DE10214082A1 - New polypeptide allergens from fungi, useful in desensitizing vaccines and for diagnosis, also related nucleic acids, vectors and transformed cells - Google Patents

Abstract

Polypeptides allergens from fungi (I): (a) containing at least 11 consecutive amino acids (aa) from sequences (A1) or (A8), of 173 or 267 aa, respectively; or (b) comprising sequences (A4), (A5) or (A6), of 16, 11 and 10 aa, respectively, are new. Independent claims are also included for: (1) polypeptides (Ia) comprising at least 11 consecutive aa from a sequence (11) of 266 aa; (2) vaccine for desensitizing patients against mold allergens containing at least one (I) or (Ia); (3) polynucleotides (II) containing at least 8 consecutive nucleotides (nt) from sequences (N2), (N7) or (N12) of 519, 918 and 1026 nt, respectively; (4) transformation vectors containing (II); (5) host cell containing the vector of (4); and (6) method for recombinant production of (I) or (Ia) by culturing cells of (5).

Description

Various substances cause allergic reactions in certain people. Not only are allergies known to components of animals, such as cat hair, but also allergies to plants or parts of plants, such as bee pollen. But are known also allergies to microorganisms, such as mold.

The present invention relates to the main allergen of the Cladosporium mold herbarum. In the context of the present invention, it was found that this Surprisingly, the main allergen is a mannitol dehydrogenase.

One aspect of the present invention relates to a polypeptide which has at least 10 consecutive amino acids from the amino acid sequence with the sequence ID No. 1. The amino acid sequence is shown in FIG. 1, as is the associated DNA sequence. The invention also relates to the polypeptides with the Seq. ID No. 4, 5 and 6.

A polypeptide according to the invention preferably has at least one epitope. Under a Epitope is an area to which antibodies can bind. Basically there is linear epitopes. In this case, the amino acids forming the epitope lie side by side However, the so-called conformation epitopes are more common. These are created by folding the Polypeptide formed. Amino acids that are not adjacent in the sequence can come into spatial proximity due to the three-dimensional folding of the polypeptide and this surface structure is bound by an antibody.

The epitopes are preferably specific for a mold. Basically, with the help suitable techniques, for example using adjuvants, antibodies against almost all amino acid sequences are generated. However, the polypeptides according to the invention play play an essential role in diagnostics and therapy. Therefore, the invention Polypeptides prefer specific epitopes, the epitopes being specific for one Are mold. Frequently, proteins or polypeptides have a certain Organism, similarities to a corresponding protein or polypeptide comes from a related organism. It is therefore possible that antibodies against an Epitope of a certain mold are directed, also with a corresponding epitope of a related mold react.

The more specific an epitope, the less antibodies against it react with one Epitope of a homologous polypeptide from a related organism. The Polypeptides according to the invention therefore preferably have those epitopes that are specific for a mold. The polypeptides particularly preferably have epitopes which are specific for a mold from the genus Cladosporium. Most notably the polypeptides preferably have an epitope which is specific for Cladosporium herbarum. Antibodies directed against such an epitope do not react with others Polypeptides.

In the context of the present invention it was found that the polypeptide with the Amino acid sequence ID No. 1 encoded for a mannitol dehydrogenase. Subject of present invention are also parts of this amino acid sequence with at least 11 amino acids. The polypeptides according to the invention preferably have at least 20 consecutive amino acids from the sequence with the sequence ID No. 1. preferred are those polypeptides which have at least 50 consecutive amino acids, and those polypeptides that are at least 100 consecutive are very particularly preferred Have amino acids from the amino acid sequence with the sequence ID No. 1.

The invention also relates to an N-terminus polypeptide with the sequence PGQQATKHESLLDQLS (Seq. ID No. 4) and two polypeptides from the C-terminal end with the Sequence LDTGLSDFVVK (Seq. ID No. 5) and MGRDGLAKEL (Seq. ID No. 6).

Another object of the present invention is a vaccine used for desensitization can be used by patients against a mold allergy. In the Desensitization becomes patients who suffer from an allergy with a small amount of one Antigen brought into contact, whereby neutralizing IgG antibodies are to be formed. Through these neutralizing antibodies, the antigens when the patient with the Antigen has come into contact. This will make the bond from allergic Avoided IgE-type triggering antibodies. The invention Polypeptides can therefore be used to make a vaccine. You can do this the recombinantly or chemically produced polypeptides into a suitable one Vaccine formulation to be incorporated. The vaccine formulation can be used in addition to Polypeptides also common additives and formulation aids, as well as adjuvants contain.

