WO2005010037A1 - Polypeptides, nucleic acids coding therefor, from the mold fungus cladosporium herbarum and the production and use thereof in diagnosis and therapy - Google Patents
Polypeptides, nucleic acids coding therefor, from the mold fungus cladosporium herbarum and the production and use thereof in diagnosis and therapy Download PDFInfo
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- WO2005010037A1 WO2005010037A1 PCT/EP2004/008098 EP2004008098W WO2005010037A1 WO 2005010037 A1 WO2005010037 A1 WO 2005010037A1 EP 2004008098 W EP2004008098 W EP 2004008098W WO 2005010037 A1 WO2005010037 A1 WO 2005010037A1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/35—Allergens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to allergens that could be isolated for the first time from the Cladosporium herbarum mold.
- Cladosporium is a fungus genus that belongs to the molds. Cladosporium species are very common and are preferred in swamps, forests and gardens because they like to grow on rotten plants or foliage. Cladosporium species can also be found in greenhouses and in poorly cleaned refrigerators. Cladosporium species can also grow on textiles such as linen. Cladosporium species can cause allergic reactions such as Cause a runny nose, cough, sneezing fits, nettle fever or asthma (mold allergy).
- One aspect relates to a polypeptide which is characterized in that it has at least 10 consecutive amino acids from the amino acid sequence with the sequence ID No. 1.
- the sequence of the polypeptide according to the invention is also shown in FIG. 1.
- the polypeptides according to the invention have a minimum length of 10 consecutive amino acids. However, longer peptides with at least 15, preferably at least 20, more preferably at least 30 and am are preferred most preferred at least 50 consecutive amino acids.
- the polypeptide according to the invention and the nucleic acid coding therefor were isolated from a mold of the genus Cladosporium, namely Cladosporium herbarum.
- a polypeptide according to the invention preferably has at least one epitope.
- An epitope is an area on the surface of a polypeptide or protein to which antibodies can bind.
- the epitopes are preferably specific for molds, in particular for Cladosporium herbarum. It is possible to generate antibodies against almost all amino acid sequences using suitable techniques, for example by using adjuvants. In the present case, however, those epitopes against which antibodies are formed without the addition of adjuvants are of interest. These are antibodies, which are mainly produced in humans by coming into contact with the mold.
- polypeptides according to the invention play an important role in particular in diagnostics and therapy. For this reason, those polypeptides which have specific epitopes are preferred according to the invention. Specifically here means that these epitopes only react with antibodies which recognize almost exclusively only those epitopes which are present on a polypeptide according to the invention.
- the antibodies which bind to the epitopes of the polypeptides according to the invention should generally not have cross-reactivity. Cross-reactivity occurs when antibodies not only enter into an antigen-antibody reaction with a specific epitope, but when these antibodies also react with other antigens (epitopes) that have a certain structural similarity to the polypeptides according to the invention.
- the extent of the cross-reactivity depends on the intended use of the polypeptide according to the invention. It may be desirable to have one Select epitope that has some cross reactivity. For example, if it is to be determined whether there is an allergic reaction to various molds, it may be desirable to select an epitope that cross-reacts with other epitopes from more or less related molds.
- epitope that can cross-react with certain endogenous structures. This is particularly important for chronic allergic reactions, e.g. in severe allergen-independent asthma.
- the polypeptides according to the invention were cloned from Cladosporium herbarum. When performing a search for similar sequences in gene banks, it was found that there is a certain, albeit slight, homology to the so-called “translationally controlled tumor protein” (hereinafter "TCTP"). This protein is also called “histamine releasing factor”.
- TCTP translationally controlled tumor protein
- This protein is also called “histamine releasing factor”.
- the polypeptide according to the invention is therefore referred to as the “Cladosporium herbarum homologue of human TCTP” (hereinafter “ClaTCTP”).
- the human TCTP molecule was first found as a protein overexpressed in tumors.
- the cDNA clone coding for ClaTCTP was isolated according to the invention from a new cDNA bank produced from Cladosporium herbarum mRNA in lambda ZAP by immunological screening with various patient sera. A primary clone was first identified that expresses a fusion protein. A reading frame was determined by sequencing and the sequence according to the invention could finally be obtained by PCR.
- the polypeptides according to the invention are an allergen that comes from a mold and that should belong to a protein group that is highly conserved in all eukaryotes. Due to the similarity of ClaTCTP to the human histamine releasing factor, this allergen is likely to play a special role in the pathogenesis of allergic diseases. It is assumed that allergic diseases caused primarily by sensitization triggered against fungal allergens, allergy-independent reactions occur in the further course of the disease. It is also assumed that a patient suffering from allergic diseases can produce IgE antibodies against his own human TCTP, which leads to the maintenance of the inflammatory allergic process even without further allergen intake. This can lead to permanent clinical pictures such as allergen-independent asthma.
- polypeptides For the intended use of the polypeptides according to the invention, it can be advantageous to characterize the epitopes located on these polypeptides in more detail. It is possible, for example, to determine which parts of a polypeptide can be particularly suitable for immunological reactions with the aid of suitable computer programs which are commercially available or from the Internet or with the aid of hydrophilicity / hydrophobicity determinations. This is a theoretical prediction, which is then usually further checked using suitable experimental test methods.
- Pepscan method can be used to determine whether epitopes are present on short polypeptides.
- short polypeptides with about 6 to 20 amino acids are chemically synthesized and coupled to carrier structures. These polypeptides are then reacted with different sera and it is checked whether there is a reaction between antibodies which are in the sera and the polypeptides.
- panels When checking, you have to use several different, as precisely defined serums as possible (so-called panels). It is necessary to use control sera (for example, non-conspicuous blood donors), and sera that usually originate from the most clinically defined patients must be used. Based on the reactivities, it can then be precisely determined whether an epitope specifically reacts only with a certain group of patients but does not react with control sera.
- polypeptides In particular if conformation epitopes are to be characterized in more detail, it is advantageous to use longer polypeptides. These longer polypeptides are preferably produced recombinantly and then reacted with the various serum panels. The recombinantly produced polypeptides are also advantageously coupled to a solid support, for example microtiter plates or polystyrene beads. The determination of the specificity of an epitope is familiar to a person skilled in the art. The appropriate methods are described in various textbooks. The sequences according to the invention can contain both T-cell epitopes and B-cell epitopes.
- a method for identifying T cell epitopes is described, for example, in the publication "Identification of Multiple T Cell Epitopes on Bet v I, the Major Birch Pollen Allergen, Using Specific T Cell Clones and Overlapping Peptides" by Ebner et al., The Journal of Immunology, Vol. 150 (1993), pp. 1047-1054.
- the determination of conformational B-cell epitopes is described, for example, by Simon-Nobbe et al. in the work "IgE-binding epitopes of enolases, a class of highly conserved fungal allergens" in J. Allergy Clin. Immunol., Vol. 106, November 2000, pp. 887-895.
- the techniques described in this work can be used for the identification of T-cell epitopes or B-cell epitopes on the polypeptides according to the invention.
- the longer polypeptides according to the invention can be produced without difficulty using recombinant techniques, that is to say by expression in a host cell.
- the desired sequence is cloned into a suitable expression vector and this vector is introduced into the host cell, for example by transformation, electroporation or other suitable techniques.
- the host cells can be selected from bacteria, for example E. coli or B. subtilis, or from fungal cells. Yeasts, such as Saccharomyces cerevisiae or various Pichia species, are suitable here. However, other known host cells can also be used, such as different Aspergillus species, or it can also be expressed in the homologous system, that is to say in Cladosporium. Expression in insect cells or cell cultures can also be considered.
- Shorter polypeptides can advantageously also be provided with the aid of the chemical solid-phase synthesis known per se.
- the polypeptides according to the invention can be used as a vaccine.
- Such vaccines can be used to desensitize patients to a mold allergy. Desensitization involves contacting allergy sufferers with a small amount of an antigen to produce neutralizing IgG antibodies. These neutralizing antibodies are then bound to the antigens with which the patient has come into contact. This avoids the antigen-antibody binding of IgE-type antibodies which trigger allergic reactions.
- the use of a polypeptide as a vaccine is therefore first of all precise characterize the epitope to which the IgE-type antibodies bind. Furthermore, it must be determined using methods known per se whether this epitope can be blocked with antibodies of the IgG type.
- a polypeptide which contains such an epitope can then be used as a vaccine, the vaccine also being able to contain conventional additives, such as formulation aids, but also suitable adjuvants.
- conventional additives such as formulation aids, but also suitable adjuvants.
- the person skilled in the art can optimize the suitable concentrations of the polypeptide for the vaccine by routine measures which are customary per se. Conventional concentrations are preferably used according to the invention.
- Another object of the present invention is a test kit for the diagnostic detection of a disease, which contains a polypeptide according to the invention.
- a disease is usually an allergy.
- the polypeptides are used in a suitable diagnostic detection system. This can be a radioimmunoassay (RIA), but is preferably an ELISA (enzyme linked immunosorbent assay).
- RIA radioimmunoassay
- ELISA enzyme linked immunosorbent assay
- the polypeptides according to the invention can also be used in Western blots.
- test kits which can be used to detect Cladosporium and are used, for example, in food analysis, are reacted with liquids in which the antigens derived from Cladosporium can be found. It can be determined whether the food is free of allergenic components that can be attributed to Cladosporium. It should be noted here that Cladosporium herbarum can grow even at relatively low temperatures, i.e. up to about + 6 ° C, and can therefore represent an undesirable contamination in areas of food technology, for example in poorly cleaned refrigerators or cold rooms or cold stores. Detection of even small amounts of Cladosporium herbarum can play an important role in the monitoring of food and its quality control, especially with regard to patients who are allergic to mold.
