EP1497427A2 - Recepteur de l'helicokinine issu d'insectes - Google Patents

Recepteur de l'helicokinine issu d'insectes

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Publication number
EP1497427A2
EP1497427A2 EP03746285A EP03746285A EP1497427A2 EP 1497427 A2 EP1497427 A2 EP 1497427A2 EP 03746285 A EP03746285 A EP 03746285A EP 03746285 A EP03746285 A EP 03746285A EP 1497427 A2 EP1497427 A2 EP 1497427A2
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
polypeptide
host cell
dna
chemical compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03746285A
Other languages
German (de)
English (en)
Inventor
Horst-Peter Antonicek
Katrin Schnizler
Marcus Weidler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer CropScience AG
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Bayer CropScience AG
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Filing date
Publication date
Application filed by Bayer CropScience AG filed Critical Bayer CropScience AG
Publication of EP1497427A2 publication Critical patent/EP1497427A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/14—Ectoparasiticides, e.g. scabicides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

  • the invention relates to polypeptides which exert the biological activity of a helicokinin receptor, as well as polynucleotides which code for these polypeptides and in particular their use for finding active substances for crop protection.
  • a chemical compound found in a high throughput screening or other way e.g. B. modulating on a target protein from insect pests, can be tested directly on a homologous target protein, which was cloned from one or more mammalian species, for selectivity in order to exclude broadly active toxic compounds. Those are particularly good as target proteins To look at proteins from insect pests, for example, which do not occur in higher organisms, such as mammals.
  • Receptors for biologically active peptides in insects are particularly good target proteins and thus targets for the development of new insecticides.
  • the biological effect of these peptides is mediated by binding to specific receptor proteins in insect cells. Many of these endocrine or neuronal peptides interact with G protein-coupled receptors (GPCRs; King and Wilson, 1999) that activate heterotrime G proteins after the binding of a corresponding peptide ligand (Vanden Broeck et al., 1997).
  • GPCRs G protein-coupled receptors
  • Antagonists that can bind to peptide receptors and that can be derived from natural peptides or that have a completely new chemical structure can interfere with normal insect development, growth, behavior or homeostasis, for example, and in this way insects -generate specific and receptor-specific novel insecticides.
  • peptides that can influence juvenile hormone biosynthesis can influence juvenile hormone biosynthesis (allatotropins and allatostatins, Tobe et al., 1994), insulin-like peptides (Lagueux et al., 1990), peptides that regulate the water balance (Coast, 1998), or those peptides that can control muscle activity (Holman, 1986; for an overview, see Gäde, 1997b) can be isolated from different species.
  • Peptides from the kinine group have been isolated from a number of insect species from various families, including Dictyoptera, Orthoptera and Lepidoptera (Coast, 1998; Blackburn et al., 1995).
  • the first members of this family of peptides were isolated based on their ability to induce contractions in the isolated intestine in cockroaches (Holman, 1991).
  • Kinins are particularly potent, diuretic peptides in insects that stimulate the secretion of primary urine in the Malpighian organs (Coast, 1998).
  • the biological functions of the peptides can be checked in various tests, which measure, for example, muscle activity (Holman, 1991) or the excretion of water and electrolytes (Ramsey, 1954). Some of these biologically active peptides have been described that in vivo an experimentally induced exaggerated effect can lead to the death of the insect pests (Seinsche et al., 2000).
  • Receptors of kinin peptides are described in mollusks from the pond snail Lymnea stagnalis (Cox et al., 1997) and in Acari from a tick species (Boophilus microplus, Holmes et al, 2000). Only in Drosophila melanogaster is a receptor for a Drosophila kinin described from insects (Terhzaz et al., 1999). The cellular response to kinin stimulation in insects is an increase in the intracellular calcium concentration, which ultimately leads to an influx of chloride ions into the lumen of the Malpighian vessels (Coast, 1998).
  • Drosophila melanogaster from the Diptera family is an important model organism for insect genetics, but does not play a major role in agriculture as a harmful insect.