Another object of the present invention is the use of at least one Polypeptide according to the invention for the diagnostic detection of a disease. At a Such a disease is usually an allergy. The polypeptides are in a suitable diagnostic detection system. This can be one Radioimmunassay (RIA) act, or preferably also an ELISA (Enzyme Linked Immunosorbent assay). The usual configuration of such a diagnostic test is known.

Another aspect of the present invention relates to nucleotides from the nucleotide sequence with the sequence ID No. 2. The nucleotide sequence with the sequence ID No. 2 is also shown in FIG. 1. This is the gene for the mannitol dehydrogenase from Cladosporium herbarum.

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 areas of application, the nucleotides be longer, in this case the polynucleotides have at least 100 consecutive ones Nucleotides selected from Sequence ID No. 2.

The polynucleotides according to the invention can be used to detect a mannitol dehydrogenase serve. The presence of a gene coding for these mannitol is preferred. Detected dehydrogenase from Cladosporium herbarum. These are the procedures are known nucleic acid amplification methods. This comes for example the NASBA method in question or, more preferably, the polymerase chain reaction (PCR).

With the help of such a procedure, the presence of the mold Cladosporium can herbarum can be detected. Such applications are not only medical Diagnostics of interest, but also in other areas, such as hygiene and examining food. It must be taken into account here that Cladosporium herbarum can grow up to about + 6 ° C even at relatively low temperatures and therefore represent an undesirable contamination in areas of food technology can. For the monitoring of food, the detection of even small quantities can be done from Cladosporium herbarum play an essential role.

In a further aspect of the present invention, a method for producing a polypeptide according to the invention disclosed. First, with the help of the invention Polynucleotides and with the help of the polymerase chain reaction a gene from a mold, preferably from Cladosporium herbarum. This polynucleotide can then be used in a suitable vector is inserted, with which a host cell is transformed. suitable Vectors multiply in the host cell and the polypeptides are expressed. Both Host cells can be customary host cells. Bacterial come into question here Host cells such as Escherichia coli or Bacillus subtilis or yeasts such as Sacharomyces cerevisiae or Pichia pastoris.

In the context of the present invention, IgE immunoblots were made from crude extract Cladosporium herbarum tested with 62 patient sera. This showed an immunoreactive 29 kD molecular weight protein recognized by 61% of patient sera. The Patients were pre-selected in the skin test or blood test (RAST) and responded positively to Cladosporium herbarum extract. No other allergen in the Cladosporium herbarum extract responds with such a large percentage of patient sera. It is therefore assumed that this protein is the main allergen of Cladosporium herbarum.

In the two-dimensional separation by isoelectric focusing and SDS-PAGE this protein is shown as a spot of 29 kD and the isoelectric point at pH = 5.8.

The protein was purified to homogeneity in a conventional method (Example 1). The Yield was 1 mg. The homogeneously purified protein was now in the IgE immunoblot with a Pool of six patients tested and was strongly positive (Example 2).

The protein purified to homogeneity became N-terminal by automated Edman degradation sequenced and internal peptide sequences were determined after CNBr degradation.

N-terminal and internal peptide sequences after trypsin digestion were by Edman degradation of about 50 µg protein is determined by cutting out a spot from the two-dimensional Electrophoresis were obtained.

Table 1 shows a list of all peptide sequences found. The "single letter code" used. The amino acids highlighted in black in Table 1 could in the sequence of Mannitol dehydrogenase from Cladosporium fulvum can be found.