- nucleotide sequence which codes for the polypeptides according to the invention.
- Nucleotide sequence has the sequence ID No. 2.
- the nucleotide sequence with the sequence ID No. 2 is shown in the attached FIG. 2, it codes for the complete polypeptide according to the invention.
- a polynucleotide according to the invention has at least eight consecutive nucleotides, preferably at least 12, more preferably at least 20 and most preferably at least 50 consecutive nucleotides.
- the nucleotides must be even longer, in which case the polynucleotides have at least 100 consecutive nucleotides selected from Sequence ID No. 2.
- the polynucleotides according to the invention can serve for the specific detection of a gene coding for the polynucleotides according to the invention.
- detection methods are known nucleic acid amplification methods.
- NASBA nucleic acid sequence based amplification
- PCR polymerase chain reaction
- nucleic acid sequences depends on what is to be detected by the method.
- suitable primers By selecting suitable primers, the presence or absence of minute amounts of nucleic acids from Cladosporium herbarum can be detected very specifically. This can play a role, for example, in food analysis or product verification. Appropriate diagnostic procedures can be used to ensure that certain products are free from allergens. If not only species of Cladosporium are to be detected, but also the presence or absence of related fungi is to be detected, primers are selected from a range which have a high degree of homology to the sequences according to the invention.
- nucleic acid diagnostics it is therefore preferred to use those areas that are highly conserved if the antigen does not only consist of Cladosporium and / or very closely related species, but also if other mold species are to be detected or the corresponding DNA is to be provided. Areas that have little homology to each other are better suited for fine diagnosis, i.e. the distinction between Cladosporium herbarum and other species that are relatively closely related.
- FIG. 1 shows the complete cDNA sequence of the polypeptide ClaTCTP according to the invention and the amino acid sequence derived therefrom in the one-letter code and the nucleotide sequence in the adjacent 5 'and 3' regions.
- FIG. 2 shows the cDNA sequence of the open reading frame, the derived amino acid sequence being listed in the three-letter code.
- FIG. 3 shows a sequence alignment of the polypeptide sequence of ClaTCTP according to the invention produced with the aid of the "Clustal V" program, which was compared with a selection of TCTP sequences, namely those of the yeast S.cerevisiae and the sequences from humans, mice, Arabidopsis and alfalfa ,
- Clustal V's algorithm divides the sequence into clusters by checking the distances between all pairs.
- the clusters are assigned to each other, first individually and then collectively, to provide a general compilation.
- the various compilation algorithms were described in Higgins, DG and Sharp, PM (1989) "Fast and sensitive multiple sequence alignments on a microcomputer", CABIOS, vol. 5, no. 2, pages 151-15 ff. From FIG. 3 it can be seen that only a few “gaps” had to be introduced in order to obtain the optimal sequence alignment.
- the sequence identity between ClaTCTP and the human TCTP is approximately 42.2% identity. Because of this homology, an immunological one can be found in certain areas Cross reactivity may occur. If one takes into account not only identical amino acids when determining homology, but also similar amino acids [(D, E); (K, R); (S, T); (1LVF)], the degree of similarity increases to approximately 64%.
- FIG. 4 shows a summary of the amino acid composition, the theoretically calculated molecular weight and the theoretically calculated isoelectric point of ClaTCTP and, for comparison, the corresponding values of the human TCTP. It is striking that the electrophoretic mobility of ClaTCTP in SDS / PAGE is abnormal and pretends that the molecular weight is too high. This is not uncommon for proteins and depends on the individual binding ability of the molecule for SDS (sodium dodecyl sulphate), which in turn is determined by the individual amino acid sequence.
- SDS sodium dodecyl sulphate
- the insert of the plasmid clone from ClaTCTP was recloned by PCR.
- the two E. coli expression plasmids pMW172 (5'Nde I; 3'Stu I) and pHISparallel2 (5 'Barn HI; 3' Stu I) and primers provided with appropriate linkers were used. All primers used are listed in Tab. 1.
- sequence of the cDNA was checked again for its correctness by sequencing in order to exclude PCR artifacts.
- the expression clone of ClaTCTP in pHISparallel2 was induced in E.coli BL21 with IPTG and a soluble protein extract was produced from the E.coli cells.
- the recombinant protein was soluble in the extraction buffer and not in the inclusion bodies.
- the extract was separated by SDS / PAGE and stained with Coomassie. A clearly overexpressed protein band was found that was missing in the control.
- the apparent molecular weight of the recombinant His-tagged protein (short: "histag-ClaTCTP2) was about 30 kD. This protein band was reactive in immunoblot with the same patient sera used to screen the clone library (not shown).
- the protein extract thus analyzed was now purified by Ni chelate chromatography on chelating Sepharose Fast Flow (Pharmacia Biotech, Uppsala, Sweden) as described by Pharmacia. The procedure was as follows.
- the rTEV digested sample is dialyzed against starting buffer to allow binding to the column
- the dialyzed sample is applied to the column. Incubation 30 min / room temperature.
- the flow through contains the recombinant protein without His day washing the column with 10 ml starting buffer eluting the Ni-binding impurities with 500 mM imidazole.
- the histag-claTCTP which was purified almost to homogeneity, was cleaved with recombinant TEV protease and purified again by Ni chelate chromatography. The protein was purified for homogeneity.
- This homogeneously purified recombinant ClaTCTP was now used to find further sera from a group of 44 patient sera, which react with the recombinant ClaTCTP in immunoblotting or to determine the frequency of the reaction to this allergen.
- the sera had been preselected with a crude extract from Cladosporium herbarum for the presence of immunoreactive bands in the range between 20 and 30 kD. 5 of the 44 patient sera showed a clear reactivity with the recombinant non-fusion ClaTCTP.
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Abstract
The invention relates to polypeptides made of a protein that was isolated from Cladosporium Herbarum and which is relevant in allergic reactions, to nucleic acids coding therefor and to the use thereof in diagnosis and therapy.
Description
Polypeptide, hierfür kodierende Nucleinsäuren aus dem Schimmelpilz Cladosporium herbarum sowie deren Herstellung und Verwendung in Diagnostik und Therapie Polypeptides, nucleic acids encoding them from the Cladosporium herbarum mold and their production and use in diagnostics and therapy
Allergische Reaktionen gewinnen eine immer größere Bedeutung. Einerseits nehmen die durch Allergien hervorgerufenen Erkrankungen vor allem in den westlichen Industrieländern immer mehr zu, andererseits werden weiter verbesserte Diagnose- und Therapiemöglichkeiten zur Verfügung gestellt.Allergic reactions are becoming increasingly important. On the one hand, the diseases caused by allergies are increasing, especially in the western industrialized countries, on the other hand, further improved diagnostic and therapeutic options are made available.
Die vorliegende Erfindung betrifft Allergene, die erstmals aus dem Schimmelpilz Cladosporium herbarum isoliert werden konnten.The present invention relates to allergens that could be isolated for the first time from the Cladosporium herbarum mold.
Cladosporium ist eine Pilzgattung, die zu den Schimmelpilzen gehört. Cladosporium-Arten sind sehr häufig und kommen bevorzugt in Sumpfgebieten, im Wald und in Gärten vor, da sie gerne auf verfaulten Pflanzen bzw. auf Laub wachsen. Man trifft Cladosporium-Arten aber auch in Gewächshäusern und in schlecht gereinigten Kühlschränken an. Auch auf Textilien, wie beispielsweise Leinenstoffen, können Cladosporium-Arten wachsen. Cladosporium-Arten können allergische Reaktionen, wie z.B. Fließschnupfen, Husten, Niesanfälle, Nesselfieber oder Asthma auslösen (Schimmelpilzallergie).Cladosporium is a fungus genus that belongs to the molds. Cladosporium species are very common and are preferred in swamps, forests and gardens because they like to grow on rotten plants or foliage. Cladosporium species can also be found in greenhouses and in poorly cleaned refrigerators. Cladosporium species can also grow on textiles such as linen. Cladosporium species can cause allergic reactions such as Cause a runny nose, cough, sneezing fits, nettle fever or asthma (mold allergy).
Es besteht ein Bedürfnis, Mittel bereitzustellen, die bei Allergieproblemen eingesetzt werden können, und zwar insbesondere bei der Diagnostik, Therapie oder auch bei der Verhinderung von allergischen Reaktionen, beispielsweise in der Lebensmittelanalytik.There is a need to provide agents that can be used for allergy problems, in particular in diagnostics, therapy or also in the prevention of allergic reactions, for example in food analysis.
Der Gegenstand der vorliegenden Erfindung ist in den Patentansprüchen näher umschrieben.The subject matter of the present invention is described in more detail in the patent claims.
Ein Aspekt betrifft ein Polypeptid, das dadurch gekennzeichnet ist, daß es wenigstens 10 aufeinanderfolgende Aminosäuren aus der Aminosäuresequenz mit der Sequenz ID Nr. 1 aufweist. Die Sequenz des erfindungsgemäßen Polypeptids ist auch in Fig. 1 dargestellt.One aspect relates to a polypeptide which is characterized in that it has at least 10 consecutive amino acids from the amino acid sequence with the sequence ID No. 1. The sequence of the polypeptide according to the invention is also shown in FIG. 1.
Die erfindungsgemäßen Polypeptide weisen eine Mindestlänge von 10 aufeinanderfolgenden Aminosäuren auf. Bevorzugt sind aber längere Peptide mit wenigstens 15, bevorzugt wenigstens 20, noch bevorzugter wenigstens 30 und am
bevorzugtesten wenigstens 50 aufeinanderfolgenden Aminosäuren. Das erfindungsgemäße Polypeptid und die dafür kodierende Nucleinsäure wurden aus einem Schimmelpilz der Gattung Cladosporium, nämlich Cladosporium herbarum isoliert.The polypeptides according to the invention have a minimum length of 10 consecutive amino acids. However, longer peptides with at least 15, preferably at least 20, more preferably at least 30 and am are preferred most preferred at least 50 consecutive amino acids. The polypeptide according to the invention and the nucleic acid coding therefor were isolated from a mold of the genus Cladosporium, namely Cladosporium herbarum.
