JP2003504051A - DNA encoding β-tubulin and use thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates to a DNA encoding β-tubulin derived from a Strongylidae nematode, a polypeptide encoded by the DNA, a diagnosis of anthelmintic drug resistance in the nematode, and a parasitic nematode species. The invention relates to the use of DNA for the identification of bacteria, the use of β-tubulin as a component of a vaccine, and to a method of identifying novel anthelmintic or antibiotic compounds.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to β derived from nematodes of the genus Strongyidae.
-Tubulin-encoding DNA, polypeptides encoded by this DNA, use of DNA for diagnosis of anthelmintic drug resistance of these nematodes and identification of species of these nematodes, as a component of vaccines Of .beta.-tubulin and a method for identifying new anthelmintic or antibiotic compounds.

Parasitic helminths (worms) are a health problem for humans and animals and cause considerable economic loss. In 1996, spending on anthelmintic agents worldwide was over US $ 2 billion. The most important anthelmintics currently used can be divided into three groups according to their mechanism of action: The cyclic amidines pyrantel and morantel act with the imidazothiazoles tetramisole and levamisole as cholinergic compounds on the parasite nervous system.

2. Benzimidazoles are inhibitors of microtubule polymerization and lead to tubulin degradation followed by loss of many cellular functions such as intracellular trafficking and cell division.

3. Macrocyclic lactones are bound by glutamine-acting chlorine channels to open,
Therefore, it acts as an inhibitor of the nervous system of nematodes and arthropods.

Microtubules are composed of tubulin subunits. Tubulin is α-
And β-tubulin, and are dimeric proteins present in the dynamic equilibrium between tubulin and microtubules. This equilibrium can be influenced by exogenous substances designed as microtubule inhibitors. Some of these inhibitors, such as benzimidazole, act by their binding to tubulin, so that they prevent self-association of these subunits into growing microtubules, while At the end, microtubule degradation continues. This results in dysfunction in intracellular processes that are important for survival, and ultimately in death of cells and complete organisms (Lacey, E.
1990) Mode of action of benzimidazole
s. Parasitology Today 6 , 112-115). Such microtubule inhibitors include various classes of compounds that are synthetically produced or produced by different organisms.

The binding of microtubule inhibitors to tubulin from various organisms shows great differences in binding affinity. Therefore, the anthelmintic agents oxfendal and thiabendall show a high affinity for tubulin from Ascaridia galli, but a weak affinity for tubulin from mammals such as sheep (Dawson). et al (1983) Pu
refrigeration and characterisation of
ubulin from the parasitic nematode, A
scarida galli, Molecular and Bioche
medical Parasitology 7 , 267-277). The selective toxicity of benzimidazoles can be explained by this selective affinity (Lac
ey, E.E. (1988) The Role of the Cytoskele
tal protein, tubulin, in the mode of a
action and mechanism of drug resistor
ce to benzimidazole, International Jo
(Urnal for Parasitology 18 , 885-936).

[0007]   The widespread use of these anthelmintic agents has led to all three classes, especially in livestock.
Which caused a non-negligible resistance problem (Bauer et al. (199
4) Anthelmintic resistance in nematod
es of farm animals. A seminal organis
ed for the European Commission, Brass
els, Belgium, 8 to 9 November 1993, pp.
17-24). The most commonly used type of anthelmintic is benzimidazole.
Resistance to benzimidazole is associated with the parasites of sheep, cattle, pigs and horses.
In case it is reported worldwide. Benzimidazole is a nematode, tapeworm
It is a broad-spectrum anthelmintic agent with action against trematodes. German stallion
Nursing studies have shown that in more than 80% of cases, small studies on benzimidazole
It showed resistance to strong strongids (Ul
lrich et al. (1988) Benzimidazole resi
stance in small strengths (Cyathost
Ominae): distribution in horse stock
in Northrhine-Westphalia, Berliner Mu
enchner Tiereezriche Wochenschrift 101 , 406-408).

[0008] Therefore, for effective treatment with anthelmintic agents it is very important to have information about possible resistance of parasitic helminths within a single horse or herd of horses. In order to test the possible resistance of the parasite helminthic populations of the small strongild, a diagnostic procedure based on the activity of anthelmintic agents at the stage of parasite development (egg, larva) has already been developed (Coles et al. (1992) World Associ
ation for the Advancement of Veterin
ary Parasitology (WAVP) Methods
for the detection of anthelmintic re
science in nematodes of veternary
importance, Veterinary Parasitology, 4 4, 35-44). The "larval development assay" (LDA) describes the inhibitory effect of anthelmintic agents in the nonparasitic first larval stage as a function of the anthelmintic agent concentration used.
Similarly, a second approach, the "egg hatch assay" (EHA), uses the anthelmintic inhibitory effect on larval hatching as a function of the anthelmintic concentration used. The drawback of both approaches is the low reproducibility of the results. Moreover, both approaches are time consuming and labor intensive.

Moreover, the sensitivity of both approaches is low. The presence of resistance is only detected when 25% or more of the population is resistant (Roos et al. (1995) New ge.
netic and practical implications of s
selection for anthelmintic resistance
in parasitic nematodes, Parasitology
Today 11 , 148-150).

Therefore, there is an urgent need for sensitive, rapid, and reproducible diagnostic procedures. In sheep parasites Haemonchus-contortus (Haemonch us contortus) and Teradorusagia Sur Kamushin Kuta (Tel adorsagia circumcincta), the operation based on the PCR technique is described. This is codon 2 of the β-tubulin isotype 1 gene.
Based on at least one point mutation in 00 (Elard et al.
(1999) PCR diagnosis of benzimidazole
-Susceptibility or -resistance in na
tural populations of the small rumin
ant parasite, Teladorsaga circumcinc ta , Veterinary Parasitology 80 , 231-23
7). A point mutation at codon 200 results in an amino acid substitution of phenylalanine with tyrosine and correlates with the benzimidazole resistance of the mutein (Kwa et al. (1995) β-tubulin genes.
from parasitic nematode Haemonchus contortus modulad drug residency in
Caenorhabditis elegans , Journal of M
molecular Biology 246 , 500-510).

From the study in Hemoncus contortus, it is known that the sequence of nematodes encoding tubulin is species-specific (WO92 / 03549).

Different species of equine small stronguilds differ in their epidemiological frequency.
The most important and most frequently found species worldwide are Cylicocyclus nassatus , Cyatostomum coronatum .
And Cyathostomum catina tum . The resistance of these species to benzimidazole has been described in various countries, especially in Germany (Burger, H.-J.
and Bauer, C.I. (1987) Efficiency of four a
nthermintics against benzimidazole-r
Esistence cythosomes of horse, Vetera
inary Record 120, 293-296).

The nucleic acid sequence of the β-tubulin cDNA from the small strongild species according to the present invention had greater than 95% identity. However, the identity of the sheep parasite with the known β-tubulin sequences is only 75.4-82.6%.

The deduced amino acid sequences are very similar within the sequences according to the invention. This is also the case for the putative β-tubulin amino acid sequence of sheep parasites.
The identity here is 95-99.8%. Amino acid substitutions occur only at very rare positions. Highlighted here is codon 200, where a phenylalanine to tyrosine change occurs.

However, non-coding β-tubulin sequences from various previously published helminthic species do not show significant identity. It is therefore surprising that not only the coding sequences but also some of the non-coding sequences of the various species of the small strongilds of the present application presented here have a high degree of identity.

[0016] Thus, these regions are also suitable for distinguishing various species of the small Strongyld from other nematode species. PCR primers derived from these intron regions can also be used for the specific detection of small strongilds in samples containing genetic material from other helminthic organisms.

Β-tubulin derived from C. elegans or a part thereof is known to have prophylactic and immunological potential (Buguio et al. (1993) Cha.
lacterization and biological biological activation
ies of anti- Brugia pahangi tubulin m
onoclonal antibodies, International J
individual for Parasitology, 7 , 913-924). The small stronguild β-tubulin encoded by the above DNA can be used as a vaccine, just as a monoclonal antibody can be used against β-tubulin.

Inhibitors of the interaction of tubulin or its subunits, such as benzimidazoles, cortidine and taxol, are important lead structures in a range of therapeutic agents directed against human, animal or plant diseases. The importance of tubulin as a target for these compounds extends to and clearly demonstrates its potential in the search for new active compounds for the control of these diseases.

The present invention relates to the family Strongilaceae, especially the subfamily Cyathost.
Ominae) nematode-derived β-tubulin-encoding DNA or a fragment of this DNA. The β-tubulin DNA may in this case be genomic DNA or cDNA. The DNA sequences to which the present invention pertains are
It can be considered a new member of the tubulin gene family of the order Streptococcus, especially the parasitic nematode of the subfamily Siatostominae. Very particularly, the present invention relates to DNA sequences encoding β-tubulin from the parasitic nematodes of the genera Siathostomum and Silicosicles.

Similarly, the present invention relates to a DNA sequence having 85% or higher identity to a polynucleotide encoding one of the amino acid sequences as shown in SEQ ID NO: 2, 4, 6, 8 or 10. .

Similarly, the present invention provides a preferred DNA having 95% or greater identity to a polynucleotide encoding one of the amino acid sequences as set forth in SEQ ID NO: 2, 4, 6, 8 or 10. Regarding arrays.