Table 1 Peptide sequences of the C. herbarum NADP-dependent mannitol dehydrogenase N-terminal sequence PGQQATKHESLLDQXSXK

• a) Starting material: raw extract separated by means of 2-dimensional SDS gel
• b) Analysis method: Edman sequencing

PGQQATKHESLLDQLSLKG

a) Starting material: natively purified protein

b) Analysis method: Edman sequencing

Peptide 1 HESLLDQLSLK

• a) Starting material: crude extract separated using 2-dim. SDS-gel
• b) Analysis method: MS / MS
• c) Note: overlaps with the N-terminal sequence

Peptide 2 VVVVTGASGP

• a) Starting material: crude extract separated using 2-dim. SDS-gel
• b) tryptic digestion
• c) Analysis method: sequencing

WVVVVTGASKR

• a) Starting material: crude extract separated using 2-dim. SDS-gel
• b) tryptic digestion
• c) Analysis method: MS / MS

Peptide 3 QVDSYE

• a) Starting material: crude extract separated using 2-dim. SDS-gel
• b) tryptic digestion
• c) Analysis method: sequencing

Peptide 4 LDTGLSDFVVK

• a) Starting material: crude extract separated using 2-dim. SDS-gel
• b) tryptic digestion
• c) Analysis method: MS / MS

Peptide 5 MGRDGLAKEL

• a) Starting material: natively purified protein
• b) CnBr digestion
• c) Analysis method: sequencing

The peptide sequences were compared by computer-assisted homology search with all protein sequences listed in the databases (Swissprot, Genbank etc.). All peptides showed homology to the mannitol dehydrogenase family. The mannitol dehydrogenase to which the peptides have the highest sequence similarity is the mannitol dehydrogenase from Cladosporium fulvum. The position of these peptides in the sequence can be seen in the "alignment" (Table 2). Protein alignment of NADP-MtDH from C. fulvum with peptide sequences from C. herbarum

C. fulvum MtDH: accession number: AAK67169 (Seq. ID No. 3)

Length of the coding sequence:
267 amino acids (AA)
801 base pairs (bp)

MW: 28.6kD
pl: 6.33

Table 2

Table 3 shows the arrangement of the polypeptides with the Seq. ID No. 4, 5 and 6 based on the Homology shown with C. fulvum.

Based on these results, the enzyme activity of the protein purified for homogeneity was examined. The experiments show that the highly purified major allergen of Cladosporium herbarum is actually a mannitol dehydrogenase, which catalyzes the following metabolic reaction: fructose + NADPH + H ⁺ ⇔ mannitol + NADP ⁺ . It was also found that NADH is not active as a co-substrate and that fructose-6-phosphate is also not active as a substrate. Fructose-6-phosphate acts as an inhibitor on the reaction. The method of determining activity is described in Example 3.

There have now been the N-terminal and an internal peptide sequence of mannitol dehydrogenase used by Cladosporium herbarum to design PCR primers by back translation. The Selection of the primers is summarized in Table 3.

DNA sequence of the oligos derived from the peptides Oligo 1

• - Derived from the N-terminal sequence of mannitol dehydrogenase (MtDH) from C. herbarum
• - Oligo sequence: 5 'CA (A / G) CA (A / G) GC (I / C) AC (I / C) AA (A / G) CA (C / T) GA 3'

Oligo 2

• - Derived from peptide 4 of MtDH from C. herbarum
• - Oligo sequence: 5 'AC (A / G) AA (A / G) TC (A / G) CT (I / C) AG (I / C) CC (A / G) GT (A / G) TC 3'

The primers are mixtures of synthetic oligonucleotides.

(A / G). , , means that at this point both adenine and guanine in the Oligonucleotides occur. Likewise with (C / T) and (I / C), where I for the base inosine stands.

Table 3

A PCR reaction with DNA from a Cladosporium herbarum cDNA library constructed by the inventors was carried out with these primers shown in Table 3 (Achatz G et al., 1995, Mol. Immunol., 32; 213-27). The result was a band of 636 bp. This was sequenced by automated DNA sequencing according to Sanger (1977, Proc. Natl. Acad. Sci. USA, 74; 5463-7) using the PCR primers as sequencing primers. Seq.ID No. 2 was determined. The protein sequence derived from this DNA sequence (Seq. ID No. 1) is 87% identical to the protein sequence of mannitol dehydrogenase from Cladosporium fulvum. If one also takes into account the substitution by chemically related amino acids (e.g. IV, isoleucine valine etc.), this value increases to 92%. Under the plausible assumption that the mannitol dehydrogenase from Cladosporium herbarum as well as the mannitol dehydrogenase from Cladosporium fulvum has a total length of 267 amino acids, peptide sequencing on the one hand and DNA sequencing on the other hand already made 65% of the amino acid sequence of the main allergen (mannitol dehydrogenase ) determined from Cladosporium herbarum. The entire previously known sequence of this protein and its alignment with the homologous sequence from Cladosporium fulvum is shown in FIG. 2.

example 1 protein purification 1. Ammonium sulfate precipitation

With an ammonium sulfate concentration of 50%, a pre-fractionation can be achieved with mannitol dehydrogenase (MtDH) remaining in the supernatant.

