subkloniert und se¬ quenziert, wodurch ein Insert, HMhk-D, gefunden wurde, das die in Fig. 1 angege¬ bene Sequenz aufweist. Diese Sequenz umfaßt die Nukleotidsequenz und die davon abgeleitete Aminosäuresequenz eines Autoantigens. Durch übliches PCR- Verfahren wurden Fragmente von HMhk-D hergestellt. Diese wurden mit vorste¬ henden Seren inkubiert, wodurch die Bindungsstelle der Autoantikörper auf die in Fig. 2 angegebene Sequenz des Autoantigens eingegrenzt wurde.The present invention is based on the knowledge of the applicant that specific autoantibodies are present in sera from persons with a tendency to thrombosis. The applicant has such sera for screening a human lambda phage expression library, for example gt 1 1, Clontech # HL 1 123 b (cf. Huynh, TV et al., DNA Cloning, (1985), IRL Press Ltd., Oxford, England, Volume 1) and positive phages were obtained. The inserts of these phages were subcloned and sequenced, whereby an insert, HMhk-D, was found which has the sequence indicated in FIG. 1. This sequence comprises the nucleotide sequence and the amino acid sequence of an autoantigen derived therefrom. Fragments of HMhk-D were prepared by conventional PCR methods. These were incubated with the above sera, as a result of which the binding site of the autoantibodies was limited to the sequence of the autoantigen indicated in FIG. 2.
Der vorstehende Ausdruck "funktionelles Derivat oder Fragment der Aminosäurese¬ quenz von Fig. 2" umfaßt jegliches Derivat oder Fragment dieser Aminosäurese¬ quenz, gegen die ein Autoantikörper gebildet werden kann. Insbesondere betrifft der Ausdruck Epitope in der Aminosäuresequenz, die als Autoantigene wirken können. Auch kann die Aminosäuresequenz von Fig. 2 Additionen, Substitutionen und/oder Deletionen von ein oder mehreren Aminosäuren aufweisen, was auch für die funktionellen Derivate oder Fragmente gilt.The above expression "functional derivative or fragment of the amino acid sequence of FIG. 2" includes any derivative or fragment of this amino acid sequence against which an autoantibody can be formed. In particular, the term refers to epitopes in the amino acid sequence that can act as autoantigens. The amino acid sequence of FIG. 2 can also have additions, substitutions and / or deletions of one or more amino acids, which also applies to the functional derivatives or fragments.
Ein weiterer Gegenstand der Erfindung ist eine für ein vorstehendes Autoantigen 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 invention is a nucleic acid coding for the above autoantigen. 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. 2 oder einen Teil davon,(a) the DNA of Figure 2 or a portion thereof,
(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.
Die DNA von Fig. 2 wurde bei der DSM (Deutsche Sammlung von Mikroorganis- men und Zellkulturen) als pX-DIV1 unter DSM 9963 am 6. Mai 1995 hinterlegt.The DNA of Fig. 2 was obtained from the DSM (German Collection of Microorganism and cell cultures) as pX-DIV1 under DSM 9963 on May 6, 1995.
Zur Herstellung einer erfindungsgemäßen DNA ist es günstig, eine humane Lamb- da-Phagen-Expressionsbibliothek, z.B. Λgt1 1 Clontech # HL 1 123 b (vgl. vor¬ stehend) mit Seren von Patienten mit einer Thromboseneigung zu screenen. Von positiven Phagen können dann die Inserts subkloniert und sequenziert werden, wodurch die Autoantigen-kodierenden Sequenzen erkannt werden können. Weiter¬ hin können die Bindungsstellen für die Autoantikörper eingegrenzt werden. Hierzu eignet es sich, durch ein PCR-Verfahren Fragmente der Phagen-Inserts herzustellen und diese mit den Seren zu inkubieren.To produce a DNA according to the invention, it is favorable to use a human lambda phage expression library, e.g. Λgt1 1 to screen Clontech # HL 1 123 b (see above) with sera from patients with a tendency to thrombosis. The inserts can then be subcloned from positive phages and sequenced, as a result of which the autoantigen-coding sequences can be recognized. Furthermore, the binding sites for the autoantibodies can be limited. For this purpose, it is suitable to use a PCR method to produce fragments of the phage inserts and to incubate them with the sera.