The present invention relates in particular to a DNA sequence coding for β-tubulin derived from the parasitic nematodes of the genera Silicosicles and Siathostomium, very particularly those derived from the parasitic nematodes of the species Silicocyclus nassatas. Sequence, preferably SEQ ID NO:
3, 5, 7, 9 or 11 or their sequences derived from Siatostrom coronatum, preferably according to SEQ ID NO: 1.

Similarly, the present invention relates to these sequences at least 1 at codon 200.
It relates to the above DNA sequence with one point mutation or one nucleotide substitution. These point mutations result in changes in the amino acid sequence encoded by this DNA, such as the substitution of the amino acid phenylalanine by tyrosine, and correlate with the resistance of tubulin with the appropriate mutations to benzimidazole.

The invention likewise relates to DNA sequences which are complementary to the above-mentioned DNA or fragments of this DNA, and fragments of these DNA sequences. These DN
The A sequence or fragments thereof are as described above or in SEQ ID NO: 1, 3, 5,
DNA sequences listed under 7, 9 or 11 or oligonucleotides derived from sequences that are 85% identical to them, preferably sequences that are 95% identical to them, and derived from strands complementary to these, Containing an oligonucleotide capable of hybridizing to

The present invention, as shown in SEQ ID NO: 12 to SEQ ID NO: 51, in this case preferably hybridizes to the above DNA sequence, preferably in the region of the non-coding sequence part of the β-tubulin gene. To an oligonucleotide consisting of or containing one of the sequences.

Similarly, the invention preferably comprises an oligonucleotide consisting of or containing one of the sequences as shown in SEQ ID NO: 12 to SEQ ID NO: 51, which hybridizes to the coding region of the above sequence. Regarding

The invention likewise relates to RNA sequences which are complementary to the above-mentioned DNA or fragments of this DNA, and fragments of these RNA sequences. These RNs
The A sequence or fragments thereof are as described above or in SEQ ID NO: 1, 3, 5,
7, 9 or 11 corresponding to a region of a DNA sequence, a sequence complementary thereto or a DNA sequence 85% identical thereto, preferably 95% identical thereto,
And it contains a ribooligonucleotide capable of hybridizing to these.

The invention likewise provides an expression construct containing one of the above DNA sequences, and a DNA
To a DNA sequence linked to it that enables the expression of These are, for example:
It contains at least one promoter or also an enhancer for constitutive or inducible expression. E. Suitable promoters for expression in E. coli include natural hybrid or bacteriophage promoters, preferably λ-phage, hsp, omp or eg in WO 98/5625, German Patent 3430683 or European Patent 0173149. Are promoters of the group of synthetic promoters such as those mentioned.

The invention likewise relates to a vector containing one of the above-mentioned DNA sequences and allowing the expression of β-tubulin or a fragment thereof according to the invention in a host cell.

The present invention likewise relates to a host cell containing the above DNA, an expression construct as described above, or a vector and allowing the expression of β-tubulin or a fragment thereof.

The invention likewise relates to the polypeptides encoded by the above-mentioned DNA sequences or one of the fragments of these DNA sequences, and fragments of these polypeptides.

The present invention is in this case preferably SEQ ID NO: 1, 3, 5, 7, 9 or 11
To a polypeptide encoded by a DNA sequence having 85%, preferably 95% identity to these sequences, or a DNA sequence of a fragment of this DNA.

The invention also encodes the codon 20 as described above, encoded by the above DNA sequence.
Polypeptides containing at least one point mutation at 0 and resistant to benzimidazole, and fragments of these polypeptides.

The present invention very particularly preferably in this case relates to a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or one of its fragments.

The invention in this case relates to a polypeptide, in particular a purified or recombinantly produced polypeptide.

The present invention relates to full length polypeptides and also corresponding fragments of these polypeptides, eg certain motifs or domains. These fragments have different lengths and are, for example, 5,10,25,50,100.
, 150, 200, 250 or 300 amino acids may be contained.

The invention likewise relates to a fusion protein containing a polypeptide as described above. The fusion protein in this case contains an additional polypeptide component not related to β-tubulin (eg a polypeptide having an enzymatic activity such as LexA, B42, glutathione S-transferase, His tag, alkaline phosphatase or an epitope tag). You may.

The present invention also relates to a method for producing the above polypeptide in a suitable prokaryotic or eukaryotic expression system. Expression can in this case be effected permanently or transiently as described above in the corresponding cell line or in the corresponding host cell in each case. Suitable prokaryotic expression systems, known host - vector systems, such as bacteria (e.g., Streptomyces (Streptomyces), Bacillus subtilis (Bacil lus sutilis), Salmonella typhimurium (Salmonell a typhimurium), Serratia marcescens (Serratia marcescens ) And especially E. coli).

Expression in eukaryotic systems is preferably carried out in baculovirus systems, especially those which allow the introduction of post-translational modifications.

The invention likewise relates to D from nematodes of the family Strongillaceae, preferably the subfamily Syathostominae, particularly preferably the genera Siathostomum and Silicocycles, very particularly preferably the species Siatomostoma coronatum and Silicocycles nassatas.
It relates to the use of the above DNA for the detection of NA. The invention is in this case complementary to DNA encoding β-tubulin or its complementary strand, and this D
It relates to the above oligonucleotides which can hybridize to NA. Preferably, these oligonucleotides are intron regions, ie non-coding DNA.
Hybridizes to the sequence.

The invention comprises: a) a sample in a Northern or Southern blot assay; b) for the detection of a nematode as described above, in which the DNA of the nematode is specifically identified and amplified using primers and PCR techniques. To the use of these oligonucleotides or parts thereof as PCR primers in diagnostic procedures.

The invention preferably relates in this case to an oligonucleotide consisting of the sequence shown in SEQ ID NO: 12 to SEQ ID NO: 51, or containing one of the sequences.

Similarly, the present invention relates to a Strongyridae, preferably the subfamily Siatostominae, particularly preferably the genera Silicocycles and Siathostomae, very particularly preferably the species, which encodes β-tubulin or a fragment thereof which is resistant to benzimidazole. It relates to the use of the above-mentioned DNA for the detection of DNA from the nematodes of Silicos crus nasustus and Siathostom coronatum. The present invention provides a DNA encoding β-tubulin which is resistant to benzimidazole in this case.
Or it relates to the above-mentioned oligonucleotide which is complementary to the complementary strand of this DNA and is capable of specifically hybridizing to this DNA.

The invention also provides: a) a sample in a Northern or Southern blot assay, b) a benzimidazole-resistant strain, wherein the nematode DNA is specifically identified and amplified using primers and PCR techniques. Relates to the use of these oligonucleotides or parts thereof as PCR primers in diagnostic procedures for the detection of nematodes.

The invention preferably relates in this case to an oligonucleotide consisting of the sequence shown in SEQ ID NO: 12 to SEQ ID NO: 51, or containing one of the sequences.

The present invention also relates to a method for detecting nematodes of the family Strongillaceae, preferably the subfamily Siatostominae, particularly preferably the genera Silicocycles and Siathostomae, very particularly preferably the species Silicocicleus nassatas and Siatostromum coronatum. It relates to a method wherein said oligonucleotide specifically hybridizes to a DNA sequence derived from said organism and which can be amplified using PCR technology. Hybridization is preferably performed in the non-coding region (intron) of the β-tubulin gene.

Detection of an organism as described above is carried out by, for example, a) an oligonucleotide probe capable of hybridizing to the above DNA encoding β-tubulin or a complementary strand thereof, or a 5′- or 3′-adjacent region thereof. Or obtaining a primer, b) contacting the oligonucleotide probe or primer with an appropriately prepared probe-containing DNA, and c) detecting hybridization of the oligonucleotide or primer (eg, using the polymerase chain reaction), d) Sequencing the detected sequence of the β-tubulin gene, and e) a DNA sequence according to the invention as described above, preferably SEQ ID NOs: 1, 3, 5
, 7, 9, or 11 and comparing this sequence with the DNA sequence shown in FIG.

The present invention also relates to the family Strongilaceae, which is resistant to benzimidazole,
A method for the detection of nematodes of the subfamily Siathostomae, particularly preferably the genera Silicosicles and Siathostomae, very particularly preferably the species Silicosicles nassatas and Siatostromum coronatum, wherein the oligonucleotide is derived from the organism, and It relates to a method for specifically hybridizing to a DNA sequence that can be amplified using PCR technology. Hybridization is preferably
It is performed in the non-coding region (intron) of the β-tubulin gene.

Detection of an organism as described above is carried out by, for example, a) an oligonucleotide probe capable of hybridizing to the above DNA encoding β-tubulin or a complementary strand thereof, or a 5′- or 3′-adjacent region thereof. Or obtaining a primer, b) contacting the oligonucleotide probe or primer with an appropriately prepared probe-containing DNA, and c) detecting hybridization of the oligonucleotide or primer (eg, using the polymerase chain reaction), d) Sequencing the detected sequence of the β-tubulin gene and e) at least one point mutation at codon 200 leading to resistance of β-tubulin encoded by these sequences to benzimidazole. A previously described book with mutations DNA sequence according to the invention, preferably SEQ ID NO: 1,
Comparing this sequence with the DNA sequence shown in 3, 5, 7, 9 or 11.
Can be implemented by:

The invention likewise relates to the detection and identification of nematodes of the family Strongillaceae, preferably the subfamily Syathostominae, particularly preferably the genera Silicocycles and Siathostomae, very particularly preferably the species Silicocycles nassatas and Siathostomium coronatum. Of the diagnostic test kits mentioned above, among which the above oligonucleotides can be made available and can be used in a detection method of the aforementioned species. Similarly, the present invention provides a sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, a sequence complementary thereto, a sequence having at least one point mutation at codon 200, or a sequence having these sequences. Make available an oligonucleotide that specifically hybridizes to the fragment. In this case, SEQ ID NO: 12 to SEQ ID NO:
Oligonucleotides consisting of or containing the sequence shown in 51 are particularly preferred.