  • the present invention is therefore based on the object of binding further receptors to which endocrine or neuronal peptides from insects, in particular from economically important insect pests, and via the binding of which the biological functions of these peptides can be mediated, and test systems based thereon with a high throughput to provide test compounds (high throughput screening assays; HTS assays).
  • polypeptides which exercise at least one biological activity of a helicokinin receptor and comprise an amino acid sequence which have at least 70% identity, preferably at least 80% identity, particularly preferably at least 90% identity, very particularly preferably have at least 95% identity, with a sequence according to SEQ ID NO: 2 over a length of at least 20, preferably at least 25, particularly preferably at least 30 consecutive amino acids and very particularly preferably over their total length.
  • the degree of identity of the amino acid sequences is preferably determined with the aid of the GAP program from the GCG program package, version 9.1 under standard settings (Devereux et al., 1984).
  • polypeptides refers to both short amino acid chains, commonly referred to as peptides, oligopeptides, or oligomers, and longer amino acid chains, commonly referred to as proteins. It includes amino acid chains that can be modified either by natural processes, such as post-translational processing, or by chemical processes that are state of the art. Such modifications can occur at various locations and multiple times in a polypeptide, such as, for example, on the peptide backbone, on the amino acid side chain, on the amino and / or on the carboxy terminus.
  • acetylations include, for example, acetylations, acylations, ADP ribosylations, amidations, covalent linkages with Flavins, heme portions, nucleotides or nucleotide derivatives, lipids or lipid derivatives or phosphatidylinositol, cyclizations, disulfide bridging, demethylations, cystine formations, formylations, gamma-carboxylations, glycosylations, hydroxylations, iodinations, methylations, myristoylations,, oxidations , proteolytic processing, phosphorylation, selenoylation and tRNA-mediated addition of amino acids.
  • polypeptides of the invention can be in the form of "mature” proteins or as parts of larger proteins, e.g. as fusion proteins. Furthermore, they can have secreting or "leader” sequences, pro sequences, sequences which enable easy purification, such as multiple histidine residues, or additional stabilizing amino acids.
  • polypeptides according to the invention do not have to be complete receptors, but can also only be fragments thereof, as long as they at least still have a biological activity of a complete helicokinin receptor.
  • polypeptides according to the invention can have deletions or amino acid substitutions compared to the corresponding region of naturally occurring receptors, as long as they still exert at least a biological activity of the complete helicokinin receptors.
  • Conservative substitutions are preferred.
  • conservative substitutions include variations in which one amino acid is replaced by another amino acid from the following group:
  • Aromatic residues Phe, Tyr and Trp.
  • biological activity of a helicokinin receptor means the binding of helicokinin to a peptide receptor.
  • Preferred forms of the polypeptides according to the invention are helicokinin receptors from lepidopters, in particular from Heliothis virescens.
  • a particularly preferred embodiment of the polypeptides according to the invention is the Helicoldnin receptor from Heliothis virescens, which has the amino acid sequence according to SEQ ID NO: 2.
  • the present invention also relates to polynucleotides which code for the polypeptides according to the invention.
  • polynucleotides according to the invention are in particular single-stranded or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA).
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • Preferred embodiments are fragments of genomic DNA, which may contain introns, and cDNAs.
  • a preferred embodiment of the polynucleotides according to the invention is a cDNA which has a polynucleotide sequence according to SEQ ID NO: 1.
  • Polynucleotides which hybridize to the sequences according to SEQ ID NO: 1 under stringent conditions are also encompassed by the present invention.
  • hybridize describes the process in which a single-stranded nucleic acid molecule is base paired with a complementary strand.
  • DNA fragments from insects other than Heliothis virescens, for example, which code for polypeptides with the biological activity of helicokinin receptors can be isolated.
  • Hybridization solution 6X SSC / 0% formamide
  • preferred hybridization solution 6X SSC / 25% formamide
  • Hybridization temperature 34 ° C
  • preferred hybridization temperature 42 ° C
  • 2nd washing step 2X SSC at 45 ° C; preferred 2nd washing step: 0.6X SSC at 55 ° C; particularly preferred 2nd washing step: 0.3X SSC at 65 ° C.