50 mM Tris-HCl, pH 7.5 was added to the extract. Proteases were taken with 1 Roche tablet Complete 100 ml extract and 2 mM EDTA inhibited.

The precipitation was carried out by adding solid, ground ammonium sulfate and was in two Steps performed, first 0-30%, then 30-50%. The precipitation was for at least 45 min., Equilibrated before the extract was centrifuged at 12,000 g. The supernatant was filtered and further purified using hydrophobic interaction chromatography (HIC).

2. HIC (phenyl sepharose)

The supernatant from the ammonium sulfate precipitation was adjusted to pH 6.5 with 3 M sodium acetate set. The column (8 ml Source 15 PHE, PHARMACIA) was washed with 1.2 M ammonium sulfate, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA equilibrated and at a flow rate of 1 ml / min with the Load sample. After washing the column with 20 ml of buffer, the gradient was over 40 ml of 1.2 M ammonium sulfate to 0.6 M ammonium sulfate eluted.

The mannitol dehydrogenase (MtDH) fractions were pooled and used for the anion exchanger prepared. The volume is concentrated using Centricon centrifuge tubes (Millipore); Buffer exchange for 50 mM Tris-HCl, pH 7.5 using PD-10 Desalting Columns (Amersham-Pharmacia).

3. Anion exchanger (Q-Sepharose)

The column (8 ml Source 15 Q, PHARMACIA) was equilibrated with 15 mM Tris-HCl, pH 7.5. eluted was with a NaCl gradient of 0-300 mM over 100 ml.

Example 2 Immunoblot of the natively purified MtDH after separation in the SDS gel

Native purified MtDH was separated in a reducing SDS gel (Laemmli UK, Nature, 1970; 27: 680-5) according to the molecular weight. The protein was then transferred to a PVDF membrane in a Western blot (Towbin H et al., Proc. Natl. Acad. Sci USA, 1979; 76: 4350-4). After saturation of free binding sites (30 min in blocking buffer: 50 mM Na phosphate pH 7.5, 0.5% Tween20, 0.5% BSA, 0.05% NaN ₃ ), the membrane was incubated with patient serum (diluted 1:10 in blocking buffer) , The membrane was then washed (3 × 10 min with blocking buffer) to remove non-specifically bound antibodies. The detection of specifically bound IgE-Ab was carried out with the help of a ¹²⁵ I-labeled rabbit-anti-human IgE antibody. After exposing the membrane to an X-ray film, the result could be read.

Results

• 1. The natively purified MtDH reacts specifically with the IgE antibodies of C. herbarum Allergy sufferers. A prominent IgE-reactive band at 29 kD is shown.
• 2. The same result, namely a prominent IgE-reactive band at 29 kD, is obtained when a C. herbarum extract is separated in the SDS gel and then in the immunoblot is incubated with patient serum.

Example 3 Immunoblot of the neatly purified MtDH after 2-dimensional separation

Native purified MtDH was separated under denaturing conditions in an isoelectric focusing (O'Farrel PH, J. Biol. Chem., 1975; 250: 4007-21) according to the net charge (isoelectric point) of the protein. The protein separated in this way was then subjected to SDS gel electrophoresis (Laemmli UK, Nature, 1970; 27: 680-5), which additionally resulted in a separation by molecular weight. The protein was transferred to a PVDF membrane in a Western blot (Towbin H et al., Proc. Natl. Acad. Sci USA, 1979; 76: 4350-4). After saturation of free binding sites (30 min in blocking buffer: 50 mM Na phosphate pH 7.5, 0.5% Tween 20, 0.5% BSA, 0.05% NaN ₃ ), the membrane was incubated with patient serum (diluted 1:10 in blocking buffer ). The membrane was then washed (3 × 10 min with blocking buffer) to remove non-specifically bound antibodies. The detection of specifically bound IgE-Ab was carried out using a ¹²⁵ I-labeled rabbit-anti-human IgE antibody. After exposure of the membrane to an X-ray film, the following results could be seen:

Results

• 1. The natively purified MtDH reacts specifically with the IgE antibodies of C. herbarum Allergy sufferers. A prominent IgE-reactive spot with a molecular weight of 29 kD and an isoelectric point of 5.8.
• 2. The same result, namely a prominent IgE-reactive spot on one Molecular weight of 29 kD and an isoelectric point of 5.8 are obtained when a C. Herbarum total extract separated in a two-dimensional gel and then in an immunoblot is incubated with patient serum. In addition, an IgE-reactive is found in the total extract Protein with a molecular weight of 29 kD and an isoelectric point of 5.6. This Protein could be an isoform of MtDH.