The invention likewise relates to a nematode of the genus Strongillaceae, preferably the subfamily Siatostominae, particularly preferably the genera Silicocycles and Siathostomae, very particularly preferably the species Silicocycles nassatas and Siathostom coronatum, which is resistant to benzimidazoles. It relates to a diagnostic test kit for the detection of species, among which the above oligonucleotides are made available and can be used in detection methods of the species mentioned. Similarly, the invention provides SEQ ID NOs: 1, 3,
Available oligonucleotides that specifically hybridize to the sequence shown in 5, 7, 9 or 11, a sequence complementary thereto, a sequence having at least one point mutation at codon 200, or a fragment of these sequences Let In this case, oligonucleotides consisting of or containing the sequences shown in SEQ ID NO: 12 to SEQ ID NO: 51 are particularly suitable.

The invention likewise relates to a diagnostic test kit as described above, in which the oligonucleotides made available in this kit are provided with a detectable marker. Such detectable markers may include, among others, enzymes, enzyme substrates, complements, enzyme inhibitors, fluorescent markers, chromophores, luminescent markers and radioisotopes.

The present invention likewise relates to β from nematodes of the family Strongillaceae, preferably the subfamily Theatostominae, particularly preferably the genera Silicocycles and Siathostomae, very particularly preferably the species Silicocicleus nassatas and Siatostromum coronatum.
-With respect to an antibody which specifically reacts with an epitope of tubulin.

The present invention likewise particularly relates to β-from the nematodes of the family Strongillaceae, preferably the subfamily Cyatostominae, particularly preferably the genera Silicocycles and Siathostomae, very particularly preferably the species Silicocycles nassatas and Siathostomium coronatum. It relates to a monoclonal antibody which specifically reacts with an epitope of tubulin.

[0055]   The invention likewise relates to the use of the above antibodies as nematicides.

The present invention also relates to a Strongyridae, preferably the subfamily Siatostominae, particularly preferably the genera Silicocycles and Siathostomum, for the production of a vaccine containing at least one of the above β-tubulin polypeptides or fragments thereof, Very particularly preferably relates to the use of the above β-tubulin polypeptides or fragments thereof from the nematodes of the species Silicocicles nassatas and Siathostom coronatum. The vaccine can in this case generate an immune response that is specific for the β-tubulin.

In a preferred embodiment, the vaccine comprises an antigenic determinant, such as SEQ ID NO: 2,4.
A polypeptide having the amino acid sequence shown in 6, 8 or 10 or the above DN
It contains the individual determinants of the polypeptide encoded by A or one of its fragments.

The invention likewise provides an immunogenic composition for immunization of a mammal, which comprises at least one of the above β-tubulin polypeptides according to the invention or a fragment thereof, or at least one of the above antibodies. It relates to a manufacturing method.

Similarly, the present invention relates to the production of an immunogenic composition which is administered to a host against β-tubulin from a parasitic nematode for the activation of a prophylactic immune response in the host. A β-tubulin according to the invention, preferably SEQ ID NOs: 1, 3, 5,
It relates to the use of a nucleic acid encoding the sequence shown in 7, 9 or 11 or a fragment thereof or a sequence homologous thereto, the above expression vector.

Similarly, the present invention relates to a vector (nucleic acid encoding β-tubulin according to the present invention, preferably the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, a fragment thereof or a homologue thereof. And a promoter sequence which controls the expression of β-tubulin according to the invention which is functionally linked to said nucleic acid sequence and which produces an immune response and a vehicle suitable for pharmaceutical purposes.
e) an immunogenic composition comprising e).

The present invention likewise relates to a method for identifying a substance that modulates an interaction of tubulin or an interaction of subunits of tubulin. The method comprises the use of tubulin, preferably tubulin from parasite nematodes, particularly preferably the eye, β-tubulin from nematode parasite nematodes, very particularly preferably parasites of the family Strongillaceae. Based on β-tubulin from reptiles, most preferably β-tubulin from parasitic nematodes of the subfamily Siatostominae. A particularly preferred group of β-tubulins used in this method are β-tubulins from the parasitic nematodes of the genera Silicocycles and Siathostomum.

The present invention relates to the identification of agents, such as small organic molecules, that modulate the interaction of tubulin protein molecules or subunits thereof. Preferably,
The present invention relates to the identification of compounds that inhibit interactions.

Similarly, the invention provides that: a) the substances to be tested are contacted with tubulin under conditions which allow the interaction of the tubulin molecules and the binding of the test substance to tubulin; By detecting the resulting binding by determining the ability of the tubulin protein molecules to act, and c) the ability of the tubulin protein molecules to interact with each other in the presence of the test substance, with respect to each other in the absence of the test substance. Comparing with the ability to act, according to the above method.

The present invention likewise relates to a method for identifying substances that modulate the ability of tubulin molecules to interact with each other. Particularly preferably, the invention is in this case
A or a fragment thereof, in particular SEQ ID NO: 1, 3, 5, 7, 9
Or the amino acids consisting of or comprising the sequence set forth in 11 and having 85%, preferably 95% identity to them and shown in SEQ ID NO: 2, 4, 6, 8 or 10. A method of using one of the above polypeptides encoded by a β-tubulin encoding sequence.

Similarly, the present invention is a method for identifying a substance that modulates the ability of tubulin to interact with each other as described above, wherein the method used is a test substance using a cell-based test system. To a method based on detecting modulation of tubulin interaction in the presence of A preferred embodiment of such a test system is the "two-hybrid system" (US Pat. No. 5,283,317, Z
ervos et al. (1993) Cell 72 , 223-232; WO
94/10300). This system is suitable for recording or describing the interaction of two proteins by the interaction that results in a detectable signal.
Also, such a system can be adapted to test systems with high throughput.

Similarly, the present invention is a method for identifying a substance that modulates the ability of tubulin to interact with each other, wherein the method used is a tubing in the presence of a test substance using a cell-free test system. A method based on detecting modulation of phosphorus interactions. A particularly preferred embodiment of such a test system is the “Scintillation Proximity Assay (SPA)” (EP 015 473). This test system is based on the detection of the interaction of ligands with receptors, eg tubulin molecules, bound to microspheres or beads, which microspheres or beads are equipped with scintillate molecules. The signal is detected when the receptor-ligand complex degrades.

The invention likewise relates to an as yet undescribed substance which has been identified using the above method and which is suitable for modulating, preferably inhibiting, the interaction of tubulin molecules.

The invention likewise relates to a process as described above for the manufacture of a medicament for use in the prophylactic or therapeutic treatment of an animal or human that has been or has already been attacked by nematodes. To the use of an as yet undescribed substance identified using one of: The medicament according to the invention contains at least one substance identified by one of the above methods and can be administered nasally, transdermally, parenterally or enterally.

For a better understanding, the meaning of certain languages and terms used in the description, examples and appended claims are explained in more detail below.

The term “fragment” with respect to proteins and DNA refers to SEQ ID NOs: 1-1.
1 describes the nucleic acid or amino acid sequences described under 1, their complementary sequences or parts of sequences which are identical to them by up to 85%, preferably up to 95%. DNA
And fragments of the polypeptide sequence contain at least 5 nucleotides or amino acids, but likewise contain up to 447 amino acids or 1343 amino acids.
Up to 4 nucleotides, or 256 in the case of the sequence shown in SEQ ID NO: 11
It may contain up to 5 nucleotides.

The terms “homology”, “identity” or “similarity” relate to sequence similarity between two peptides or between two nucleic acids. Homology can in each case be determined by comparing the positions in each sequence to each other. If a position in the compared sequences is occupied by the same base or amino acid, then the two molecules at this position are homologous. A measure of homology between sequences is a function of the number of corresponding or homologous positions that the sequences share with each other. A "heterologous" sequence has less than 40% identity, but preferably less than 25% identity.

The term “homology” especially means that a DNA segment of at least 15 base pairs in length or a strand complementary to the DNA is at least 85%, preferably 95% identical to the corresponding DNA. To do. Homology can be determined using, among other things, computer programs such as the GCG program (Devereux et al.
al. (1983), Nucleic Acids Res. 12 , 387-
395).

“Homology” also exists when a DNA segment can hybridize to the DNA strand or its complementary strand.

The terms “hybridize” or “hybridization” describe the process by which a single-stranded nucleic acid molecule undergoes base pairing with a complementary DNA strand, the ability of a single-stranded nucleic acid molecule to be stringent to hybridization conditions. Dependent.

The term “stringency” relates to hybridization conditions. "High stringency" is given when base pairing is made difficult. "Low stringency" is given when base pairing is facilitated.