Eine erfindungsgemäße DNA kann in einem Vektor bzw. Expressionsvektor vor¬ liegen. Beispiele solcher sind dem Fachmann bekannt. Im Falle eines Expressions¬ vektors für E.coli sind dies z.B. Λgt1 1 , pGEMEX, pUC-Derivate, pGEX-2T, pET3b, pXal und pQE31 . 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 Insektenzellen eignet sich besonders der Baculovirus-Expressionsvektor pAc SG His NT-A.A DNA 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. Λgt1 1, pGEMEX, pUC derivatives, pGEX-2T, pET3b, pXal and pQE31. 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 pAc SG His NT-A is particularly suitable for expression in insect cells.
Der Fachmann kennt geeignete Zellen, um eine erfindungsgemäße, in einem Expressionsvektor vorliegende DNA zu exprimieren. Beispiele solcher Zellen um¬ fassen die E.coli-Stämme Y1089, HB101 , DH1 , x1776, JM101 , JM 109, BL21 und XL1 blue, 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 for expressing a DNA according to the invention which is present in an expression vector. Examples of such cells include the E. coli strains Y1089, HB101, DH1, x1776, JM101, JM 109, BL21 and XL1 blue, 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 DNA 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 DNA in Form eines Fusions¬ proteins exprimiert werden kann.The person skilled in the art knows how a DNA 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 DNA according to the invention can be expressed in the form of a fusion protein.
Des weiteren kennt der Fachmann Bedingungen, transformierte bzw. transfizierte Zellen zu kultivieren. Auch sind ihm Verfahren bekannt, das exprimierte Auto¬ antigen zu isolieren und zu reinigen. Ein vorstehendes, rekombinant hergestelltes Autoantigen, das auch ein Fusionsprotein sein kann, ist somit ebenfalls Gegen¬ stand der Erfindung.Furthermore, the person skilled in the art knows conditions, transformed or transfected Cultivate cells. He is also aware of processes for isolating and purifying the expressed autoantigen. An above, recombinantly produced autoantigen, which can also be a fusion protein, is therefore also the subject of the invention.
Erfindungsgemäße Autoantigene zeichnen sich dadurch aus, daß sie in Personen mit einer Thromboseneigung spezifische Autoantikörper erkennen. Sie eignen sich daher als Mittel zur Feststellung (Diagnose) einer Thromboseneigung. Eine solche Feststellung kann durch übliche Nachweisverfahren, insbesondere einen Western Blot, einen ELISA, eine Immunpräzipitation oder durch Immunfluoreszenz, erfolgen. Hierzu können die erfindungsgemäßen Autoantigene, wenn es angebracht ist, markiert sein oder in Kombination mit markierten, gegen sie gerichteten Anti¬ körpern eingesetzt werden. Auch können erfindungsgemäße Autoantigene in einem Biosensor-Verfahren verwendet werden.Autoantigens according to the invention are distinguished in that they recognize specific autoantibodies in people with a tendency to thrombosis. They are therefore suitable as a means of determining (diagnosing) a tendency to thrombosis. Such a determination can be made by conventional detection methods, in particular a Western blot, an ELISA, an immunoprecipitation or by immunofluorescence. For this purpose, the autoantigens according to the invention can, if appropriate, be labeled or be used in combination with labeled antibodies directed against them. Autoantigens according to the invention can also be used in a biosensor method.
Ferner können erfindungsgemäße Autoantigene in einem Kit vorliegen. Dieser kann die Autoantigene, wie in vorstehender Form angegeben, zusammen mit üblichen Zusatzstoffen, wie Puffer, Trägermaterial und Kontrollen, enthalten. Ein solcher Kit ist ebenfalls Gegenstand der Erfindung.Furthermore, autoantigens according to the invention can be present in a kit. This can contain the autoantigens, as stated in the above form, together with conventional additives, such as buffers, carrier material and controls. Such a kit is also the subject of the invention.
Darüberhinaus eignen sich erfindungsgemäße Autoantigene auch für therapeuti¬ sche Zwecke. Beispielsweise können durch sie Autoantikörper affinitätschromato- graphisch aus dem Kreislauf von Patienten mit einer Thromboseneigung entfernt werden. Auch ist an eine Entfernung von für eine Thromboseneigung spezifischen Autoantikörper-produzierenden Lymphozyten durch Kopplung der erfindungs¬ gemäßen Autoantigene an Zell-Toxine zu denken.In addition, autoantigens according to the invention are also suitable for therapeutic purposes. For example, they can be used to remove autoantibodies from the circulation of patients with a tendency to thrombosis using affinity chromatography. A removal of autoantibody-producing lymphocytes specific for a tendency to thrombosis by coupling the autoantigens according to the invention to cell toxins is also contemplated.