Ein erfindungsgemäßes Polypeptid weist bevorzugt wenigstens ein Epitop auf. Unter einem Epitop versteht man einen Bereich an der Oberfläche eines Polypeptids oder Proteins, an den Antikörper binden können. Es gibt lineare Epitope, bei denen die das Epitop bildenden Aminosäuren nebeneinander auf der Polypeptidsequenz angeordnet sind. Häufiger sind aber sogenannte Konformationsepitope. Diese werden durch die dreidimensionale Faltung eines Polypeptids gebildet. Dabei können Aminosäuren, die in der Polypeptidsequenz nicht nebeneinander angeordnet sind, aufgrund der dreidimensionalen Faltung des Polypeptids in räumliche Nähe kommen und eine Oberflächenstruktur bilden, an die Antikörper binden können.A polypeptide according to the invention preferably has at least one epitope. An epitope is an area on the surface of a polypeptide or protein to which antibodies can bind. There are linear epitopes in which the amino acids forming the epitope are arranged side by side on the polypeptide sequence. So-called conformation epitopes are more common. These are formed by the three-dimensional folding of a polypeptide. Amino acids which are not arranged next to one another in the polypeptide sequence can come into spatial proximity due to the three-dimensional folding of the polypeptide and form a surface structure to which antibodies can bind.
Bevorzugt sind die Epitope spezifisch für Schimmelpilze, insbesondere für Cladosporium herbarum. Es ist möglich, mit Hilfe geeigneter Techniken, beispielsweise durch Verwendung von Adjuvantien, Antikörper gegen fast alle Aminosäuresequenzen zu erzeugen. Im vorliegenden Fall sind aber solche Epitope interessant, gegen die Antikörper ohne die Zugabe von Adjuvantien gebildet werden. Es handelt sich hierbei um Antikörper, die vor allem bei Menschen dadurch gebildet werden, daß diese in Kontakt mit dem Schimmelpilz kommen.The epitopes are preferably specific for molds, in particular for Cladosporium herbarum. It is possible to generate antibodies against almost all amino acid sequences using suitable techniques, for example by using adjuvants. In the present case, however, those epitopes against which antibodies are formed without the addition of adjuvants are of interest. These are antibodies, which are mainly produced in humans by coming into contact with the mold.
Die erfindungsgemäßen Polypeptide spielen insbesondere bei der Diagnostik und Therapie eine wesentliche Rolle. Deshalb sind erfindungsgemäß solche Polypeptide bevorzugt, die spezifische Epitope aufweisen. Spezifisch bedeutet hier, daß diese Epitope nur mit solchen Antikörpern reagieren, die nahezu ausschließlich nur solche Epitope erkennen, die auf einem erfindungsgemäßen Polypeptid vorhanden sind. Die Antikörper, die an die Epitope der erfindungsgemäßen Polypeptide binden, sollen im allgemeinen nicht eine Kreuzreaktivität aufweisen. Eine Kreuzreaktivität liegt dann vor, wenn Antikörper nicht nur mit einem bestimmten Epitop eine Antigen-Antikörper-Reaktion eingehen, sondern wenn diese Antikörper auch mit anderen Antigenen (Epitopen) reagieren, die eine gewisse strukturelle Ähnlichkeit zu den erfindungsgemäßen Polypeptiden aufweisen.The polypeptides according to the invention play an important role in particular in diagnostics and therapy. For this reason, those polypeptides which have specific epitopes are preferred according to the invention. Specifically here means that these epitopes only react with antibodies which recognize almost exclusively only those epitopes which are present on a polypeptide according to the invention. The antibodies which bind to the epitopes of the polypeptides according to the invention should generally not have cross-reactivity. Cross-reactivity occurs when antibodies not only enter into an antigen-antibody reaction with a specific epitope, but when these antibodies also react with other antigens (epitopes) that have a certain structural similarity to the polypeptides according to the invention.
Das Ausmaß der Kreuzreaktivität hängt ab von dem beabsichtigten Einsatzzweck des erfindungsgemäßen Polypeptids. Es kann durchaus wünschenswert sein, ein solches
Epitop auszuwählen, das eine gewisse Kreuzreaktivität aufweist. Wenn beispielsweise ermittelt werden soll, ob eine allergische Reaktion gegen verschiedene Schimmelpilze vorliegt, kann es wünschenswert sein, ein Epitop auszuwählen, das kreuzreagiert mit anderen Epitopen aus mehr oder weniger verwandten Schimmelpilzen.The extent of the cross-reactivity depends on the intended use of the polypeptide according to the invention. It may be desirable to have one Select epitope that has some cross reactivity. For example, if it is to be determined whether there is an allergic reaction to various molds, it may be desirable to select an epitope that cross-reacts with other epitopes from more or less related molds.
Es kann auch wünschenswert sein, ein Epitop auszuwählen, das mit bestimmten körpereigenen Strukturen kreuzreagieren kann. Dies spielt vor allem bei chronischen allergischen Reaktionen eine Rolle, z.B. beim schweren Allergen-unabhängigen Asthma.It may also be desirable to choose an epitope that can cross-react with certain endogenous structures. This is particularly important for chronic allergic reactions, e.g. in severe allergen-independent asthma.
Wenn aber ermittelt werden soll, ob eine Reaktion auf einen spezifischen Vertreter von Cladosporium vorliegt, dann kann es bevorzugt sein, ein solches Epitop auszuwählen, das sehr spezifisch ist, bei dem also keine oder nahezu keine Kreuzreaktionen mit anderen Epitopen auftreten.However, if it is to be determined whether there is a reaction to a specific representative of Cladosporium, then it may be preferred to select such an epitope that is very specific, in which no or almost no cross-reactions with other epitopes occur.
Die erfindungsgemäßen Polypeptide wurden aus Cladosporium herbarum kloniert. Bei Durchführung einer Recherche nach ähnlichen Sequenzen in Genbanken wurde gefunden, daß eine gewisse, allerdings geringe Homologie zu dem sogenannten "translationally controlled tumor protein" (im folgenden "TCTP") besteht. Dieses Protein wird auch als "histamine releasing factor" bezeichnet. Im Rahmen der vorliegenden Erfindung wird daher das erfindungsgemäße Polypeptid bezeichnet als das "Cladosporium herbarum Homolog des humanen TCTP" (im folgenden kurz "ClaTCTP"). Das menschliche TCTP Molekül wurde zuerst als ein in Tumoren überexprimiertes Protein gefunden. Der cDNA Klon kodierend für ClaTCTP wurde erfindungsgemäß aus einer neuen, aus Cladosporium herbarum mRNA hergestellten cDNA-Bank in Lambda-ZAP durch immunologisches Screening mit verschiedenen Patientenseren isoliert. Dabei wurde zunächst ein Primärklon identifiziert, der ein Fusionsprotein exprimiert. Durch Sequenzierung wurde ein Leserahmen ermittelt und durch PCR konnte schließlich die erfindungsgemäße Sequenz erhalten werden.The polypeptides according to the invention were cloned from Cladosporium herbarum. When performing a search for similar sequences in gene banks, it was found that there is a certain, albeit slight, homology to the so-called "translationally controlled tumor protein" (hereinafter "TCTP"). This protein is also called "histamine releasing factor". In the context of the present invention, the polypeptide according to the invention is therefore referred to as the "Cladosporium herbarum homologue of human TCTP" (hereinafter "ClaTCTP"). The human TCTP molecule was first found as a protein overexpressed in tumors. The cDNA clone coding for ClaTCTP was isolated according to the invention from a new cDNA bank produced from Cladosporium herbarum mRNA in lambda ZAP by immunological screening with various patient sera. A primary clone was first identified that expresses a fusion protein. A reading frame was determined by sequencing and the sequence according to the invention could finally be obtained by PCR.
Bei den erfindungsgemäßen Polypeptiden handelt es sich um ein Allergen, das von einem Schimmelpilz stammt und das zu einer in allen Eukaryoten hoch konservierten Proteingruppe gehören dürfte. Aufgrund der Ähnlichkeit von ClaTCTP zu dem menschlichen histamine releasing factor spielt dieses Allergen voraussichtlich bei der Pathogenese allergischer Erkrankungen eine besondere Rolle. Es wird davon ausgegangen, daß bei allergischen Erkrankungen, die primär durch eine Sensibilisierung
gegen pilzliche Allergene ausgelöst werden, im weiteren Krankheitsverlauf Allergen- unabhängige Reaktionen auftreten. Es wird weiterhin davon ausgegangen, daß ein unter allergischen Erkrankungen leidender Patient gegen das eigene humane TCTP IgE- Antikörper produzieren kann, was zu einer Aufrechterhaltung des entzündlichen allergischen Prozesses auch ohne weitere Allergenzufuhr führt. Dies kann dann zu permanenten Krankheitsbildern wie Allergen-unabhängiges Asthma führen.The polypeptides according to the invention are an allergen that comes from a mold and that should belong to a protein group that is highly conserved in all eukaryotes. Due to the similarity of ClaTCTP to the human histamine releasing factor, this allergen is likely to play a special role in the pathogenesis of allergic diseases. It is assumed that allergic diseases caused primarily by sensitization triggered against fungal allergens, allergy-independent reactions occur in the further course of the disease. It is also assumed that a patient suffering from allergic diseases can produce IgE antibodies against his own human TCTP, which leads to the maintenance of the inflammatory allergic process even without further allergen intake. This can lead to permanent clinical pictures such as allergen-independent asthma.