The term “complementary” refers to the ability of purine and pyrimidine nucleotides to form base pairs with each other via hydrogen bridges. Complementary base pairs are guanine and cytosine, adenine and thymine, and adenine and uracil, among others.

Those skilled in the art will appreciate that due to the degenerate genetic code (ie, the 64 codon code for 20 amino acids), many “silent” substitutions of nucleotide base pairs alter the identity of the protein product encoded thereby. Without knowing that it can be introduced into the sequence that encodes it. All such substitutions should be included within the scope of the present invention.

The term “specifically hybridize” refers to at least about 6,12,20,30,50,100,150,200,300 of the one β-tubulin gene described above.
, 350, 400 or 440 contiguous nucleotides, preferably according to the invention which hybridizes to the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 or one of its homologous or complementary sequences. The ability of the nucleic acid molecule to hybridize, ie, 10 times more molecules hybridize to a nucleic acid (eg, mRNA or genomic DNA) of a cell encoding a protein other than β-tubulin.
It preferably relates to the ability of 100-fold more molecules to hybridize, particularly preferably 100-fold more molecules to hybridize.

The term “plasmid” relates to extrachromosomal genetic elements. The underlying plasmids used for the present invention may be obtained commercially, freely obtained or derived from such plasmids according to known methods.

The term “vector” is used to introduce exogenous DNA into a host cell D
Describe the NA element. Vectors contain nucleotide sequences that encode one or more polypeptides. Vectors that can control the expression of the genes they contain are
It is called an "expression vector".

The term “gene”, within the scope of the present invention, relates to a nucleic acid which comprises an open reading frame encoding one of the above β-tubulin polypeptides. Exons and possible intron sequences are also included here.

The terms “interact” or “interaction” describe a detectable interaction between molecules. The term "binding" is further included herein.

The term “modulate” relates to both stimulation and inhibition or inhibition of biochemical processes. In the context of the present invention, "modulation" refers to the inhibition or suppression of an interaction between tubulin polypeptides or subunits or fragments thereof, or of interactions that can be exhibited, for example, in irreversible binding of tubulin polypeptides to each other. Means irritation.

The term “nucleic acid” relates to polynucleotides, such as deoxyribonucleic acid (DNA) or, where appropriate, ribonucleic acid (RNA). Similarly, the term also includes analogs of RNA or DNA made from nucleotide analogs, and in that case single-stranded ("sense" or "antisense") and double-stranded polynucleotides.

The term “promoter” refers to a specific D, operably linked to a promoter.
It relates to a DNA sequence which regulates the expression of NA. The term also includes “tissue-specific” promoters, ie, promoters that control the expression of a particular DNA only in certain cells (eg, cells of certain tissues). Similarly, “tissue non-specific” promoters and promoters that confer or induce constitutive expression are included.

The terms “protein”, “polypeptide” and “peptide” are interchangeable in their use in the context of the present specification when they relate to a gene product.

A “fusion protein” is common to tubulin sequences.
or a fusion of the first amino acid sequence encoding one of the above β-tubulin polypeptides with a second amino acid sequence having no basic homology. The second amino acid sequence may in this case be from the same organism as the first one,
Otherwise, it may be derived from other organisms (intergenic). Generally, a fusion protein can be represented by the formula X-tubulin-Y, where "tubulin" refers to one of the above polypeptides and X and Y are unrelated to the tubulin amino acid sequence. Represents a polypeptide. X or Y may in each case be absent independently of one another.

The terms “cell” or “host cell” can be used interchangeably in the context of the specification presented herein. It is understood that these terms refer not only to individual cells, but also to the progeny of such cells. Due to certain modifications (eg, mutations) in subsequent generations, such progeny may not be identical to stem cells, but are further encompassed by the present invention.

The term “intron” describes those sequences of DNA, described above, preferably genomic, that are transcribed but the associated flanking sequences (exons) are removed from the transcript by “splicing”. .

Nucleic Acids As already mentioned, one aspect of the invention is the nematode of the family Strongylaceae, in particular the subfamily Siathostomae, very particularly the genera Siathostomum and Silicocycles, especially the species Silicocycles nassatas and Siathostom coronatum. 85%, preferably 95% homologous to a nucleic acid encoding a β-tubulin polypeptide or a fragment thereof, or a [blank] in SEQ ID NO: 1, 3, 5, 7, 9 and 11 And a homologous thereto, encoding β-tubulin or a fragment thereof according to one of the sequences as set forth in SEQ ID NOs: 2, 4, 6, 8 or 10. SEQ ID NO: 3 represents the degenerate sequence of a nucleic acid from Silicosicles nassatas encoding β-tubulin, where “r” represents purine (guanine or adenine) and “y” represents pyrimidine (thymine, Or uracil or cytosine),
And "w" represents adenine or thymine, or uracil. Therefore,
SEQ ID NO: 3 contains a number of sequences that can be present in an organism of the species Silicocicleus nassatas. The sequences shown in SEQ ID NOs: 5, 7 and 11 are β-
Three constant sequences encoding tubulin are shown, which are representative and preferred embodiments of the DNA shown in SEQ ID NO: 3.

Similarly, part of the present invention optionally comprises a length of at least 2,5,10,2.
An oligonucleotide encoding a β-tubulin polypeptide containing 5, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids. Such oligonucleotides can be used as primers or antisense molecules (ie, as non-coding nucleic acids), and at least about 6,12,24,30,60,100,120,150 or 21.
It contains 0 base pairs, while the coding nucleic acid contains about 200,300,400,50
It contains 0,600,700,800,900,1000,1100,1200 or 1300 base pairs.

The present invention also relates to those that specifically hybridize under stringent conditions to the nucleic acid represented by one of the sequences shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11. Will be described. Correspondingly, stringent conditions are, for example, a wash step with 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by 2 × SSC at 50 ° C., and are well known to those skilled in the art (eg, Current. protocols in M
olecular Biology, John Willey & Sons,
N. Y. (1989), 6.3.1-6.3.6). Therefore, the salt concentration in the washing step has lower stringency (2xSSC, 50 ° C).
Alternatively, it may be chosen to be higher (0.2 × SSC, 50 ° C.). Furthermore, the temperature in the wash step can vary from conditions for low stringency (eg about 22 ° C.) to higher stringency (eg about 65 ° C.). Both salt concentrations and temperatures can be varied and matched to each other.

Particularly suitable oligonucleotides that can be used for the identification and characterization of existing, eg genomic, DNA, as primers or for hybridization are described in SEQ ID NO: 12-51. However, also other oligonucleotides may be derived from the sequences shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 which can then be used as primers or for hybridization. . Particularly suitable for the identification of the species or genera in which the DNA present can be designated are those oligonucleotides which hybridize in the region of the intron of the DNA present (genomic). The intron of the genomic DNA encoding β-tubulin that can be isolated from the above nematodes is described in SEQ ID NO: 11 by the example of Silicocycles nassatas. Thus, introns are those sequences located between coding exons. In a preferred embodiment, the primers or hybridization probes described above contain labeling groups which allow the detection of the oligonucleotide, ie eg radioisotopes, fluorescent groups, enzymes or enzyme cofactors.

Such oligonucleotides can be used in diagnostic test kits to determine the origin (ie, organism) of the DNA present. The oligonucleotides have SEQ ID NOs: 1, 3, 5, 7, 9 and 1 to recognize specific sequences.
DNAs shown in 1 [blank] are suitable for their fragmentation, their homologous sequences and their specific hybridization with their complementary sequences. Therefore, they also allow recognition and identification of those sequences encoding β-tubulin that result in the expression of benzimidazole resistant β-tubulin by virtue of one or more point mutations at codon 200. As a result, the sequences of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, preferably SEQ ID NOs: 12-5.
These oligonucleotides derived from the embodiment described in 1 are suitable for the identification of frequently existing nematode species of the subfamily Siatostominae and for recognition of the existing resistance to benzimidazoles. .

Oligonucleotides according to the present invention can be made by standard methods well known to those skilled in the art, such as de novo DNA synthesis.

The nucleic acids described herein are in whole cells or in cell lysates in partially purified or biologically pure form, ie where other cellular components or chemical precursors and by-products are of DNA. It can also be present if it is removed in the case of chemical synthesis.

The β-tubulin-encoding nucleic acids described above can be obtained starting from mRNAs present in many eukaryotic cells. It is also possible to obtain the DNA according to the present invention starting from the genomic DNA derived from the nematode cells (see also the following example). The gene encoding β-tubulin can be obtained from, for example, a cDNA library or a genomic DNA library. cDNA may be obtained by isolating total mRNA of cells, eg nematode cells.
Starting from the mRNA, double-stranded cDNA can then be made and inserted into a suitable plasmid or vector. The DNA according to the invention can be produced by amplification using the known polymerase chain reaction (PCR) or alternatively by de novo DNA synthesis (J. Sambrook et al.
． (1989) Molecular Cloning, 2nd Edition
, Chap. 14, see).