Des weiteren eignen sich erfindungsgemäße Nukleinsäuren, insbesondere DNAs, für diagnostische Zwecke aller Art. Auch können solche Nukleinsäuren für thera¬ peutische Maßnahmen verwendet werden. Beispielsweise können die Nukleinsäu¬ ren in übliche Expressionsvektoren inseriert werden und diese in Personen mit einer Thromboseneigung eingeschleust werden. Durch Expression der Nukleinsäuren werden Autoantigene erhalten, die dann die Autoantikörper abfangen können.Furthermore, nucleic acids according to the invention, in particular DNAs, are suitable for diagnostic purposes of all kinds. Such nucleic acids can also be used for therapeutic measures. For example, the nucleic acids can be inserted into conventional expression vectors and these can be introduced into people with a tendency to thrombosis. By expression of the nucleic acids will receive autoantigens that can then intercept the autoantibodies.
Kurze Beschreibung der Zeichnungen:Brief description of the drawings:
Fig. 1 zeigt die Nukleotidsequenz und die davon abgeleitete Aminosäurese¬ quenz eines erfindungsgemäßen, durch HMhk-D kodierten Autoanti¬ gens, und1 shows the nucleotide sequence and the amino acid sequence derived therefrom of an autoantigen according to the invention encoded by HMhk-D, and
Fig. 2 zeigt die Nukleotidsequenz und die davon abgeleitete Aminosäurese¬ quenz der eingegrenzten Autoantikörper-Bindungsstelle des durch HMhk-D kodierten Autoantigens.2 shows the nucleotide sequence and the amino acid sequence derived therefrom of the delimited autoantibody binding site of the autoantigen encoded by HMhk-D.
Die folgenden Beispiele erläutern die Erfindung.The following examples illustrate the invention.
Beispiel 1 : Herstellung einer erfindungsgemäßen DNAExample 1: Preparation of a DNA according to the invention
Eine humane Lambda-Phagen-Expressionsbibliothek,Λgt1 1 Clontech # HL 1 123 b (vgl. vorstehend) wurde mit dem Serum einer eine Thromboseneigung aufweisen¬ den Person HM gescreent. Es wurden positive Phagen erhalten. Die Inserts (EcoRI- Fragmente) dieser Phagen wurden in einem Vektor, pUC 18 (vgl. Yanisch-Perron, C. et al., Gene 33,(1985), 103) subkloniert und dann sequenziert. Es wurde ein Insert, HMhk-D, identifiziert, das die in Fig. 1 angegebene Sequenz aufweist. Diese umfaßt die Nukleotid (Aminosäure)sequenz eines erfindungsgemäßen Autoanti¬ gens.A human lambda phage expression library, Λgt1 1 Clontech # HL 1 123 b (see above) was screened with the serum of a person HM who had a tendency to thrombosis. Positive phages were obtained. The inserts (EcoRI fragments) of these phages were subcloned in a vector, pUC 18 (cf. Yanisch-Perron, C. et al., Gene 33, (1985), 103) and then sequenced. An insert, HMhk-D, was identified which has the sequence given in FIG. 1. This comprises the nucleotide (amino acid) sequence of an autoantigen according to the invention.
Durch ein übliches PCR-Verfahren wurden Fragmente von HMhk-D hergestellt.Fragments of HMhk-D were produced by a conventional PCR method.
Diese Fragmente wurden mit dem Serum vorstehender Person inkubiert. BeiThese fragments were incubated with the serum from the subject. at
Verwendung der Primer:Use of the primers:
GGC CTG CAG TTA GTC ATC TTT TGG CTT GGGC CTG CAG TTA GTC ATC TTT TGG CTT G
Pstl-LinkerPstl linker
GGC GAA TTC AGC TCC AAA GCA CCT AAGGGC GAA TTC AGC TCC AAA GCA CCT AAG
EcoRI-Linker wurde ein Fragment, HMhk-DIV, erhalten, das mit dem Serum reagierte. Dieses Fragment ist ohne seine beiden Linker in Fig. 2 angegeben. Seine Sequenz umfaßt die Nukleotid (Aminosäure)sequenz der eingegrenzten Autoantikörper-Bindungs¬ stelle des durch HMhk-D kodierten Autoantigens.EcoRI linker was obtained a fragment, HMhk-DIV, which reacted with the serum. This Fragment is shown in Fig. 2 without its two linkers. Its sequence comprises the nucleotide (amino acid) sequence of the delimited autoantibody binding site of the autoantigen encoded by HMhk-D.