  • the present invention encompasses polynucleotides which have an at least 70% identity, preferably at least 80% identity, particularly preferably at least 90% identity, very particularly preferably at least 95% identity, with a sequence according to SEQ ID NO: 1 over a length of at least 20, preferably at least 25, particularly preferably at least 30 consecutive nucleotides and very particularly preferably over their total length.
  • the degree of identity of the polynucleotide sequences is preferably determined with the aid of the GAP program from the GCG program package, version 9.1 under standard settings.
  • the present invention furthermore relates to DNA constructs which comprise a polynucleotide according to the invention and a heterologous promoter.
  • heterologous promoter refers to a promoter which has different properties than the promoter which controls the expression of the gene in question in the original organism.
  • promoter as used herein generally refers to expression control sequences.
  • heterologous promoters depend on whether pro- or eukaryotic cells or cell-free systems are used for expression.
  • heterologous promoters are the early or late promoter of SV40, adenovirus or cytomegalovirus, the lac system, the trp system, the main operator and promoter regions of the phage lambda, the control regions of the fd coat protein, the promoter of the 3-phosphoglycerate kinase coding gene, the promoter of the gene coding for acid phosphatase and the promoter of the gene coding for the ⁇ -mating factor of the yeast.
  • the invention furthermore relates to vectors which contain a polynucleotide according to the invention or a DNA construct according to the invention. All plasmids, phasmids, cosmids, YACs or artificial chromosomes used in molecular biological laboratories can be used as vectors.
  • the present invention also relates to host cells which contain a polynucleotide according to the invention, a DNA construct according to the invention or a vector according to the invention.
  • host cell refers to cells that do not naturally contain the polynucleotides of the invention.
  • Suitable host cells are both prokaryotic cells, such as bacteria of the genera Bacillus, Pseudomonas, Streptomyces, Streptococcus, Staphylococcus, preferably E. coli, and eukaryotic cells, such as yeast, mammalian, amphibian, insect or plant cells.
  • Preferred eukaryotic host cells are HEK-293, Schneider S2, Spodoptera Sf9, Kc, CHO, COS1, COS7, HeLa, C127, 3T3 or BHK cells and in particular Xenopus oocytes.
  • the invention furthermore relates to antibodies which bind specifically to the abovementioned polypeptides or receptors.
  • Such antibodies are produced in the usual way.
  • such antibodies can be produced by injecting a substantially immunocompetent host with an amount of the invention effective for antibody production Polypeptide or a fragment thereof and by subsequent recovery of this antibody.
  • an immortalized cell line which produces monoclonal antibodies can be obtained in a manner known per se.
  • the antibodies can optionally be labeled with a detection reagent.
  • Preferred examples of such a detection reagent are enzymes, radioactively labeled elements, fluorescent chemicals or biotin.
  • fragments can also be used which have the desired specific binding properties.
  • antibody as used herein also extends to parts of complete antibodies, such as Fa-, F (ab ') 2 - or Fv fragments, which still have the ability to target the epitopes of the polypeptides according to the invention tie.
  • the polynucleotides according to the invention can be used in particular for the production of transgenic invertebrates. These can be used in test systems which are based on an expression of the polypeptides according to the invention which differs from the wild type. Furthermore, on the basis of the information disclosed herein, it is possible to produce transgenic invertebrates in which the modification of other genes or promoters causes a change in the expression of the polypeptides according to the invention.
  • transgenic invertebrates are produced, for example, in Drosophila melanogaster by gene transfer mediated by P element (Hay et al., 1997) or in Caenorhabditis elegans by gene transfer mediated by transposon (e.g. by Tel; Plasterk, 1996).
  • the invention thus also relates to tansgenic invertebrates which contain at least one polynucleotide according to the invention, preferably transgenic invertebrates of the species Drosophila melanogaster or Caenorhabditis elegans, and their transgenic progeny.
  • the transgenic invertebrates preferably contain the polypeptides according to the invention in a form which differs from the wild type.