Vectors and Plasmids The invention also relates to one of the nucleic acid sequences according to the invention which is operably linked to transcriptional regulatory sequences.
Including an expression vector containing "Operably linked" means that the nucleic acid sequence is linked to regulatory sequences so that the expression of the protein encoded by the nucleic acid sequence can be controlled. "Transcriptional regulatory sequences" include, for example, promoters, enhancers and other regulatory elements. The expression vector contains, for example, the gene encoding β-tubulin according to the present invention or a fragment thereof.
These vectors can be used for integration into cells in which the corresponding polypeptides or also fusion proteins are subsequently produced. E. Suitable promoters for the expression of the protein according to the invention in E. coli include the natural hybrid or bacteriophage promoters. Preferably, they are phage lambda promoters, omp promoters or synthetic promoters (also WO98 / 15625, German Patent 3430683, European Patent 01731).
No. 49, cf.). Suitable vectors include commercially available products such as the pET series (pET3a, pET23a, pET28a and His).
An expression vector of pGEX with a tag or His tag, pET3a) or a glutathione synthetase fusion can be obtained. Next, the expression vector
For example, DE3 lysogenic E. E. coli strains such as BL21 (DE3), HMS174
(DE3) or AD494 (DE3) can be transformed.

Expression of β-Tubulin Polypeptides The present invention also includes cells containing the nucleic acid sequences according to the present invention (eg inserted in a vector or in the genome). These host cells may be prokaryotic or eukaryotic cells. Suitable prokaryotic expression systems are for example Streptomyces spp., Bacillus subtilis, Salmonella typhimurium, Serratia marcescens and preferably E. It is a bacterial system such as E. coli.

Suitable eukaryotic expression systems are those which allow baculovirus systems, particularly preferably post-translational modifications.

[0101]   Other eukaryotic expression systems (eg yeast, insect cells) can be used as well.

Polypeptides The present invention likewise provides a DNA according to the invention, preferably SEQ ID NOs: 1, 3, 5, 7
, 9 and 11 and fragments thereof or β-tubulin polypeptides encoded by homologous DNA sequences as described above. A preferred embodiment of these β-tubulins is SEQ ID NO: 2, 4, 6,
It is described in the sequences shown in 8 and 10. In a preferred embodiment, said polypeptide is a purified polypeptide that is free of contaminating proteins of the cells in which it is produced.

The polypeptide may be a full-length protein, or a protein of at least 5,10,
25, 50, 75, 100, 125, 150, 200, 250, 300, 350
Alternatively, those fragments, motifs or domains containing 400 amino acids. Polypeptide fragments can be obtained and tested by testing the polypeptides encoded by the nucleic acid fragments obtained from the sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9 and 11.

Polypeptide fragments may also be chemically synthesized by known methods.

The present invention also includes the polypeptide encoded by the degenerate sequence set forth in SEQ ID NO: 3. The different possible bases at particular positions in the DNA sequence result in different polypeptides with the amino acids encoded by the codons obtained in each case. The polypeptide encoded by the degenerate sequence set forth in SEQ ID NO: 3 is set forth in SEQ ID NO: 4 and the variable amino acids are "
Marked by "Xaa".

Preferred embodiments of polypeptides are set forth in SEQ ID NOs: 2, 4, 6, 8 and 10.

[0107]   The invention includes all methods for producing the polypeptides according to the invention.

It is well known to those skilled in the art that the polypeptide of the present invention can be obtained by various methods, for example, chemical methods such as solid phase method. The use of recombinant methods is recommended for obtaining large quantities of proteins.

The steps to be emphasized for the preparation of recombinant β-tubulin are: Natural, synthetic or semi-synthetic DNA encoding β-tubulin according to the invention
Getting in. 2. The β-according to the present invention, either as such or as a fusion protein
Incorporation of this DNA into an expression vector suitable for expressing tubulin. 3. Transformation of suitable, preferably prokaryotic, host cells with this expression vector. 4. Propagation of host cells transformed in a manner suitable for expressing β-tubulin according to the invention. 5. Harvesting cells and purification of β-tubulin by suitable known methods.

For example, the expression vector may be λDE3 lysogenic E. E. coli strains such as BL21 (D
E3), HM S174 (DE3) or AD494 (DE3). After growth of cells under standard conditions well known to those skilled in the art, expression is I
Induced with PTG. After the induction of cells, the culture was performed at a temperature of 18 to 37 ° C for 3 to 24
Will be carried out for hours. When cells are disrupted and the expressed protein is chromatographic, Ni-NTA in the case of a protein expressed with a His tag
It is purified by the FPLC method on a column and also by ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis or also immunoaffinity purification which is specific for the polypeptides according to the invention.

A homologue or fragment of a polypeptide according to the invention may be mutagenized,
For example, it can be generated by site-directed (point) mutagenesis, or deletion.

The polypeptides according to the invention may also be chemically modified, for example with glycosyl groups, lipids, phosphates, acetyl groups or similar groups. Covalent derivatives include groups that modify and amino acid side chains of the polypeptide or the N-terminus or C
It can be obtained by coupling with a terminal functional group.

In the expression of the polypeptides according to the invention, it may be advantageous to change certain codons in order to allow optimal expression. This is the use of certain codons in heterologous expression systems ("codon usage").
Is different from one of the organisms according to the invention. Furthermore, deletion of the 5'- or 3'-untranslated region is possible if some destabilizing sequence motifs (eg ATTTA) are present in the 3'region of the cDNA.

Fusion Proteins The polypeptides according to the invention can also be present as part of fusion proteins. Such fusion proteins are fully encompassed by the present invention. The fusion protein is useful in situations where it is desired to obtain an immunogenic fragment of β-tubulin (eg EP 0259149; Schlien.
ger et al. (1992) J. Virol. 66 , 2). Under certain circumstances, the fusion protein facilitates expression of the polypeptide. For example, a polypeptide according to the present invention may be made as a glutathione S-transferase (GST) fusion protein. Such a GST fusion protein facilitates purification of the polypeptide (eg, Current protocols in
Molecular Biology, eds. Ausubel et al.
(See John Willey & Sons, NY 1991). A fusion protein is, for example, a "leader" sequence used for purification, the purification of which is [chromatographic means eg on a Ni ²⁺ NTA column].
[Blank] may contain sequences that give poly-His sequences at the N-terminus (but also at the C-terminus) of the protein (eg Hachuli et al.
al. (1987) J. Chromatography 411 , 177,).

[0115]   Techniques for producing such fusion proteins are well known to those of skill in the art.

Antibodies Another aspect of the invention relates to antibodies which specifically react with the β-tubulin polypeptide according to the invention.

For example, the anti-protein or anti-peptide serum or monoclonal antibody is
It can be made according to standard protocols by use of immunogens derived from β-tubulin polypeptides according to the invention (eg Antibo).
die: A. Laboratory Manual ed. by Harlo
w and Lane (Cold Spring Harbor Press,
1988)).

A mammal, such as a mouse, hamster or rabbit, is immunized with an immunogenic form or immunogenic fraction of a polypeptide according to the invention, eg with a polypeptide capable of producing an antibody response. It Appropriate techniques are well known to those of ordinary skill in the art. Thus, the immunogenic fraction of β-tubulin is administered in the presence of adjuvant. In the process of immunization, antibody titers in plasma or serum are treated with, for example, E.
It can be observed by testing with a LISA assay or other immunoassay.

In a preferred embodiment, the antibody according to the invention is a β-tubulin polypeptide according to the invention, for example the polypeptide shown in SEQ ID NO: 2, 4, 6, 8 or 10 or SEQ ID NO: 1,3. , 5, 7, 9 or 11 or immunospecific for the antigenic determinants of those polypeptides encoded by sequences up to 85% identical thereto, preferably sequences up to 95% identical. .

After immunization of the mammal, polyclonal anti-β-tubulin antibody can be isolated from serum. In the production of monoclonal antibodies, antibody-producing cells (lymphocytes)
Was obtained from the immunized animal, and to obtain hybridoma cells,
Fused with immortal cells such as myeloma cells according to known methods (eg, K
oehler and Milstein (1975) Nature 256 ,
495-497; Kozbar et al. (1983) Immunolog
y Today 4 , 72; Cle et al. (1985) Monoclo
nal Antibodies and Cancer Therapy, Al
an R.A. Liss, Inc. pp. 77-96).

The “antibody” described herein also includes fragments of antibodies that specifically react with β-tubulin according to the present invention. The antibody is fragmented and the fragments are tested using conventional techniques.

Preferred embodiments include detectable labels (eg radioisotopes, fluorescent groups,
An antibody as described above carrying an enzyme or enzyme cofactor).

Antibodies that specifically bind to β-tubulin polypeptides according to the present invention can also be used for immunohistochemical staining of tissue samples to detect specific β-tubulin expression. Anti-β-tubulin antibodies can also be used for diagnostic purposes, eg for immunoprecipitation or immunoblotting.

Diagnostic Test Procedures Similarly, the present invention makes available nucleic acid molecules that can be used for diagnostic purposes.

These are nucleic acid molecules as described above, SEQ ID NO: 1, 3, 5, 7, 9 or 1
1 or a DNA sequence complementary thereto. For example, the oligonucleotides set forth in SEQ ID NOS: 12-51 are available, which include a sense or antisense sequence encoding β-tubulin, and an intron sequence portion described as an example in SEQ ID NO: 11. It can hybridize.

In this procedure, the cell's nucleic acid is allowed to hybridize and the DN
The A probe is contacted with the oligonucleotide and hybridization of the sample is detected with the oligonucleotide.