Beispiel 2: Expression einer erfindungsgemäßen DNAExample 2: Expression of a DNA according to the invention
1. Expression in E.coli1. Expression in E. coli
1 .1 Das Insert HMhk-D von Beispiel 1 wurde in die EcoRI-Stelle des Expressions¬ vektors pXal (vgl. Produktinformation von Boehringer Mannheim) inseriert, wodurch das Expressionsplasmid pX-D erhalten wurde. Dieses kodiert für ein Fusionsprotein aus einem ß Gal-Anteil (N-terminaler Fusionspartner) und dem erfindungsgemäßen Autoantigen von Fig. 1 (C-terminaler Fusions¬ partner). pX-D wurde zur Transformation von E.coli XL1 blue (vgl. Bullock, W.O. et al., Bio Techniques 5, (1987), 376-278) verwendet. Es wurde das vorstehende Fusionsprotein erhalten. Sein Nachweis erfolgte durch einen gegen den ß Gal-Anteil gerichteten Antikörper bzw. durch einen gegen das Autoantigen gerichteten Antikörper.1 .1 The insert HMhk-D from Example 1 was inserted into the EcoRI site of the expression vector pXal (cf. product information from Boehringer Mannheim), whereby the expression plasmid pX-D was obtained. This codes for a fusion protein composed of a β-Gal portion (N-terminal fusion partner) and the autoantigen according to the invention from FIG. 1 (C-terminal fusion partner). pX-D was used to transform E.coli XL1 blue (see Bullock, W.O. et al., Bio Techniques 5, (1987), 376-278). The above fusion protein was obtained. It was detected by an antibody directed against the ßGal portion or by an antibody directed against the autoantigen.
1.2 Das Insert HMhk-DIV von Beispiel 1 wurde zwischen die EcoRI- und Pstl- Stellen von pXal (vgl. vorstehend 1.1 ) inseriert, wodurch das Expressions¬ plasmid pX-DIV1 erhalten wurde. Dieses Expressionsplasmid wurde bei der DSM unter DSM 9963 am 6. Mai 1995 hinterlegt. pX-DIV1 kodiert für ein Fusionsprotein aus einem ß Gal-Anteil (N-terminaler Fusionspartner) und dem erfindungsgemäßen Autoantigen von Fig. 2 (C-terminaler Fusions¬ partner). pX-DIV1 wurde zur Transformation von E.coli XL1 blue (vgl. vor¬ stehend 1 .1 ) verwendet. Es wurde das vorstehende Fusionsprotein erhalten. Sein Nachweis erfolgte wie vorstehend unter 1 .1 .1.2 The insert HMhk-DIV from Example 1 was inserted between the EcoRI and PstI sites of pXal (cf. 1.1 above), whereby the expression plasmid pX-DIV1 was obtained. This expression plasmid was deposited with the DSM under DSM 9963 on May 6, 1995. pX-DIV1 codes for a fusion protein composed of a β Gal portion (N-terminal fusion partner) and the autoantigen according to the invention from FIG. 2 (C-terminal fusion partner). pX-DIV1 was used for the transformation of E. coli XL1 blue (cf. above 1 .1). The above fusion protein was obtained. It was verified as in 1.1 above.
2. Expression in Insektenzellen2. Expression in insect cells
Das Insert HMhk-D von Beispiel 1 wurde in die EcoRI-Stelle des Expressionsvektors pAc SG His NT-A, B, C (vgl. Produktinformation der Firma PharMingen, Dianova, Raboisen) inseriert, wodurch das Expressionsplasmid pAc-D erhalten wurde. Dieses kodiert für ein Fusionsprotein aus einem Histidin-Anteil (N-terminaler Fusionspartner) und dem erfindungsgemäßen Autoantigen von Fig. 1 (C-terminaler Fusionspartner). pAc-D wurde zur Transformation von Sf9-Insektenzellen (vgl. Produktionformation vorstehend) verwendet. Es wurde das vorstehende Fusions¬ protein erhalten. Sein Nachweis erfolgte durch einen gegen den Histidin-Anteil gerichteten Antikörper bzw. durch einen gegen das Autoantigen gerichteten Antikörper.The HMhk-D insert from Example 1 was inserted into the EcoRI site of the expression vector pAc SG His NT-A, B, C (see product information from PharMingen, Dianova, Raboisen), whereby the expression plasmid pAc-D was obtained. This codes for a fusion protein composed of a histidine portion (N-terminal fusion partner) and the autoantigen according to the invention from FIG. 1 (C-terminal fusion partner). pAc-D was used to transform Sf9 insect cells (see production formation above). The above fusion protein was obtained. It was detected by an antibody directed against the histidine portion or by an antibody directed against the autoantigen.