Für den bestimmungsgemäßen Einsatz der erfindungsgemäßen Polypeptide kann es vorteilhaft sein, die auf diesen Polypeptiden befindlichen Epitope näher zu charakterisieren. Es ist beispielsweise möglich, mit Hilfe von geeigneten Computerprogrammen, die kommerziell oder aus dem Internet erhältlich sind oder mit Hilfe von Hydrophilie/Hydrophobie-Bestimmungen zu ermitteln, welche Teile eines Polypeptids besonders geeignet sein können für immunologische Reaktionen. Hierbei handelt es sich um eine theoretische Voraussage, die anschließend üblicherweise mit geeigneten experimentellen Testmethoden weiter überprüft wird.For the intended use of the polypeptides according to the invention, it can be advantageous to characterize the epitopes located on these polypeptides in more detail. It is possible, for example, to determine which parts of a polypeptide can be particularly suitable for immunological reactions with the aid of suitable computer programs which are commercially available or from the Internet or with the aid of hydrophilicity / hydrophobicity determinations. This is a theoretical prediction, which is then usually further checked using suitable experimental test methods.
Um zu bestimmen, ob auf kurzen Polypeptiden Epitope vorhanden sind, kann man sich der sogenannten Pepscan-Methode bedienen. Hierzu werden kurze Polypeptide mit etwa 6 bis 20 Aminosäuren chemisch synthetisiert und an Trägerstrukturen gekoppelt. Diese Polypeptide werden dann mit verschiedenen Seren umgesetzt und es wird überprüft, ob eine Reaktion zwischen Antikörpern, die sich in den Seren befinden, und den Polypeptiden auftreten. Bei der Überprüfung muß man mehrere verschiedene, möglichst genau definierte Seren einsetzen (sogenannte Panels). Es ist erforderlich, daß Kontrollseren (beispielsweise nicht auffällige Blutspender) eingesetzt werden und des müssen Seren eingesetzt werden, die üblicherweise von möglichst genau klinisch definierten Patienten herstammen. Aufgrund der Reaktivitäten kann man dann genau bestimmen, ob ein Epitop spezifisch nur mit einer bestimmten Gruppe von Patienten reagiert, mit Kontrollseren aber nicht reagiert.The so-called Pepscan method can be used to determine whether epitopes are present on short polypeptides. For this purpose, short polypeptides with about 6 to 20 amino acids are chemically synthesized and coupled to carrier structures. These polypeptides are then reacted with different sera and it is checked whether there is a reaction between antibodies which are in the sera and the polypeptides. When checking, you have to use several different, as precisely defined serums as possible (so-called panels). It is necessary to use control sera (for example, non-conspicuous blood donors), and sera that usually originate from the most clinically defined patients must be used. Based on the reactivities, it can then be precisely determined whether an epitope specifically reacts only with a certain group of patients but does not react with control sera.
Insbesondere wenn Konformationsepitope näher charakterisiert werden sollen, ist es vorteilhaft, längere Polypeptide einzusetzen. Diese längeren Polypeptide werden bevorzugt rekombinant hergestellt und dann mit den verschiedenen Serumpanels umgesetzt. Auch die rekombinant hergestellten Polypeptide werden vorteilhaft an einem festen Träger, beispielsweise Mikrotiterplatten oder Polystyrolkügelchen gekoppelt. Die Bestimmung der Spezifität eines Epitops ist einem Fachmann geläufig. Die dazu geeigneten Methoden sind in verschiedenen Lehrbüchern näher beschrieben.
Die erfindungsgemäßen Sequenzen können sowohl T-Zell Epitope, wie auch B-Zell Epitope enthalten. Eine Methode, wie T-Zell Epitope identifiziert werden können, wird beispielsweise in der Veröffentlichung "Identification of Multiple T Cell Epitopes on Bet v I, the Major Birch Pollen Allergen, Using Specific T Cell Clones and Overlapping Peptides" von Ebner et al., The Journal of Immunology, Vol. 150 (1993), S. 1047-1054 beschrieben. Die Bestimmung von konformationellen B-Zell Epitopen beschreibt beispielsweise Simon-Nobbe et al. in der Arbeit "IgE-binding epitopes of enolases, a class of highly conserved fungal allergens" in J. Allergy Clin. Immunol., Vol. 106, November 2000, S. 887-895. Die in diesen Arbeiten beschriebenen Techniken können für die Identifizierung von T-Zell Epitopen oder B-Zell Epitopen auf den erfindungsgemäßen Polypeptiden angewandt werden.In particular if conformation epitopes are to be characterized in more detail, it is advantageous to use longer polypeptides. These longer polypeptides are preferably produced recombinantly and then reacted with the various serum panels. The recombinantly produced polypeptides are also advantageously coupled to a solid support, for example microtiter plates or polystyrene beads. The determination of the specificity of an epitope is familiar to a person skilled in the art. The appropriate methods are described in various textbooks. The sequences according to the invention can contain both T-cell epitopes and B-cell epitopes. A method for identifying T cell epitopes is described, for example, in the publication "Identification of Multiple T Cell Epitopes on Bet v I, the Major Birch Pollen Allergen, Using Specific T Cell Clones and Overlapping Peptides" by Ebner et al., The Journal of Immunology, Vol. 150 (1993), pp. 1047-1054. The determination of conformational B-cell epitopes is described, for example, by Simon-Nobbe et al. in the work "IgE-binding epitopes of enolases, a class of highly conserved fungal allergens" in J. Allergy Clin. Immunol., Vol. 106, November 2000, pp. 887-895. The techniques described in this work can be used for the identification of T-cell epitopes or B-cell epitopes on the polypeptides according to the invention.
Die längeren erfindungsgemäßen Polypeptide können mit Hilfe rekombinanter Techniken, also durch Expression in einer Wirtszelle, ohne Schwierigkeiten hergestellt werden. Die gewünschte Sequenz wird hierzu in einen geeigneten Expressionsvektor kloniert und dieser Vektor wird beispielsweise durch Transformation, Elektroporation oder andere geeignete Techniken in die Wirtszelle eingebracht. Die Wirtszellen können ausgewählt werden aus Bakterien, beispielsweise E.coli oder B.subtilis oder aus Pilzzellen. In Frage kommen hier Hefen, wie Saccharomyces cerevisiae oder verschiedene Pichia-Arten. Es können aber auch andere bekannte Wirtszellen eingesetzt werden, wie verschiedene Aspergillus-Arten, oder es kann auch im homologen System, also in Cladosporium exprimiert werden. Es kommt auch die Expression in Insektenzellen oder Zellkulturen in Betracht.The longer polypeptides according to the invention can be produced without difficulty using recombinant techniques, that is to say by expression in a host cell. For this purpose, the desired sequence is cloned into a suitable expression vector and this vector is introduced into the host cell, for example by transformation, electroporation or other suitable techniques. The host cells can be selected from bacteria, for example E. coli or B. subtilis, or from fungal cells. Yeasts, such as Saccharomyces cerevisiae or various Pichia species, are suitable here. However, other known host cells can also be used, such as different Aspergillus species, or it can also be expressed in the homologous system, that is to say in Cladosporium. Expression in insect cells or cell cultures can also be considered.
Kürzere Polypeptide können vorteilhafterweise auch mit Hilfe der an sich bekannten chemischen Festphasensynthese bereitgestellt werden.Shorter polypeptides can advantageously also be provided with the aid of the chemical solid-phase synthesis known per se.
In einem weiteren Aspekt der vorliegenden Erfindung können die erfindungsgemäßen Polypeptide als Impfstoff eingesetzt werden. Derartige Impfstoffe können zur Desensibilisierung von Patienten gegen eine Schimmelpilzallergie verwendet werden. Bei einer Desensibilisierung werden Patienten, die an einer Allergie leiden, mit einer geringen Menge eines Antigens in Kontakt gebracht, wodurch neutralisierende IgG-Antikörper gebildet werden sollen. Diese neutralisierenden Antikörper werden dann an die Antigene, mit denen der Patient in Kontakt gekommen ist, gebunden. Dadurch wird die Antigen- Antikörper-Bindung von allergischen Reaktionen auslösenden Antikörpern vom IgE-Typ vermieden. Für die Verwendung eines Polypeptids als Impfstoff wird daher zunächst genau
das Epitop zu charakterisieren sein, an das die Antikörper vom IgE-Typ binden. Weiterhin muß mit an sich bekannten Methoden festgestellt werden, ob dieses Epitop mit Antikörpern vom IgG-Typ blockiert werden kann. Ein Polypeptid, das ein derartiges Epitop enthält, kann dann als Impfstoff eingesetzt werden, wobei der Impfstoff auch übliche Zusatzstoffe, wie Formulierungshilfen, aber auch geeignete Adjuvantien enthalten kann. Die geeigneten Konzentrationen des Polypeptids für den Impfstoff kann der Fachmann durch an sich übliche Routinemaßnahmen optimieren. Erfindungsgemäß bevorzugt werden übliche Konzentrationen eingesetzt.In a further aspect of the present invention, the polypeptides according to the invention can be used as a vaccine. Such vaccines can be used to desensitize patients to a mold allergy. Desensitization involves contacting allergy sufferers with a small amount of an antigen to produce neutralizing IgG antibodies. These neutralizing antibodies are then bound to the antigens with which the patient has come into contact. This avoids the antigen-antibody binding of IgE-type antibodies which trigger allergic reactions. The use of a polypeptide as a vaccine is therefore first of all precise characterize the epitope to which the IgE-type antibodies bind. Furthermore, it must be determined using methods known per se whether this epitope can be blocked with antibodies of the IgG type. A polypeptide which contains such an epitope can then be used as a vaccine, the vaccine also being able to contain conventional additives, such as formulation aids, but also suitable adjuvants. The person skilled in the art can optimize the suitable concentrations of the polypeptide for the vaccine by routine measures which are customary per se. Conventional concentrations are preferably used according to the invention.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Testkit zum diagnostischen Nachweis einer Erkrankung, das ein erfindungsgemäßes Polypeptid enthält. Bei einer derartigen Erkrankung handelt es sich üblicherweise um eine Allergie. Die Polypeptide werden in einem geeigneten diagnostischen Nachweissystem eingesetzt. Es kann sich hierbei um einen Radioimmunassay (RIA) handeln, bevorzugt handelt es sich aber um einen ELISA (enzyme linked immunosorbent assay). In einer anderen Alternative, die insbesondere für differenzierte diagnostische Aussagen vorteilhaft sein kann, können die erfindungsgemäßen Polypeptide auch in Western Blots eingesetzt werden.Another object of the present invention is a test kit for the diagnostic detection of a disease, which contains a polypeptide according to the invention. Such a disease is usually an allergy. The polypeptides are used in a suitable diagnostic detection system. This can be a radioimmunoassay (RIA), but is preferably an ELISA (enzyme linked immunosorbent assay). In another alternative, which can be particularly advantageous for differentiated diagnostic statements, the polypeptides according to the invention can also be used in Western blots.