  • the present invention furthermore relates to processes for producing the polypeptides according to the invention.
  • host cells which contain a polynucleotide according to the invention can be cultivated under suitable conditions.
  • the polynucleotide to be expressed can be adapted to the "codon usage" of the host cells.
  • the desired polypeptides can then be isolated from the cells or the culture medium in a conventional manner.
  • the polypeptides can also be produced in in vitro systems.
  • a rapid method for isolating the polypeptides according to the invention begins with the expression of a fusion protein, whereby the fusion partner can be affinity-cleaned in a simple manner.
  • the fusion partner can be, for example, glutathione S-transferase.
  • the fusion protein can then be purified on a glutathione affinity column.
  • the fusion partner can be separated by partial proteolytic cleavage, for example on linkers between the fusion partner and the polypeptide according to the invention to be purified.
  • the linker can be designed to include target amino acids, such as arginine and lysine residues, which define sites for trypsin cleavage. Standard cloning techniques using oligonucleotides can be used to create such linkers.
  • detergent extractions are preferably carried out in the cleaning processes, for example using detergents which act as secondary and Do not influence the tertiary structures of the polypeptides, or influence them only slightly, such as non-ionic detergents.
  • the purification of the polypeptides according to the invention can include the isolation of membranes starting from host cells which express the polynucleotides according to the invention. Such cells preferably express the polypeptides according to the invention in a sufficient number of copies, so that the amount of polypeptides in a membrane fraction is at least 10 times higher than that found in comparable membranes - by cells which naturally express helicokinin receptors; the amount is particularly preferably at least 100 times, very particularly preferably at least 1000 times higher.
  • isolation or purification means that the polypeptides of the invention are separated from other proteins or other macromolecules of the cell or tissue.
  • a composition containing the polypeptides according to the invention is preferably enriched at least 10-fold and particularly preferably at least 100-fold with respect to the protein content of polypeptides according to the invention compared to a crude extract from host cells.
  • the purity or the protein content of the preparations can be determined in a manner known per se, for example by SDS polyacrylamide gel electrophoresis.
  • polypeptides according to the invention can also be affinity-purified without fusion partners using antibodies which bind to the polypeptides.
  • the present invention furthermore also relates to processes for producing the polynucleotides according to the invention.
  • the polynucleotides according to the invention can be produced in the usual way.
  • the polynucleotides can be synthesized completely chemically. It is also possible to chemically synthesize only short pieces of the polynucleotides according to the invention and to label such oligonucleotides radioactively or with a fluorescent dye.
  • the marked Oligonucleotides can be used to search cDNA libraries made from insect mRNA or genomic libraries made from insect genomic DNA. Clones to which the labeled Ohgonuldeotide hybridize are selected to isolate the DNA in question. After characterization of the isolated DNA, the polynucleotides according to the invention are obtained in a simple manner.
  • the polynucleotides according to the invention can also be produced by means of PCR methods using chemically synthesized Ohgonuldeotide.
  • oligonucleotide (s) as used herein means DNA molecules consisting of 10 to 50 nucleotides, preferably 15 to 30 nucleotides. They are chemically synthesized and can be used as probes.
  • new active substances for crop protection or pharmaceutical active substances for the treatment of humans and animals such as chemical compounds which, as modulators, in particular as agonists or antagonists, change the properties of the helicokinin receptors according to the invention can be identified become.
  • a recombinant DNA molecule which comprises at least one polynucleotide according to the invention is introduced into a suitable host cell.
  • the host cell is cultivated in the presence of a compound or a sample which comprises a multiplicity of compounds under conditions which allow the expression of the receptors according to the invention.
  • a change in the receptor properties can be detected, for example, as described in Example 2 below. In this way it is possible to find insecticidal substances, for example.
  • Receptors preferably change the concentration of intracellular cAMP or intracellular calcium via an interaction with G proteins after activation. Changes in the receptor properties due to chemical compounds can Therefore, after heterologous expression, for example by measuring the intracellular cAMP concentrations directly via ELISA assay systems (Biomol, Hamburg, Germany) or RIA assay systems (NEN, Schwalbach, Germany) in HTS format. Indirect measurement of the cAMP concentration is possible with the help of reporter genes (eg luciferase), the expression of which depends on the cAMP concentration (Stratowa et al, 1995). The coexpression of receptors with special G proteins, e.g.