Beispiel 3: Nachweis von Autoantikörpern durch ein erfindungsgemäßes Auto¬ antigenExample 3: Detection of autoantibodies by an autoantigen according to the invention
Das exprimierte Fusionsprotein von Beispiel 2, 1 .1 bzw. Beispiel 2, 2. wurde einer Polyacrylamid-Gelelektrophorese unterzogen und auf eine PVDF-Membran über¬ tragen. Die Membran wurde nach üblichen Verfahren abgesättigt und mit Seren von vorstehender Person HM wie auch von Personen ohne Thromboseneigung in einer Verdünnung von 1 :100 1 h bei 25°C inkubiert. Nach mehreren Wasch¬ schritten mit PBS (0,05% Tween 20) wurde ein käuflicher POD-konjugierter Anti¬ human Ig-Antikörper (1 :1000, DAKO) zugegeben und ebenfalls 1 h inkubiert. Die Nachweisreaktion erfolgte nach mehreren Waschschritten mit dem POD-Substrat AEC (Aminoethylcarbazol, Sigma) nach Herstellerangaben, bis Banden sichtbar waren.The expressed fusion protein from Example 2, 1 .1 or Example 2, 2. was subjected to a polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was saturated by conventional methods and incubated with sera from the above person HM as well as from people without a tendency to thrombosis in a dilution of 1: 100 at 25 ° C. for 1 h. After several washing steps with PBS (0.05% Tween 20), a commercially available POD-conjugated anti-human Ig antibody (1: 1000, DAKO) was added and also incubated for 1 h. The detection reaction took place after several washing steps with the POD substrate AEC (aminoethylcarbazole, Sigma) according to the manufacturer's instructions until bands were visible.
Es zeigte sich, daß das erfindungsgemäße Autoantigen spezifische Autoantikörper im Serum einer eine Thromboseneigung aufweisenden Person erkennt. It was found that the autoantigen according to the invention recognizes specific autoantibodies in the serum of a person who has a tendency to thrombosis.

Claims

Patentansprüche claims
1 . Autoantigen, umfassend die Aminosäuresequenz von Fig. 2 oder ein funktio- nelles Derivat oder Fragment davon.1 . Autoantigen comprising the amino acid sequence of Fig. 2 or a functional derivative or fragment thereof.
2. DNA, kodierend für das Autoantigen nach Anspruch 1 .2. DNA coding for the autoantigen according to claim 1.
3. DNA nach Anspruch 2, wobei die DNA umfaßt:3. DNA according to claim 2, wherein the DNA comprises:
(a) die DNA von Fig. 2 oder einen Teil davon,(a) the DNA of Figure 2 or a portion thereof,
(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.
4. Expressionsplasmid, umfassend die DNA nach Anspruch 2 oder 3.4. Expression plasmid comprising the DNA of claim 2 or 3.
5. Transformante, enthaltend das Expressionsplasmid nach Anspruch 4.5. Transformant containing the expression plasmid according to claim 4.
6. Verfahren zur Herstellung des Autoantigens nach Anspruch 1 , umfassend die Kultivierung der Transformante nach Anspruch 5 unter geeigneten Bedingungen.6. A method for producing the autoantigen according to claim 1, comprising culturing the transformant according to claim 5 under suitable conditions.
7. Verwendung des Autoantigens nach Anspruch 1 als Reagens zur Diagnose und/oder Therapie.7. Use of the autoantigen according to claim 1 as a reagent for diagnosis and / or therapy.
8. Verwendung der DNA nach Anspruch 2 oder 3 als Reagens zur Diagnose.8. Use of the DNA according to claim 2 or 3 as a reagent for diagnosis.
9. Kit, umfassend das Autoantigen nach Anspruch 1 und übliche Zusatzstoffe. 9. Kit comprising the autoantigen according to claim 1 and conventional additives.
PCT/DE1996/000904 1995-05-19 1996-05-17 Autoantigen which can be used to detect a tendency to thrombosis WO1996036708A1 (en)

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Citations (2)

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Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0424554A (en) * 1990-05-18 1992-01-28 Takara Shuzo Co Ltd Method and kit for detecting calpastatin abnormality disease
WO1995033060A1 (en) * 1994-05-31 1995-12-07 Rhone-Poulenc Rorer S.A. Method of cancer treatment by p53 protein control

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