Wenn die erfindungsgemäßen Testkits zum Nachweis einer Erkrankung verwendet werden, befinden sich die Antikörper gegen die nachzuweisenden Epitope in der Regel in Körperflüssigkeiten, insbesondere Serum. Testkits, die zum Nachweis von Cladosporium dienen können und beispielsweise in der Lebensmittelanalytik eingesetzt werden, werden mit solchen Flüssigkeiten umgesetzt, in denen sich die von Cladosporium herstammenden Antigene befinden können. Dabei kann festgestellt werden, ob die Lebensmittel frei von allergenen Bestandteilen sind, die auf Cladosporium zurückzuführen sind. Beachtet werden sollte hier, daß Cladosporium herbarum auch bei verhältnismäßig niedrigen Temperaturen, also bis zu etwa +6°C wachsen kann und daher in Bereichen der Lebensmitteltechnologie eine unerwünschte Kontamination, beispielsweise in schlecht gereinigten Kühlschränken oder Kühlräumen bzw. Kühllagerhäusern darstellen kann. Für die Überwachung von Lebensmitteln und deren Qualitätskontrolle kann der Nachweis auch von geringen Mengen von Cladosporium herbarum eine wesentliche Rolle spielen, insbesondere im Hinblick auf Patienten, die eine Schimmelpilzallergie haben.If the test kits according to the invention are used to detect a disease, the antibodies against the epitopes to be detected are usually in body fluids, in particular serum. Test kits, which can be used to detect Cladosporium and are used, for example, in food analysis, are reacted with liquids in which the antigens derived from Cladosporium can be found. It can be determined whether the food is free of allergenic components that can be attributed to Cladosporium. It should be noted here that Cladosporium herbarum can grow even at relatively low temperatures, i.e. up to about + 6 ° C, and can therefore represent an undesirable contamination in areas of food technology, for example in poorly cleaned refrigerators or cold rooms or cold stores. Detection of even small amounts of Cladosporium herbarum can play an important role in the monitoring of food and its quality control, especially with regard to patients who are allergic to mold.
Ein weiterer Aspekt der vorliegenden Erfindung betrifft Polynucleotide aus einer Nucleotidsequenz, die für die erfindungsgemäßen Polypeptide kodiert. Eine solche
Nucleotidsequenz hat die Sequenz ID Nr. 2. Die Nucleotidsequenz mit der Sequenz ID Nr. 2 ist in der anliegenden Figur 2 dargestellt, sie kodiert für das vollständige erfindungsgemäße Polypeptid.Another aspect of the present invention relates to polynucleotides from a nucleotide sequence which codes for the polypeptides according to the invention. Such Nucleotide sequence has the sequence ID No. 2. The nucleotide sequence with the sequence ID No. 2 is shown in the attached FIG. 2, it codes for the complete polypeptide according to the invention.
Ein erfindungsgemäßes Polynucleotid weist wenigstens acht aufeinanderfolgende Nucleotide, bevorzugt wenigstens 12, noch bevorzugter wenigstens 20 und am bevorzugtesten wenigstens 50 aufeinanderfolgende Nucleotide auf. Für manche Ausführungsformen müssen die Nucleotide noch länger sein, in diesem Fall weisen die Polynucleotide wenigstens 100 aufeinanderfolgende Nucleotide auf, ausgewählt aus der Sequenz ID Nr. 2.A polynucleotide according to the invention has at least eight consecutive nucleotides, preferably at least 12, more preferably at least 20 and most preferably at least 50 consecutive nucleotides. For some embodiments, the nucleotides must be even longer, in which case the polynucleotides have at least 100 consecutive nucleotides selected from Sequence ID No. 2.
Die erfindungsgemäßen Polynucleotide können zum spezifischen Nachweis eines Gens kodierend für die erfindungsgemäßen Polynucleotide dienen. Bei diesen Nachweisverfahren handelt es sich um an sich bekannte Nucleinsäureamplifikationsmethoden. Es kommen beispielsweise NASBA (nucleic acid sequence based amplification) in Frage oder bevorzugter die Polymerase Kettenreaktion (PCR). Ein Überblick geeigneter Nachweisverfahren findet sich in dem Lehrbuch Bioanalytik (Lottspeich/Zorbas, Spektrum akademischer Verlag Heidelberg/Berlin, 1998). Auf dieses Lehrbuch und die darin verwiesenen Sekundärliteraturstellen wird hiermit ausdrücklich verwiesen.The polynucleotides according to the invention can serve for the specific detection of a gene coding for the polynucleotides according to the invention. These detection methods are known nucleic acid amplification methods. For example, NASBA (nucleic acid sequence based amplification) or, more preferably, the polymerase chain reaction (PCR) are possible. An overview of suitable detection methods can be found in the textbook Bioanalytics (Lottsspeicher / Zorbas, Spektrum academic publisher Heidelberg / Berlin, 1998). We hereby expressly refer to this textbook and the secondary literature references referred to in it.
Für die Auswahl geeigneter Nucleinsäuresequenzen kommt es darauf an, was mit dem Verfahren nachgewiesen werden soll. Durch Auswahl geeigneter Primer kann sehr spezifisch die Anwesenheit bzw. Abwesenheit geringster Mengen von Nucleinsäuren aus Cladosporium herbarum nachgewiesen werden. Dies kann beispielsweise bei der Lebensmittelanalytik oder auch bei der Produktüberprüfung eine Rolle spielen. Wenn sichergestellt werden soll, daß bestimmte Produkte frei sind von Allergenen, können entsprechende diagnostische Verfahren eingesetzt werden. Wenn nicht nur Spezies von Cladosporium nachgewiesen werden sollen, sondern auch das Vorhandensein bzw. die Abwesenheit von verwandten Pilzen nachgewiesen werden soll, werden Primer aus einem Bereich ausgewählt, die eine hohe Homologie zu den erfindungsgemäßen Sequenzen aufweisen. Für die Nucleinsäurediagnostik ist es daher bevorzugt solche Bereiche, die stark konserviert sind, dann zu verwenden, wenn das Antigen nicht nur aus Cladosporium und/oder sehr nahe verwandten Spezies besteht, sondern wenn auch andere Schimmelpilzspezies nachgewiesen oder die entsprechende DNA bereitgestellt werden soll.
Bereiche, die eine geringe Homologie zueinander aufweisen, eignen sich besser für die Feindiagnostik, also die Unterscheidung zwischen Cladosporium herbarum und anderen Spezies, die relativ nahe verwandt sind.The choice of suitable nucleic acid sequences depends on what is to be detected by the method. By selecting suitable primers, the presence or absence of minute amounts of nucleic acids from Cladosporium herbarum can be detected very specifically. This can play a role, for example, in food analysis or product verification. Appropriate diagnostic procedures can be used to ensure that certain products are free from allergens. If not only species of Cladosporium are to be detected, but also the presence or absence of related fungi is to be detected, primers are selected from a range which have a high degree of homology to the sequences according to the invention. For nucleic acid diagnostics, it is therefore preferred to use those areas that are highly conserved if the antigen does not only consist of Cladosporium and / or very closely related species, but also if other mold species are to be detected or the corresponding DNA is to be provided. Areas that have little homology to each other are better suited for fine diagnosis, i.e. the distinction between Cladosporium herbarum and other species that are relatively closely related.
Bei der Auswahl der geeigneten Nucleotidsequenzen, die als Primer dienen sollen, muß berücksichtigt werden, daß eine gewisse Homologie zu dem humane TCTP besteht. Je nach dem Zweck der Aussage der diagnostischen Maßnahme müssen daher entweder solche Bereiche für die Auswahl der Primer ausgewählt werden, die möglichst keine Ähnlichkeit zu dem humane TCTP haben. Wenn aber mit Hilfe der diagnostischen Mittel eine Beziehung zu den humanen TCTP-Sequenzen eine Rolle spielen kann, kann es vorteilhaft sein, gerade in diesen Bereichen solche Polypeptidsequenzen bzw. Polynucleotidsequenzen einzusetzen, die eben diese verhältnismäßig hohe Homologie aufweisen.When choosing the appropriate nucleotide sequences to be used as primers, it must be taken into account that there is a certain homology to the human TCTP. Depending on the purpose of the diagnostic measure, it is therefore necessary either to select those areas for the selection of the primers that are as similar as possible to the human TCTP. If, however, a relationship to the human TCTP sequences can play a role with the aid of the diagnostic agents, it can be advantageous to use those polypeptide sequences or polynucleotide sequences that have this relatively high homology in precisely these areas.