  • G ⁇ l5, G l6 or also chimeric G proteins in heterologous systems and the measurement of the increase in calcium, e.g. with fluorescent dyes or equorine, is an alternative screening option (Stahles et al., 1997 ; Conklin et al., 1993).
  • binding of GTP to the activated G protein can be used as a read-out system for the testing of substances.
  • the polynucleotides or polypeptides according to the invention also make it possible to find compounds which bind to the receptors according to the invention without having to measure a change in the activity of the receptors.
  • host cells which contain the polynucleotides according to the invention and express the corresponding receptors or polypeptides or the gene products themselves are brought into contact with a compound or a mixture of compounds under conditions which allow the interaction of at least one compound with the host cells, the receptors or the individual Allow polypeptides.
  • the polypeptides according to the invention can be used in labeled form in such binding experiments.
  • agonist refers to a molecule that activates the receptors of the invention.
  • modulator refers to a molecule that displaces an agonist from its binding site.
  • modulator as used herein represents the generic term for agonist or antagonist.
  • Modulators can be small organic chemical molecules, peptides or antibodies that bind to the polypeptides according to the invention.
  • modulators can be small organic chemical molecules, peptides or antibodies which bind to a molecule which in turn binds to the polypeptides according to the invention and thereby influences their biological activity. Modulators may 'constitute natural substrates and ligands mimetics.
  • the modulators are preferably small organic chemical compounds.
  • the binding of the modulators to the polypeptides according to the invention can change the cellular processes in a way which leads to the death, paralysis or sterility of the insects treated therewith.
  • In vivo tests on insects, insect larvae or insect eggs to verify the insecticidal properties of the found modulators are well known.
  • the present invention therefore also extends to the use of modulators of the polypeptides according to the invention as insecticides or medicaments - hereinafter referred to as "active ingredients”.
  • the active compounds can be converted into the customary formulations, such as solutions, emulsions, wettable powders, suspensions, powders, dusts, pastes, soluble powders, granules, suspension emulsion concentrates, active substance-impregnated natural and synthetic substances and very fine encapsulations in polymeric substances.
  • formulations are prepared in a known manner, for example by mixing the active ingredients with extenders, that is to say liquid solvents and / or solid carriers, if appropriate using surface-active agents, ie emulsifiers and / or dispersants and / or foam-generating agents. If water is used as an extender, organic solvents can, for example, also be used as auxiliary solvents.
  • extenders that is to say liquid solvents and / or solid carriers, if appropriate using surface-active agents, ie emulsifiers and / or dispersants and / or foam-generating agents.
  • surface-active agents ie emulsifiers and / or dispersants and / or foam-generating agents.
  • organic solvents can, for example, also be used as auxiliary solvents.
  • aromatics such as xylene, toluene, or alkylnaphthalenes
  • chlorinated aromatics and chlorinated aliphatic hydrocarbons such as chlorobenzenes, chlorethylenes or methylene chloride
  • aliphatic hydrocarbons such as cyclohexane or paraffins, for example petroleum fractions, mineral and vegetable oils
  • Alcohols such as butanol or glycol, and their ethers and esters
  • ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone
  • strongly polar solvents such as dimethyl formamide and dimethyl sulfoxide, and water.
  • Possible solid carriers are: e.g. Ammonium salts and natural rock powders such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth and synthetic rock powders such as highly disperse silica, aluminum oxide and silicates are suitable as solid carriers for granules: e.g. broken and fractionated natural rocks, such as calcite, marble, pumice, sepiolite, dolomite as well as synthetic granules from inorganic and organic flours and granules from organic material, such as sawdust, coconut shells, corn cobs and tobacco stems; as emulsifying and / or foaming agents are possible: e.g.