In this manner, D, which preferably encodes β-tubulin, is obtained by specific hybridization of an oligonucleotide according to the invention to a DNA probe.
Methods are available that allow the use of oligonucleotides that hybridize to the intron region of NA to distinguish between small strongilds of various species and / or other nematode species. In a particularly preferred embodiment, the oligonucleotides according to the invention enable the identification of resistance in, for example, the small strongildordo (Siathostomieae) in horses, in particular the resistance of the species Silicocyclus nassatas, Siatostoma coronatum and Siatostoma catinatum. In this operation, β
-A resistant form of tubulin is present in the DNA according to the invention which codes for it, for example in Elard et al. (1998) PCR diagnosis o
f benzimidazole-susceptibility or -r
esistanece in natural populations of
the small ruminant parasite, Telador sagia circumcincta , Veterinary Parasi
The fact that it carries at least one point mutation, which can be detected by PCR as described in topology 80 , 231-237, can be used.

The method may include humans and animals affected by nematodes, such as horses, sheep, pigs, goats, camels, buffalo, donkeys, hares, roosters, fur animals, birds (eg chickens, turkeys, ducks), freshwater. And especially useful for investigating possible therapeutic strategies in saltwater fish (eg rainbow trout, carp). It allows identification and differentiation of parasitic nematodes and recognition of their resistant populations, avoiding treatment with inactive nematicides.

The methods described herein are made available, for example, in the form of prefabricated diagnostic test kits containing at least one of the nucleic acid molecules or antibodies described above prepared in a ready-to-use dosage form.

Method for Discovering Nematicidal Substance The present invention relates to a method for identifying a new specific anthelmintic substance using tubulin or a fragment thereof.

In a preferred embodiment, β-tubulin polypeptides according to the invention are used for this. However, the method can also be practiced with tubulin from species other than those described herein. Methods of using β-tubulin polypeptides other than those according to the invention are fully encompassed by the invention. Particularly preferably, the β-tubulin polypeptide shown in SEQ ID NO: 2, 4, 6, 8 or 10 is used for the method. Thus, the present invention further makes available the often present parasite nematode-derived β-tubulin polypeptides. These can be used in various test systems for the identification of novel inhibitors of tubulin interactions, or tubulin subunit interactions.

Cell-Free Test Systems Many test systems for testing compounds and their natural extracts for their purposes have high treatment numbers to maximize the number of substances considered in a given time period. I'm aiming. Cell-free research-based test systems use purified or semi-purified proteins. They are suitable for "first-time" tests whose main aim is to detect possible effects of substances on target proteins.

Effects such as cytotoxicity are generally neglected in these in vitro systems. The test system in this case checks both the inhibitory and inhibitory effects of the substance, as well as the stimulatory effect. The effectiveness of a substance can be checked by a concentration-dependent test series. A control batch without test substance is used to test the effect.

One possibility for the identification of substances that modulate the interaction of tubulin or its subunits is the “Scintillation Proximity Assay” (SPA), see EP 015473. This test system uses the interaction of a radiolabeled ligand (eg a small organic molecule or a second radiolabeled protein molecule) with a receptor (eg tubulin). The receptors are in this case bound to globules (“microspheres”) or beads that are equipped with scintillate molecules. During the radioactive decay process, the scintillate material in the microspheres is excited by the radiolabeled subatomic particles, and a detectable photon is emitted. The test conditions are optimized so that only those particles emanating from the ligand provide a signal emanating from the ligand bound to the receptor or tubulin.

In one possible embodiment, tubulin is bound to beads with or without interacting or binding test substances. α- or β-tubulin subunits can be used here. The radiolabeled ligand may be, for example, a labeled benzimidazole or an additional labeled β-tubulin molecule.
In the case of ligand binding to immobilized tubulin, the ligand inhibits or blocks the interaction that exists between immobilized and free tubulin in order to bind itself to the area of the contact surface. I must do it. The resulting binding to immobilized tubulin can then be detected by a flash of light. Correspondingly, the complex existing between the immobilized and free labeled tubulin is destroyed by the binding of the test substance, which reduces the detected intensity of the light flash. The test system then corresponds to a complementary inhibition system.

Cell-Based Test Systems The β-tubulins that can be utilized by the present invention, but also tubulins from other species, can be used in cell-based test systems for the identification of substances that inhibit tubulin interactions. Enable development.

An example of such a test system is the “two hybrid system”. A particular example of this is the "interaction trap". This is a genetic selection for interacting proteins in yeast (eg Gyuris et al. (1993) Cdi.
1, a human G1 and S phase protein ph
osphatase that associates with Cdk ²
． Cell 75 , 791-803). The test system is designed to detect and record the interaction of two proteins in that the resulting interaction will give rise to a detectable signal.

[0138] Such a test system is also adaptable to test processing of multiple test substances within a given time period.

The system is based on the construction of two vectors, a "bait" vector and a "prey" vector. A gene encoding tubulin, preferably a gene encoding β-tubulin according to the present invention,
It is cloned in a bait vector and then expressed as a fusion protein with LexA protein, a DNA binding protein. A second gene encoding tubulin, preferably β-tubulin according to the invention, is cloned into a prey vector, in which case it is expressed as a fusion protein with the B42 prey protein. Both vectors are present in Saccharomyces cerevisiae (Sacc haromyces cerevisiae) host, which, l
It contains a copy of LexA-binding DNA 5'to the acZ or HIS3 reporter gene. Activation of transcription of the reporter gene occurs when the interaction occurs between the two tubulin (fusion) proteins. If the presence of the test substance results in the inhibition or interference of the tubulin interaction, the two tubulin (fusion) proteins can no longer interact and the product of the reporter gene is
No longer produced.

Tubulin, in particular β-tubulin or a fragment thereof according to the invention,
And by the above method it is possible to identify novel and specific antiparasitic compounds.

The compounds found by the above methods and polypeptides are for the treatment of humans or humans and animals infected with pathogenic endoparasites of agricultural animals, pets, zoo animals and laboratory and test animals. It is useful.

The compounds are active against all stages of development of normal, susceptible and also resistant strains. By treatment with agents containing one or more of these compounds, both economic losses in agricultural animals and diseases in humans and animals can be avoided or treated. The following parasites, in this case of particular interest as targets discovered active compounds: Yuharirui (Enoplida) eye, for example: (. Trichuri s spp) ( . Capillaria spp) Torikyurisu species, Kapiraria species, Torikomosoidesu species (Trichomosoides spp.), Torikinera species (Tr ichinella spp.).

[0143]桿線insects (Rhabditia) eye, for example: (. Microne ma spp) Mikuronema species, strontium di Roy Death species (Strongyloides sp
p. ).

[0144]   Roundworm (Strongylida) Eyes, for example: Strongylus species (Stro ngylus   spp. ), Triodont Horus species (Triodontopho rus   spp. ), Esophago goddontus species (Oesophagodontus   spp. ), Triconema species (Trichonema  spp. ), Giarosef
Ars species (Gyalocephalus  spp. ), Cyrindofara Links
seed(Cylindropharynx  spp. ), Potelliostomum species (Po teriostomum   spp. ), Cyclococercus species (Cyclococ ercus   spp. ), Silicostefanus spp. (Cylicostephan us   spp. ), Essogostomamu species (Oesophagostomum  
spp. ), Chabeltia species (Chabertia  spp. ), Stefanula
Seeds (Stephanurus  spp. ), Ancyloma species (Ancylo stoma   spp. ), Ancinaria species (Uncinaria  spp. ),
Bunostomum species (Bunostomum  spp. ), Globocephalus species (G lobocephalus   spp. ), Singhams species (Syngamus  s
pp. ), Theatomastoma species (Cyatostomum  spp. ), Silicon
Siculus species (Cylicocyclus  spp. ), Neostronzillus species ( Neostrongylus   spp. ), Cyst cowls (Cytoca ulus   spp. ), Niumostrondisus sp. (Pneumstrong lus   spp. ), Spicosaurus species (Spicocaulus  spp. )
， Erahostron dilus species (Elaphostrongylus  spp. ),
Parella host Longines species (Parelaphostrongylus  spp
． ), Kleno Soma species (Crenosoma  spp. ), Paracrenosoma species (P aracrenosoma   spp. ), Angiostronzillus species (Angi ostrangelus   spp. ), Ellos trondirus species (Aeluro stronglus   spp. ), Filaroides species (Filaroides   spp. ), Paraphylloides (Parafilaroides  spp
． ), Trichothron dilus species (Trichostrongylus  spp.
), Haemonchus species (Haemonchus  spp. ), Ostertagia species ( Ostertagia   spp. ), Marshallia species (Marshallag ia   spp. ), Couperia species (Cooperia  spp. ), Nematozilla
Seeds (Nematodirus  spp. ), Hyostronzillus species (Hyos trongylus   spp. ), Obeliskoides species (Obeliscoid es   spp. ), Amidostomum species (Amidostomum  spp. ),
Orlanus species (Ollulanus  spp. ).

The order Oxyurida , for example: Oxyuris spp.
spp. ), Enterobiusu species (Enterobius spp.), Pasarurasu species (Passalurus spp.), Shifashia species (Syphacia
spp. ), Aspiculuris spp., Heterakis spp.

[0146] roundworms acids (Ascaridida) eye, for example: (. Ascaris spp) (. Toxascaris spp) (. Toxocara spp) (. Parascari s spp) Ascaris species, Tokisasukarisu species, Toxocara species, Parasukarisu species, Anisakis species (Anisakis spp .), Ascaridia spp.