Figur 1 zeigt die vollständige cDNA-Sequenz des erfindungsgemäßen Polypeptids ClaTCTP und die davon abgeleitete Aminosäuresequenz im Einbuchstabencode sowie die Nucleotidsequenz in den benachbarten 5'- und 3'-Regionen.FIG. 1 shows the complete cDNA sequence of the polypeptide ClaTCTP according to the invention and the amino acid sequence derived therefrom in the one-letter code and the nucleotide sequence in the adjacent 5 'and 3' regions.
Figur 2 zeigt die cDNA-Sequenz des offenen Leserahmens, wobei die abgeleitete Aminosäuresequenz im Dreibuchstabencode aufgeführt ist.FIG. 2 shows the cDNA sequence of the open reading frame, the derived amino acid sequence being listed in the three-letter code.
Figur 3 zeigt ein mit Hilfe des Programms "Clustal V" hergestelltes Sequenz-Alignment der erfindungsgemäßen Polypeptidsequenz von ClaTCTP, die gegenübergestellt wurde einer Auswahl von TCTP-Sequenzen, nämlich diejenigen der Hefe S.cerevisiae und der Sequenzen aus Mensch, Maus, Arabidopsis und Alfalfa.FIG. 3 shows a sequence alignment of the polypeptide sequence of ClaTCTP according to the invention produced with the aid of the "Clustal V" program, which was compared with a selection of TCTP sequences, namely those of the yeast S.cerevisiae and the sequences from humans, mice, Arabidopsis and alfalfa ,
Der Algorithmus von Clustal V teilt die Sequenz in Cluster ein, indem er die Abstände zwischen allen Paaren überprüft. Die Cluster werden einander zugeordnet, zuerst individuell und dann kollektiv, um eine allgemeine Zusammenstellung bereitzustellen. Die vielfältigen Zusammenstellungsalgorithmen wurden beschrieben in Higgins, D.G. und Sharp, P.M. (1989) "Fast and sensitive multiple sequence alignments on a microcomputer", CABIOS, vol. 5, no. 2, Seiten 151-15 ff. Aus Figur 3 geht hervor, daß nur wenige "gaps" eingeführt werden mußten, um das optimale Sequenz-Alignment zu erhalten. Die Sequenzidentität zwischen ClaTCTP und dem human TCTP beträgt etwa 42,2 % Identität. Aufgrund dieser Homologie kann in bestimmten Bereichen eine immunologische
Kreuzreaktivität auftreten. Wenn man bei der Bestimmung der Homologie nicht nur identische Aminosäuren berücksichtigt, sondern auch ähnliche Aminosäuren [(D,E); (K,R); (S,T); (1LVF)], erhöht sich der Grad der Ähnlichkeit auf etwa 64 %.Clustal V's algorithm divides the sequence into clusters by checking the distances between all pairs. The clusters are assigned to each other, first individually and then collectively, to provide a general compilation. The various compilation algorithms were described in Higgins, DG and Sharp, PM (1989) "Fast and sensitive multiple sequence alignments on a microcomputer", CABIOS, vol. 5, no. 2, pages 151-15 ff. From FIG. 3 it can be seen that only a few “gaps” had to be introduced in order to obtain the optimal sequence alignment. The sequence identity between ClaTCTP and the human TCTP is approximately 42.2% identity. Because of this homology, an immunological one can be found in certain areas Cross reactivity may occur. If one takes into account not only identical amino acids when determining homology, but also similar amino acids [(D, E); (K, R); (S, T); (1LVF)], the degree of similarity increases to approximately 64%.
Figur 4 zeigt eine Zusammenfassung der Aminosäurezusammensetzung, das theoretisch berechnete Molekulargewicht und den theoretisch berechneten isoelektrischen Punkt von ClaTCTP und zum Vergleich die entsprechenden Werte des humanen TCTP. Es fällt auf, daß die elektrophoretische Mobilität von ClaTCTP in der SDS/PAGE anomal ist und ein zu hohes Molekulargewicht vortäuscht. Dies ist bei Proteinen an sich nicht ungewöhnlich und hängt von der individuellen Bindefähigkeit des Moleküls für SDS (sodium dodecyl sulphate) ab, welche wiederum durch die individuelle Aminosäuresequenz bestimmt wird.FIG. 4 shows a summary of the amino acid composition, the theoretically calculated molecular weight and the theoretically calculated isoelectric point of ClaTCTP and, for comparison, the corresponding values of the human TCTP. It is striking that the electrophoretic mobility of ClaTCTP in SDS / PAGE is abnormal and pretends that the molecular weight is too high. This is not uncommon for proteins and depends on the individual binding ability of the molecule for SDS (sodium dodecyl sulphate), which in turn is determined by the individual amino acid sequence.
Die vorliegende Erfindung wird durch die nachfolgenden Beispiele weiter erläutert.The following examples further illustrate the present invention.
Beispiel 1 : Klonierung von ClaTCTPExample 1: Cloning of ClaTCTP
Das Insert des Plasmidklons von ClaTCTP wurde durch PCR rekloniert. Dazu wurden die beiden E.coli Expressionsplasmide pMW172 (5'Nde I; 3'Stu I) und pHISparallel2 (5' Barn H I; 3' Stu I) sowie mit entsprechenden Linkern versehene Primer verwendet. Alle verwendeten Primer sind in Tab. 1 aufgeführt.
The insert of the plasmid clone from ClaTCTP was recloned by PCR. For this purpose, the two E. coli expression plasmids pMW172 (5'Nde I; 3'Stu I) and pHISparallel2 (5 'Barn HI; 3' Stu I) and primers provided with appropriate linkers were used. All primers used are listed in Tab. 1.
Tab. 1: Klonierung claTCTP und humTCTP in pHis parallel 2 und pMW172 ♦ claTCTP Restriktionsenzyme:Tab. 1: Cloning claTCTP and humTCTP in pHis parallel 2 and pMW172 ♦ claTCTP restriction enzymes:
Nach diesem Schritt wurde die Sequenz der cDNA nochmals durch Sequenzieren auf ihre Richtigkeit überprüft, um PCR-Artefakte auszuschließen. After this step, the sequence of the cDNA was checked again for its correctness by sequencing in order to exclude PCR artifacts.
Der Expressionsklon von ClaTCTP in pHISparallel2 wurde in E.coli BL21 mit IPTG induziert und aus den E.coli Zellen wurde ein löslicher Proteinextrakt hergestellt. Das rekombinante Protein war löslich im Extraktionspuffer und nicht in inclusion bodies angefallen. Der Extrakt wurde durch SDS/PAGE aufgetrennt und mit Coomassie gefärbt. Es zeigte sich eine deutlich überexprimierte Proteinbande, die in der Kontrolle fehlte. Das scheinbare Molekulargewicht des rekombinanten mit dem His-tag versehenen Proteins (kurz: "histag- ClaTCTP2) war etwa 30 kD. Diese Proteinbande war im Immunblot reaktiv mit denselben Patientenseren, die zum Screenen der Klonbank benutzt worden waren (nicht gezeigt).The expression clone of ClaTCTP in pHISparallel2 was induced in E.coli BL21 with IPTG and a soluble protein extract was produced from the E.coli cells. The recombinant protein was soluble in the extraction buffer and not in the inclusion bodies. The extract was separated by SDS / PAGE and stained with Coomassie. A clearly overexpressed protein band was found that was missing in the control. The apparent molecular weight of the recombinant His-tagged protein (short: "histag-ClaTCTP2) was about 30 kD. This protein band was reactive in immunoblot with the same patient sera used to screen the clone library (not shown).
Beispiel 2: Reinigung von ClaTCTPExample 2: Purification of ClaTCTP
Der so analysierte Proteinextrakt wurde nun durch Ni-Chelat-Chromatographie auf Chelating Sepharose Fast Flow (Pharmacia Biotech, Uppsala, Schweden) chromatographisch gereinigt wie von Pharmacia beschrieben. Dabei wurde folgendermaßen vorgegangen.The protein extract thus analyzed was now purified by Ni chelate chromatography on chelating Sepharose Fast Flow (Pharmacia Biotech, Uppsala, Sweden) as described by Pharmacia. The procedure was as follows.