Example 4

To demonstrate the results according to the invention, the enzyme activity was determined using the conventionally purified protein. The absorption of NADPH was measured at 340 nm in the photometer. Reaction mixture (1 ml) 50 mM Tris-HCl, pH 7.5
0.25 mM NADPH or NADH
D-fructose or fructose-6-phosphate (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 M)
ad 1 ml H ₂ O
Start the reaction with 0.5 µl Cla h1 results. Reaction with fructose and NADPH.
No reaction with fructose-6-phosphate and NADH. SEQUENCE LISTING

Claims (28)

1. polypeptide, characterized in that it contains at least 11 consecutive amino acids from the amino acid sequence with the sequence ID No. 1 or a sequence selected from Seq. ID No. 4, 5 and 6. 2. Polypeptide according to claim 1, characterized in that it has at least one epitope having. 3. Polypeptide according to claim 2, characterized in that the epitope is specific for a mold. 4. Polypeptide according to claim 3, characterized in that the mold to the Belongs to the genus Cladosporium. 5. Polypeptide according to claim 4, characterized in that it is in the Mold is Cladosporium herbarum. 6. Polypeptide according to one of the preceding claims, characterized in that the amino acid sequence with sequence ID No. 1 for a mannitol dehydrogenase coded. 7. Polypeptide according to one of the preceding claims, characterized in that that it has at least 20 consecutive amino acids from Sequence ID No. 1. 8. Polypeptide according to one of claims 1 to 6, characterized in that it has at least 50 consecutive amino acids from sequence ID No. 1. 9. Polypeptide according to one of claims 1 to 6, characterized in that it has at least 100 consecutive amino acids from sequence ID No. 1. 10. vaccine to desensitize patients to a mold allergy, characterized in that it contains at least one polypeptide according to one of Claims 1 to 9 contains. 11. Use of a polypeptide according to one of claims 1 to 9 for the preparation of a vaccine. 12. Use of a polypeptide according to one of claims 1 to 9 for diagnostic evidence of a disease. 13. Use according to claim 11 or 12, characterized in that it is the disease is an allergy. 14. Polynucleotide, characterized in that it is at least eight consecutive Has nucleotides from the nucleotide sequence with the sequence ID No. 2. 15. Polynucleotide according to claim 14, characterized in that it is at least 12th has consecutive nucleotides. 16. Polynucleotide according to claim 14, characterized in that it is at least 20 has consecutive nucleotides. 17. Polynucleotide according to claim 14, characterized in that it is at least 50 has consecutive nucleotides. 18. Polynucleotide according to claim 14, characterized in that it is at least 100 has consecutive nucleotides. 19. Use of a nucleotide according to any one of claims 14 to 18 for detection a mannitol dehydrogenase. 20. Use according to claim 19, characterized in that it is a Mannitol dehydrogenase is from a mold. 21. Use according to claim 20, characterized in that it is in the Mold is Cladosporium herbarum. 22. Use of a nucleotide according to one of claims 14 to 18 for the production a nucleic acid sequence coding for at least part of a mannitol Dehydrogenase. 23. Use according to one of claims 19 to 22, characterized in that at least one polynucleotide according to any one of claims 14 to 18 in a polymerase Chain reaction (PCR) is used. 24. Vector for transforming a host cell, characterized in that it is a Nucleic acid according to one of claims 14 to 18. 25. Host cell, characterized in that it contains a vector according to claim 24 has been transformed. 26. Host cell according to claim 25, characterized in that it is Escherichia coli acts. 27. Host cell according to claim 25, characterized in that it is Pichia pastoris acts. 28. Process for the recombinant production of a polypeptide according to one of the Claims 1 to 9, characterized in that a host cell according to one of the claims 25 to 27 is attracted, the polypeptide being expressed and purified.