[0147]   Nematodes (Spirurida) Eyes, for example: Gnatostoma species (Gnath ostoma   spp. ), Fisaloptera species (Physaloptera  s
pp. ), Terrasia species (Thelazia  spp. ), Gondolonema species (Go ngylonema   spp. ), Hablonema species (Habronema  spp
． ), Parabronema species (Parabronema  spp. ), Drachia species ( Drascia   spp. ), Drakancruz species (Dracunculus  
spp. ).

[0148] filamentous insects (Filariida) eye, for example: (. St ephanofilaria spp) ( . Parafil aria spp) (. Setaria spp) Stefano filarial species, para filarial species, Setaria species, lower species, (L oa spp.) Dirofilaria spp.,
Ritomosoidesu species (Litomosoides spp.), Burujia species (Br ugia spp.), Bukereria species (Wuchereria spp.), Onchocerca species (Onchocerca spp.).

[0149] thorny headed worms (Gigantorhynchida) eye, for example: (. Filicollis spp) (. Monilifor mis spp) (. Macracanthorhy nchus spp) Firikorisu species, Monirihorumisu species, pillow Kant phosphorus dregs species, Purotenorukisu species (Prothenorchis s
pp. ).

[0150]   Flagella (Mastigophora(Flagellata))   Trypanosoma (Trypanosomatidae), For example, trypanosomes
B. Brusei (Trypanosoma b. brucei), T.S. b. Moth
Mubiense (T. b. gambiense), T.S. Congorense (T. cong
olense), T.W. Kuruji (T. Cruzi), T.S. Evansi (T. evan si ), T.S. Equinum (T. equinum), T.S. Levi (T. Lewis i ), T.S. Perkae (T. percae), T.S. Simie (T. simiae),
T. Vivax (T. vivax), Reishmania Brasiliensis (Le shhmania brasiliensis ), L.S. Donovani (L. dono vani ), L.S. Tropica (L. tropica),   Trichomonadaceae (Trichomonadidae), For example, Giardia
Rumbilia (Giardia lambilia), G. Canis (G. cani s )．   Hairy root-legs (arthropods) (Sarcomastigophora (Rhiz opoda) ), For example, Entamoeba Histolytica (Entamoeba histolytica )   Hartmanera family (Hartmanellidae), For example, Acanthamoeba
seed(Acanthamoeba  sp. ), Harutomanera species (Hartmane lla   spp. )   Sporozoites (Apicomplexa (Sporozoa)), For example, Aime
Rear aserburina (Eimeria acervulina), E. Adenoy
death(E. adenoides), E. Alabamensis (E. alabahme nsis ), E. Anatis (E. anatis), E. Anselis (E. ans eris ), E. Arloingi (E. arloingi), E. Ashata (E. ashata ), E. Aubrunensis (E. auburnensis), E.
Bovis (E. bovis), E. Brunetch (E. brunetti), E. Mosquito
varnish(E. canis), E. Chinchilla (E. chinchillae), E
． Kurparium (E. clupearum), E. Columba (E. column bae ), E. Controller (E. contourta), E. Kurandaris (E. crandalis ), E. Del briekki (E. debriecki), D.
Dispelsa (E. dispersa), E. Ellipsoidales (E. ellip sodales ), E. Falciformis (E. falciformis)
, E. Fauley (E. faurei), E. Raveana (E. labbeana ), E. Leukart ((E. leucarti), E. Magna (E. magna)
, E. Maxima (E. maxima), E. Media (E. media), E. Me
Rare Grigis (E. meleagridis), E. Melia Grimitis (E. m eleagrimitis ), E. Mitis (E. mitis), E. Necatri
Cous (E. necatrix), E. Nina CoreaE. ninako hlyakimovae ), E. Orbis (E. ovis), E. Parva (E. parva ), E. Pabonis (E. pavonis), E. Perholence (E. perforans ), E. Fasani (E. phasani), E. Pyriholmi
Su (E. piriformis), E. Pracox (E. praecox)
, E. Resident (E. residua), E. Scatbra (E. scabra), E. spec. , E. Steady (E. steedai), E. Switzerland(E. s uis ), E. Tenera (E. tenella), E. Torunkata (E. trun cata ), E. Truttae (E. truttae), E. Zernii (E. z uernii ), Globidium species (Globium spec.), Isos
Paula Berry (Isospora belli), I. Canis (I. canis ), I. Ferris (I. felis), I. Ohioensis (I. ohioen sis ), I. Rivolta (I. rivolta),I. spec., I. Switzerland
(I. suis), Eospora caninum (Neospora caninum ), Cistisospora species (Cysissospora spec.), Cryptos
Polydium species (Cryptosporidium spec.),   Toxoplasmataceae (Toxoplasma daidae), For example, Toxopra
Suma Gonjii (Toxoplasma gondii)   Sarcocyst family (Sarcocystidae), For example, sarcocystis
Bobikanis (Sarcocystis bovicanis), S. Bobbi Homini
Su (S. bovihominis), S. Neubona (S. neuvona), S
． Obikanis (S. vicanis), S. Obiferis (S. ovifer is ),S. spec., S. Suiho Minis (S. suihominis)   Leukozoid (Leucozoid), For example, Leukozido Zimon Simon
Ji (Leucozytozoon simondi)   Plasmodium family (Plasmodidiae), For example, Plasmodium
Bell gay (Plasmodium berghei), P. Falciparum (P ． falciparum ), P. Malariae (P. malariae), P. Oh
Bare (P. ovale), P. Vivax (P. vivax),P. spec.   Pyroplasms (Piroplasma), For example, Babesia Argen
China (Babesia argentina), B. Bovis (B. bovis)
, B. Canis (B. canis),B. spec.， Terelia Parva (Th eileria parva ),T. spec.   Adelaina (Adeleina), For example, Hepatozun Kanis (Hepa tozoon canis ),H. spec.   The following are more important:   Myxosporean (Myxospora) And microsporidia (Microspor a ), For example, Gurgia seed (Glugea spec.) And Nosema species (No sema spec. ) And Pneumocystis carinii (Pneumocy stis carinii ), Hairs (Ciliates) (Ciliophora (C iliata) ), For example, Valantidium coli (Balantidium coli ), Ichthyophyllius species (Ichthiophthirius sp ec. ), Tricondina species (Trichondina spec.) Or epi
Stillis species (Epistylis spec.).

Furthermore, the compounds and agents discovered are protozoa of insects, for example strains, Microsporidia , in particular Nosema , very particularly the species Nosema which is a parasite of the honey bee. -It is effective against Apis ( Nosema apis ).

[0152] Example Example 1 beta-tube acquisition mRNA phosphorus cDNA and genomic DNA, C. Nassatus helminths from Quick Prep ^R Micro m
RNA kit (Biotech, Freiburg, Germany) and using C.I. Coronatum and C.I. Dynat ^R mRNA from Catinatum helminths
Obtained using the Direct kit (Dynal, Hamburg, Germany). Helminths were isolated from the equine large intestine and were microscopically distinguished according to the characteristic structure of the head and tail (RS Lichtenfeld (1
975), Helminths of domestic equids, Pr.
ocedings of the Helminthological So
ciety, Washington, 42 (Special issue), 1
-92, see).

CDNA synthesis was carried out according to C. In the case of mRNA derived from Nassatus, "reverse transcription system" (
Promega, Madison, USA) and C.I. Coronatum and C.I. In the case of mRNA derived from catinatum, superscript RTI
I reverse transcriptase (Gibco BRL Life Technologies). In the above case, an oligonucleotide with a length of 15 base pairs was used. The incubation was carried out at 42 ° C for 1 hour.

Genomic DNA was prepared using the QIA Amp-issue kit (Qiagen, Hi.
Lden, Germany) from 4 to 40 adult helminths. In this procedure, helminths were digested with Protease K for 2 hours at 55 ° C, and genomic DNA was extracted using a "spin column".

Example 2 Amplification of β-Tubulin Sequences Amplification of full length β-tubulin sequences or fragments can be performed, for example, in Ampl
iTaq Gold ^™ Polymerase (Perkin Elmer, Foster
City, California, USA).

Amplification of the β-tubulin sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 or a fragment thereof can be carried out using the primers shown in SEQ ID NO: 12-51.

C. For amplification of cDNA from coronatum, for example, SEQ ID NOs: 43 and 4
The sequence shown in FIG. The sequences shown in SEQ ID NOs: 40 and 42 are particularly suitable for amplification of cDNA derived from catinatum.

C. Amplification of Nastustus cDNA and genomic DNA of all species according to the invention was performed with 5 μl of 10 × buffer, 2.5 μl of MgCl ₂ (25 mM), ₂ μl of dNTP mixture (2 mM for each NTP), each specific primer (SEQ ID NO: 12).
-47) (50 pmol / μl) 1 μl, polymerase 0.5 μl (2.5 U)
And in a total volume of 50 μl containing 1-5 μl of DNA template. When the degenerate primer (SEQ ID NO: 48-51) is used, the concentration is 500 pmol /
1 μl of each primer was used. Annealing was performed at 46 ° C. for the degenerate primers and for specific primers the temperature was varied according to the calculated melting temperature. PCR cycles were chosen as follows: 96 min for 10 min, then 35-40 cycles with 1 min denaturation at 94 ° C, 1 min annealing, 1 min at 72 ° C and 10 min 72 ° C final step. C. Coronatum and C.I. For amplification of cDNA derived from catinatum, a "touchdown" PCR temperature program with the following profile was performed: first 15 cycles at 94 ° C for 30 seconds, then 1 minute at 60 ° C and 1 at 72 ° C.
Minutes, followed by 15 cycles of 95 ° C. for 30 seconds, 1 minute 55 ° C. and 1 minute 72
C. and finally 10 cycles of 95 ° C. for 30 seconds, then 45 ° C. for 1 minute and 72 ° C. for 1 minute. 7 for amplification of larger fragments (> 1000 base pairs)
The extension phase at 2 ° C. was extended to 2.30 minutes.