Reinigungsprozedur His-claTCTP (Immobilized Metal Affinity Chromatography, IMAC)His-claTCTP purification procedure (Immobilized Metal Affinity Chromatography, IMAC)
a) Reinigung des Histidin-qetaqqten Proteinsa) Purification of the histidine-qetaqqten protein
Die gesamte Prozedur erfolgt unter gravity flowThe entire procedure takes place under gravity flow
• Laden der Säule• Loading the column
2 ml Gel (Chelating Sepharose Fast Flow®, Pharmacia Biotech), beladen mit 0.5 Volumen 0.1 M NiS04 in einem Gelvolumen (2 ml) Starting buffer resuspendieren Starting buffer: 50 mM Na2HPO4 (pH 8.0) 300 mM NaCI 10 mM Imidazol Gel in die Säule leeren Säule mit 10 Säulenvolumen Starting buffer äquilibrieren
• Vorbereitung der Probe2 ml gel (Chelating Sepharose Fast Flow ® , Pharmacia Biotech), loaded with 0.5 volume 0.1 M NiS0 4 in a gel volume (2 ml) Resuspend starting buffer Starting buffer: 50 mM Na 2 HPO 4 (pH 8.0) 300 mM NaCI 10 mM Equilibrate Imidazol Gel into the empty column with 10 column volumes of Starting buffer • Preparation of the sample
200 ml LBamp Medium mit E.coli BL21 (His-claTCTP Klon 1 ) und 200 ml LBamp Medium mit E.coli BL21 (His-hum TCTP Klon 1 :3/2) beimpfen.Inoculate 200 ml LBamp medium with E.coli BL21 (His-claTCTP clone 1) and 200 ml LBamp medium with E.coli BL21 (His-hum TCTP clone 1: 3/2).
folgenden Schritte beziehen sich auf nur jeweils einen KlonThe following steps apply to only one clone at a time
Inkubation O/N bei 37°C 2 L LBamp Medium mit Übernachtkultur versetzen (1/10 Volumen der Übernachtkultur in frisches LBamp Medium) Inkubation bei 37°C bis OD600 = 0.6-0.8 Induktion der Proteinexpression mit 0.8 mM IPTG; Inkubation für weitere 3.5 h Zentrifugation 5000 rpm / 20 min / 4°C Überstand ableeren, Pellet O/N bei -20°C Pellet auftauen und in 60 ml Starting buffer resuspendieren Suspension in flüssigen Stickstoff frieren Suspension im 37°C Wasserbad auftauen Frieren/Tauen-Zyklus zweimal wiederholenIncubation O / N at 37 ° C Add 2 L LBamp medium with overnight culture (1/10 volume of the overnight culture in fresh LBamp medium) Incubation at 37 ° C to OD 600 = 0.6-0.8 Induction of protein expression with 0.8 mM IPTG; Incubation for a further 3.5 h centrifugation, drain 5000 rpm / 20 min / 4 ° C supernatant, thaw pellet O / N at -20 ° C pellet and resuspend in 60 ml starting buffer Freeze suspension in liquid nitrogen Thaw suspension in 37 ° C water bath Freeze / Repeat thawing cycle twice
Zugabe von Lysozym zwischen den Zyklen, bis die Suspension viskos wird bzw. die Bakterien weitgehend lysiert sind (ca. 130 μg Lysozym) Sonifizieren des Lysates (Branson Sonifier 250; 10 Pulse bei 20 %), um die genomische DNA zu scheren Zentrifugation 5000 rpm / 35 min / 4°C Filtrieren des Überstandes (0.45 μm Filter) - der Überstand enthält die lösliche ProteinfraktionAdd lysozyme between cycles until the suspension becomes viscous or the bacteria are largely lysed (approx. 130 μg lysozyme). Sonify the lysate (Branson Sonifier 250; 10 pulses at 20%) to shear the genomic DNA. Centrifugation 5000 rpm / 35 min / 4 ° C Filter the supernatant (0.45 μm filter) - the supernatant contains the soluble protein fraction
• Binden der Probe an die Säule• Bind the sample to the column
Probe in 5 ml-Schritten auf die Säule auftragen Inkubation 30 min bei Raumtemperatur - währenddessen mit sterilem Spatel mehrmals durchmischen den flow through sammeln und die nächste 5 ml Fraktion auf die Säule laden weitere 30 min inkubieren und mit Spatel durchmischen
den flow through sammeln; Prozedur wiederholen, bis die gesamte Probe aufgetragen ist.Apply the sample to the column in 5 ml increments. Incubate for 30 min at room temperature - in the meantime mix several times with a sterile spatula, collect the flow through and load the next 5 ml fraction onto the column. Incubate for another 30 min and mix with the spatula collect the flow through; Repeat the procedure until the entire sample is applied.
• Eluierung der Proteine• Elution of the proteins
Zugabe von 3 x 5 Säulenvolumen (3 x 10 ml) Starting buffer flow through in 3 separaten Fraktionen sammeln Zugabe von 2 Säulenvolumen (4 ml) Elution buffer 1 (= Starting buffer mit 100 mM Imidazol) in 1 ml Fraktionen sammeln Zugabe von 2 Säulenvolumen (4 ml) Elution buffer 2 (= Starting buffer mit 200 mM Imidazol) in 1 ml Fraktionen sammeln Zugabe von 2 Säulenvolumen (2 ml) Elution buffer 3 (= Starting buffer mit 300 mM Imidazol) in 1 ml Fraktionen sammeln Zugabe von 2 Säulenvolumen (4 ml) Elution buffer 4 (= Starting buffer mit 400 M Imidazol) in 1 ml Fraktionen sammeln Zugabe von 2 Säulenvolumen (4 ml) Elution buffer 5 (= Starting buffer mit 500 mM Imidazol) in 1 ml Fraktionen sammelnAdd 3 x 5 column volumes (3 x 10 ml) Collect starting buffer flow through in 3 separate fractions Add 2 column volumes (4 ml) Elution buffer 1 (= starting buffer with 100 mM imidazole) collect in 1 ml fractions Add 2 column volumes (4 ml) Elution buffer 2 (= Starting buffer with 200 mM imidazole) collect in 1 ml fractions Add 2 column volumes (2 ml) Elution buffer 3 (= Starting buffer with 300 mM imidazole) collect in 1 ml fractions Add 2 column volumes (4 ml) Elution buffer 4 (= Starting buffer with 400 M imidazole) collect in 1 ml fractions Add 2 column volumes (4 ml) Elution buffer 5 (= Starting buffer with 500 mM imidazole) collect in 1 ml fractions
• Proteolytischer Verdau des His-Tags mit recombinant Tobacco Etch Virus (rTEV) Protease• Proteolytic digestion of His-Tag with recombinant Tobacco Etch Virus (rTEV) protease
Verwendung von 1 ml (c = 10 mg/ml) Protein-Eluat Verwendung von rTEV Protease - kloniert und gereinigt von Universität Salzburg, Institut für Biochemie - rTEV c = 0.6 mg/ml Die Proteinprobe wird 1 :10 in Starting buffer verdünnt Für die Verdaureaktion wird das Fusionsprotein mit rTEV im Verhältnis 4:1 gemischtUse of 1 ml (c = 10 mg / ml) protein eluate Use of rTEV protease - cloned and purified by University of Salzburg, Institute of Biochemistry - rTEV c = 0.6 mg / ml The protein sample is diluted 1:10 in Starting buffer For the Digestion reaction, the fusion protein is mixed with rTEV in a ratio of 4: 1
500.0 μl Fusionsprotein (c = 1 μg/μl) = 500.0 μg 4 + 208.5 μf rTEV Protease (c = 0.6 mg/ml) = 125.1 μg 1
Inkubation O/N bei 30°C Aliquots a 250 μl (für Dialyse)500.0 μl fusion protein (c = 1 μg / μl) = 500.0 μg 4 + 208.5 μf rTEV protease (c = 0.6 mg / ml) = 125.1 μg 1 Incubation O / N at 30 ° C aliquots of 250 μl (for dialysis)
• Dialyse• dialysis
Die rTEV verdaute Probe wird gegen Starting buffer dialysiert, um eine Bindung an die Säule zu ermöglichenThe rTEV digested sample is dialyzed against starting buffer to allow binding to the column
Dialyse für 2 h bei 4°C (Puffer 3 x wechseln)Dialysis for 2 h at 4 ° C (change buffer 3 x)
• Reinigung des rTEV-verdauten Proteins durch IMAC• Purification of the rTEV-digested protein by IMAC
Die dialysierte Probe wird auf die Säule aufgetragen Inkubation 30 min / Raumtemperatur Der flow through enthält das rekombinante Protein ohne His-Tag Waschen der Säule mit 10 ml Starting buffer Eluierung der Ni-bindenden Verunreinigungen mit 500 mM Imidazol.The dialyzed sample is applied to the column. Incubation 30 min / room temperature. The flow through contains the recombinant protein without His day washing the column with 10 ml starting buffer eluting the Ni-binding impurities with 500 mM imidazole.
Beispiel 3Example 3
Das nahezu zur Homogenität gereinigte histag-claTCTP wurde mit rekombinanter TEV Protease gespalten und wieder durch Ni-Chelat-Chromatographie gereinigt. Das Protein wurde zur Homogenität gereinigt. Dieses homogene gereinigte rekombinante ClaTCTP wurde nun benützt, um aus einer Gruppe von 44 Patientenseren weitere Seren zu finden, die mit dem rekombinanten ClaTCTP im Immunblot reagieren bzw. um die Häufigkeit der Reaktion auf dieses Allergen festzustellen. Die Seren waren mit einem Rohextrakt von Cladosporium herbarum auf die Anwesenheit von immunreaktiven Banden im Bereich zwischen 20 und 30 kD vorselektiert worden. Von den 44 Patientenseren zeigten 5 eine deutliche Reaktivität mit dem rekombinanten non-fusion ClaTCTP.The histag-claTCTP, which was purified almost to homogeneity, was cleaved with recombinant TEV protease and purified again by Ni chelate chromatography. The protein was purified for homogeneity. This homogeneously purified recombinant ClaTCTP was now used to find further sera from a group of 44 patient sera, which react with the recombinant ClaTCTP in immunoblotting or to determine the frequency of the reaction to this allergen. The sera had been preselected with a crude extract from Cladosporium herbarum for the presence of immunoreactive bands in the range between 20 and 30 kD. 5 of the 44 patient sera showed a clear reactivity with the recombinant non-fusion ClaTCTP.
Parallel zur Reinigung von ClaTCTP wurde in ähnlicher Weise als Vergleichsmaterial das in der Literatur bereits beschriebene humane TCTP (MacDonald et al., Science 269, 688 (1995)) als rekombinantes Protein aufgereinigt. Es zeigte sich, daß von den eben erwähnten 5 Patienten, die mit ClaTCTP reagierten, einer (KIM) auch mit dem humanen
TCTP stark reagierte und zwei weitere Patienten (CG, FW) eine etwas schwächere Reaktion zeigten. Dieses Ergebnis belegt, daß eine Kreuzreaktivität zwischen dem aus Cladosporium herstammenden Protein und einer körpereigenen Struktur (humanes TCTP) auftreten kann. Dies kann Ursache für Erkrankungserscheinungen sein, die auf Autoimmunmechanismen zurückzuführen sind.
In parallel to the purification of ClaTCTP, the human TCTP (MacDonald et al., Science 269, 688 (1995)) already described in the literature was purified as a comparative material in a similar manner as a recombinant protein. It was shown that of the 5 patients just mentioned who reacted with ClaTCTP, one (KIM) also with the human TCTP reacted strongly and two other patients (CG, FW) showed a somewhat weaker reaction. This result shows that cross-reactivity between the Cladosporium-derived protein and an endogenous structure (human TCTP) can occur. This can be the cause of symptoms that are due to autoimmune mechanisms.
Claims
Patentansprücheclaims
1) Polypeptid, dadurch gekennzeichnet, daß es wenigstens 10 aufeinanderfolgende Aminosäuren aus der Aminosäuresequenz mit der Sequenz ID Nr. 1 aufweist.1) Polypeptide, characterized in that it has at least 10 consecutive amino acids from the amino acid sequence with the sequence ID No. 1.
2) Polypeptid nach Anspruch 1 , dadurch gekennzeichnet, daß es wenigstens 15 aufeinanderfolgende Aminosäuren aus der Aminosäuresequenz mit der Sequenz ID Nr. 1 aufweist.2) Polypeptide according to claim 1, characterized in that it has at least 15 consecutive amino acids from the amino acid sequence with the sequence ID No. 1.
3) Polypeptid nach einem der Ansprüche 1 oder 2, dadurch gekennzeichnet, daß es wenigstens ein Epitop aufweist.3) Polypeptide according to one of claims 1 or 2, characterized in that it has at least one epitope.
4) Polypeptid nach Anspruch 3, dadurch gekennzeichnet, daß das Epitop spezifisch ist für einen Schimmelpilz.4) polypeptide according to claim 3, characterized in that the epitope is specific for a mold.
5) Polypeptid nach Anspruch 4, dadurch gekennzeichnet, daß der Schimmelpilz zu der Gattung Cladosporium gehört.5) polypeptide according to claim 4, characterized in that the mold belongs to the genus Cladosporium.
6) Polypeptid nach Anspruch 5, dadurch gekennzeichnet, daß es sich bei dem Schimmelpilz um Cladosporium herbarum handelt.6) Polypeptide according to claim 5, characterized in that it is Cladosporium herbarum in the mold.
7) Impfstoff zur Desensibilisierung von Patienten gegen eine Schimmelpilzallergie, dadurch gekennzeichnet, daß er wenigstens ein Polypeptid nach einem der Ansprüche 1 bis 6 enthält.7) Vaccine for the desensitization of patients against a mold allergy, characterized in that it contains at least one polypeptide according to one of claims 1 to 6.
8) Verwendung eines Polypeptids nach einem der Ansprüche 1 bis 6 zur Herstellung eines Impfstoffes.8) Use of a polypeptide according to one of claims 1 to 6 for the preparation of a vaccine.
9) Testkit zum Nachweis einer allergischen Reaktion in Körperflüssigkeiten, dadurch gekennzeichnet, daß es wenigstens ein Polypeptid nach einem der Ansprüche 1 bis 6 enthält.
10) Verwendung eines Polypeptids nach einem der Ansprüche 1 bis 6 zur diagnostischen Bestimmung einer allergischen Erkrankung.9) Test kit for detecting an allergic reaction in body fluids, characterized in that it contains at least one polypeptide according to one of claims 1 to 6. 10) Use of a polypeptide according to one of claims 1 to 6 for the diagnostic determination of an allergic disease.
1 1 ) Polynucleotid, dadurch gekennzeichnet, daß es wenigstens 10 aufeinanderfolgende Nucleotide aus der Nucleotidsequenz mit der Sequenz ID Nr. 2 aufweist.1 1) Polynucleotide, characterized in that it has at least 10 consecutive nucleotides from the nucleotide sequence with the sequence ID No. 2.
12) Polynucleotid nach Anspruch 1 1 , dadurch gekennzeichnet, daß es wenigstens 15 aufeinanderfolgende Nucleotide aufweist.12) polynucleotide according to claim 1 1, characterized in that it has at least 15 consecutive nucleotides.
13) Polynucleotid nach Anspruch 11 , dadurch gekennzeichnet, daß es wenigstens 20 aufeinanderfolgende Nucleotide aufweist.13) Polynucleotide according to claim 11, characterized in that it has at least 20 consecutive nucleotides.
14) Testkit zum Nachweis von Nucleinsäure von Cladosporium, dadurch gekennzeichnet, daß es wenigstens ein Polynucleotid nach einem der Ansprüche 11 bis 13 enthält.14) Test kit for the detection of Cladosporium nucleic acid, characterized in that it contains at least one polynucleotide according to one of claims 11 to 13.
15) Verwendung eines Polynucleotids nach einem der Ansprüche 11 bis 13 zur Herstellung einer Nucleinsäuresequenz kodierend für wenigstens einen Teil eines Polypeptids nach einem der Ansprüche 1 bis 6.15) Use of a polynucleotide according to one of claims 11 to 13 for the production of a nucleic acid sequence coding for at least part of a polypeptide according to one of claims 1 to 6.
16) Verwendung nach Anspruch 15, dadurch gekennzeichnet, daß wenigstens ein Polynucleotid nach einem der Ansprüche 11 bis 13 in einer Polymerase Kettenreaktion eingesetzt wird.16) Use according to claim 15, characterized in that at least one polynucleotide according to one of claims 11 to 13 is used in a polymerase chain reaction.
17) Vektor zur Transformation einer Wirtszelle, dadurch gekennzeichnet, daß er ein Polynucleotid nach einem der Ansprüche 11 bis 13 beinhaltet.17) vector for transforming a host cell, characterized in that it contains a polynucleotide according to one of claims 11 to 13.
18) Wirtszelle, dadurch gekennzeichnet, daß sie mit einem Vektor gemäß Anspruch 17 transformiert wurde.18) host cell, characterized in that it was transformed with a vector according to claim 17.
19) Wirtszelle nach Anspruch 18, dadurch gekennzeichnet, daß es sich um Escherichia coli handelt.19) Host cell according to claim 18, characterized in that it is Escherichia coli.
20) Wirtszelle nach Anspruch 18, dadurch gekennzeichnet, daß es sich um eine Hefe handelt.
21) Verfahren zur rekombinanten Herstellung eines Polypeptids nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß eine Wirtszelle nach einem der Ansprüche 18 bis 20 angezogen wird, wobei das Polypeptid exprimiert und gereinigt wird.
20) Host cell according to claim 18, characterized in that it is a yeast. 21) Process for the recombinant production of a polypeptide according to one of claims 1 to 6, characterized in that a host cell according to one of claims 18 to 20 is grown, the polypeptide being expressed and purified.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DE10333217.0 | 2003-07-22 | ||
DE10333217A DE10333217A1 (en) | 2003-07-22 | 2003-07-22 | Polypeptides, encoding nucleic acids from the mold Cladosporium herbarum and their preparation and use in diagnostics and therapy |
Publications (1)
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WO2005010037A1 true WO2005010037A1 (en) | 2005-02-03 |
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PCT/EP2004/008098 WO2005010037A1 (en) | 2003-07-22 | 2004-07-20 | Polypeptides, nucleic acids coding therefor, from the mold fungus cladosporium herbarum and the production and use thereof in diagnosis and therapy |
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001068826A2 (en) * | 2000-03-14 | 2001-09-20 | Syngenta Participations Ag | Protoporphyrinogen oxidase ('protox') genes |
WO2002042481A1 (en) * | 2000-11-27 | 2002-05-30 | Dnavec Research Inc. | Paramyxovirus vector encoding angiogenesis gene and utilization thereof |
-
2003
- 2003-07-22 DE DE10333217A patent/DE10333217A1/en not_active Withdrawn
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2004
- 2004-07-20 WO PCT/EP2004/008098 patent/WO2005010037A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001068826A2 (en) * | 2000-03-14 | 2001-09-20 | Syngenta Participations Ag | Protoporphyrinogen oxidase ('protox') genes |
WO2002042481A1 (en) * | 2000-11-27 | 2002-05-30 | Dnavec Research Inc. | Paramyxovirus vector encoding angiogenesis gene and utilization thereof |
Non-Patent Citations (2)
Title |
---|
BREITENBACH MICHAEL ET AL: "The allergens of Cladosporium herbarum and Alternaria alternata", CHEMICAL IMMUNOLOGY. FUNGAL ALLERGY AND PATHOGENICITY S. KARGER PUBLISHERS INC., 79 FIFTH AVENUE, NEW YORK, NY, 10003, USA; S. KARGER AG, CH-4009, BASEL, SWITZERLAND SERIES : CHEMICAL IMMUNOLOGY (ISSN 1015-0145), 2002, pages 48 - 72, XP001183976, ISSN: 3-8055-7391-X * |
HOLLER C ET AL: "Histamine releasing factor (TCTP) is an auto-reactive IgE-binding antigen in patients allergic to Cladosporium herbarum.", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 113, no. 2 Supplement, February 2004 (2004-02-01), & 60TH ANNUAL MEETING OF THE AMERICAN ACADEMY OF ALLERGY, ASTHMA AND IMMUNOLOGY (AAAAI); SAN FRANCISCO, CA, USA; MARCH 19-23, 2004, pages S299, XP002303197, ISSN: 0091-6749 * |
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