Example 3 C. Nassatas, C.I. Coronatum and C.I. The PCR product from the amplification of cDNA or genomic DNA from catinatum was "Original TA Cl".
oning Kit "/ Invitrogen, Leek, Netherlands
with ds), i.e. it was cloned in "Original TA Cloning ^R" vectors.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 14/435 C12N 1/15 4C084 16/18 1/19 4C085 C12N 1/15 1/21 4H045 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21/02 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/53 D G01N 33/15 33/566 33/50 33/569 A 33/53 33/577 B 33/566 33/58 A 33/569 C12P 21/08 33/577 C12N 15/00 ZNAA 33/58 5/00 A // C12P 21/08 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, L, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ , DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU ZA, ZW (72) Inventor Cienider, Thomas Germany Day-30539 Hanover Inde Abbie 38 38 (72) Inventor Pape, Michaela Germany Day-30173 Hanover Elkart Array 21 F Term (reference) 2G045 AA28 AA40 CB21 DA12 DA13 DA14 DA36 FB02 FB03 FB07 4B024 AA01 AA11 BA31 BA43 CA04 DA02 DA06 EA04 GA01 GA03 GA11 HA14 HA15 4B063 QA01 QA18 QQ08 QQ42 QR32 QR38 QR55 QR77 QR80 QS24 QS25 QS28 QS34 QX02 4B064 AG01 AG27 AG31 CA19 CC24 DA01 DA13 DA20 4B065 AA26X AA90Y AA91X AA92X AB01 AB05 AC14 BA02 CA24 CA25 CA44 CA45 CA46 4C084 AA02 AA03 AA06 AA07 AA13 BA03 CA53 NA14 ZB392 4C085 AA13 AA14 DD62 4H045 AA10 AA11 AA20 AA30 BA10 CA50 DA76 DA86 EA29 EA31 EA50 FA74

Claims (45)

[Claims] 1. A DNA encoding β-tubulin derived from Cyatostominae or a fragment thereof. 2. A) a polynucleotide having at least 85% identity to a polynucleotide encoding an amino acid sequence as set forth in SEQ ID NO: 2; b) an amino acid sequence as set forth in SEQ ID NO: 4. A polynucleotide having at least 85% identity to the encoding polynucleotide; c) a polynucleotide having at least 85% identity to the polynucleotide encoding the amino acid sequence as set forth in SEQ ID NO: 6; d) A polynucleotide having at least 85% identity to a polynucleotide encoding an amino acid sequence as set forth in SEQ ID NO: 8; e) a polynucleotide encoding an amino acid sequence as set forth in SEQ ID NO: 10. Porinu with at least 85% identity Reochido; comprising the claim 1 DNA according. 3. A) a polynucleotide having at least 95% identity to a polynucleotide encoding the amino acid sequence as set forth in SEQ ID NO: 2; b) an amino acid sequence as set forth in SEQ ID NO: 4. A polynucleotide having at least 95% identity to the encoding polynucleotide; c) a polynucleotide having at least 95% identity to the polynucleotide encoding the amino acid sequence as set forth in SEQ ID NO: 6; d) A polynucleotide having at least 95% identity to a polynucleotide encoding an amino acid sequence as set forth in SEQ ID NO: 8; e) to a polynucleotide encoding an amino acid sequence as set forth in SEQ ID NO: 10 Polynes with at least 95% identity Reochido; comprising the claim 1 DNA according. 4. A DNA according to claim 1, comprising the sequence as set forth in SEQ ID NO: 1. 5. The DNA according to claim 1, which comprises the sequence as shown in SEQ ID NO: 3. 6. A DNA according to claim 1, which comprises the sequence as set forth in SEQ ID NO: 5. 7. The DNA according to claim 1, comprising the sequence as set forth in SEQ ID NO: 7. 8. A DNA according to claim 1, which comprises the sequence as set forth in SEQ ID NO: 9. 9. A sequence comprising the sequence as set forth in SEQ ID NO: 11,
The DNA according to claim 1. 10. DNA according to one of claims 1 to 3 and 5 to 9, characterized in that the DNA is derived from Cylycocycles . 11. The DNA according to claim 1, wherein the DNA is derived from Cyatostomum . 12. DNA according to one of claims 1 to 3 and 5 to 10, characterized in that the DNA is derived from Cylicocycus nassatas . 13. DNA which is characterized in that from Shiatosutomamu-Koronatamu (Cyathost omum coronatum), DNA according to one of claims 1 to 4 and 11. 14. DNA according to one of claims 1 to 13, characterized in that the DNA contains at least one base substitution at codon 200 which leads to the expression of a polypeptide with anthelmintic resistance. 15. DNA, characterized in that the DNA is complementary to the DNA according to one of claims 1 to 14 or a fragment thereof. 16. RNA, characterized in that the RNA is complementary to the DNA according to one of claims 1-15. 17. An expression construct, which enables expression of DNA.
4. A construct, characterized in that it comprises a DNA according to one of 4 and a sequence operably linked thereto. 18. A vector, characterized in that it contains the DNA according to one of claims 1-14. 19. A host cell comprising the DNA according to one of claims 1-14, the expression construct according to claim 17 or the vector according to claim 18. 20. A polypeptide encoded by the DNA according to any one of claims 1-14 or a fragment thereof. 21. The polypeptide of claim 20, consisting of or comprising the amino acid sequence as set forth in SEQ ID NO: 2. 22. The polypeptide of claim 20, consisting of or comprising the amino acid sequence as set forth in SEQ ID NO: 4. 23. The polypeptide of claim 20, consisting of or comprising the amino acid sequence as set forth in SEQ ID NO: 6. 24. The polypeptide of claim 20, consisting of or comprising the amino acid sequence as set forth in SEQ ID NO: 8. 25. The polypeptide of claim 20, consisting of or comprising the amino acid sequence as set forth in SEQ ID NO: 10. 26. A polypeptide encoded by the DNA of claim 14. 27. A method for producing a polypeptide according to one of claims 20 to 26, which comprises expression of the polypeptide or a fragment thereof in a prokaryotic or eukaryotic expression system. 28. Use of a DNA oligonucleotide according to one of claims 1 to 15 for the detection of DNA derived from Satostominae, preferably a DNA oligonucleotide which hybridizes specifically to a non-coding DNA part. 29. A DNA which is derived from Siathostomae and which specifically hybridizes to the DNA according to one of claims 1 to 15 for the detection of the DNA encoding the polypeptide according to claim 26. use. 30. A method for the detection of Siathostomae, characterized in that the DNA according to claim 28 is hybridized to the DNA according to one of claims 1 to 15 and this is amplified by means of PCR. . 31. Antiparasitic resistance, characterized in that the DNA according to claim 29 is hybridized to the DNA according to one of claims 1 to 15 and this is amplified by means of PCR. Detection method of Satostominae. 32. A DNA oligonucleotide comprising one of the sequences as set forth in SEQ ID NO: 12 to SEQ ID NO: 51, or a sequence derived from one of the DNA sequences of claims 1-15. . 33. A diagnostic test kit comprising at least one of the oligonucleotide of claim 32 and / or the antibody of claim 35 or 36. 34. The diagnostic test kit according to claim 33, wherein the DNA oligonucleotide is provided with a detectable label. 35. An antibody, characterized in that the antibody specifically reacts with an epitope of the polypeptide according to one of claims 20 to 26. 36. The antibody according to claim 3, characterized in that the antibody is monoclonal.
The antibody according to 5. 37. Use of the antibody according to claim 35 or 36 as a nematicide. 38. Use of a polypeptide according to one of claims 20 to 26 for the production of a vaccine. 39. A method for identifying a substance that modulates the interaction of tubulin. 40. A) contacting the test substance with tubulin under conditions which allow the interaction of the tubulin molecules with each other and the binding of the test substance to tubulin, and b) the resulting binding of the test substance. , Detected by determining the ability of the tubulin protein molecules to interact with each other, and c) the ability of the tubulin protein molecules to interact with each other in the presence of the test substance interacts with each other in the absence of the test substance. 40. The method according to claim 39, characterized in that it is compared with their capabilities. 41. Method according to claim 39 or 40, characterized in that the tubulin used is the polypeptide according to one of claims 20 to 26. 42. The method according to one of claims 39 to 41, characterized in that a cell-based test system is used for the detection of the modulation of tubulin interaction in the presence of the test substance. . 43. Method according to one of claims 39 to 41, characterized in that a cell-free test system is used for the detection of the modulation of tubulin interaction in the presence of the test substance. . 44. A substance identified in the method of one of claims 39-43. 45. Use of a substance according to claim 44 for the manufacture of a medicament for the preventive or therapeutic treatment of nematode attack.