  • nonionic and anionic emulsifiers such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, e.g. Alkylaryl polyglycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates and protein hydrolyzates;
  • Possible dispersants are: e.g. Lignin liquor and methyl cellulose.
  • Adhesives such as carboxymethyl cellulose, natural and synthetic polymers in the form of powders, granules or latices, such as gum arabic, polyvinyl alcohol, polyvinyl acetate, and natural phospholipids, such as cephalins and lecithins and synthetic phospholipids, can be used in the formulations.
  • Other additives can be mineral and vegetable oils.
  • Dyes such as inorganic pigments, for example iron oxide, titanium oxide, ferrocyan blue and organic dyes, such as alizarin, azo and metal phthalocyanine dyes and trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc, can be used become.
  • the formulations generally contain between 0.1 and 95% by weight of active compound, preferably between 0.5 and 90%.
  • the active compounds can preferably be used as crop protection agents, in particular for controlling insects from the order of the Lepidoptera, such as, for example, Pectinophora gossypiella, Bupalus piniarius, Cheimatobia brumata, Li hocolletis blancardella, Hyponomeuta padella, Plutella xylostella, Malacos .
  • Lepidoptera such as, for example, Pectinophora gossypiella, Bupalus piniarius, Cheimatobia brumata, Li hocolletis blancardella, Hyponomeuta padella, Plutella xylostella, Malacos .
  • the treatment of the plants and parts of plants with the active compounds takes place directly or by acting on their surroundings, living space or storage space according to the usual treatment methods, e.g. by dipping, spraying, vaporizing, atomizing, scattering, spreading and, in the case of propagation material, in particular in the case of seeds, furthermore by coating in one or more layers.
  • the active ingredients are also suitable for combating insects that farm animals such as cattle, sheep, goats, horses, pigs, donkeys, camels, buffalo, rabbits, chickens, turkeys, ducks, geese, bees, other pets, such as dogs , Cats, house birds, aquarium fish and so-called experimental animals such as hamsters, guinea pigs, rats and mice.
  • farm animals such as cattle, sheep, goats, horses, pigs, donkeys, camels, buffalo, rabbits, chickens, turkeys, ducks, geese, bees, other pets, such as dogs , Cats, house birds, aquarium fish and so-called experimental animals such as hamsters, guinea pigs, rats and mice.
  • the control of these insects is intended to reduce deaths and reduced performance (for meat, milk, wool, skins, eggs, honey, etc.), so that the use of the active ingredients enables more economical and easier animal husbandry.
  • the active ingredients are used in the veterinary sector in a known manner by enteral administration in the form of, for example, tablets, capsules, drinkers, drenches, granules, pastes, boluses, the feed-through method, suppositories, by parenteral administration, such as, for example, by injections (intramuscular, subcutaneous, intravenous, intraperitonal, etc.), implants, by nasal application, by dermal application in the form of, for example, diving or bathing (dipping), spraying (spray), pouring on (pour-on and spot-on), washing, powdering and with the help of shaped articles containing active ingredients, such as collars, ear tags, tail tags, limb tapes, holsters, marking devices, etc.
  • enteral administration in the form of, for example, tablets, capsules, drinkers, drenches, granules, pastes, boluses, the feed-through method, suppositories
  • parenteral administration such as, for example
  • the active ingredients can be formulated (for example powders, emulsions, flowable agents) which contain the active ingredients in an amount of 1 to 80% by weight, directly or after 100 to 10,000 Apply a thinner or use it as a chemical bath.
  • formulated for example powders, emulsions, flowable agents
  • polynucleotides, vectors and regulatory regions according to the invention described above can also be used to find genes which code for polypeptides which are involved in the construction of functionally similar receptors in insects.
  • functionally similar receptors are understood to include receptors which comprise polypeptides which differ in terms of the amino acid sequence from those herein described polypeptides, but have essentially the same functions.
  • SEQ ID NO: 1 shows the polynucleotide sequence of the isolated helicokinin receptor cDNA.
  • SEQ ID NO: 2 shows the amino acid sequence of the polypeptide encoded by the polynucleotide sequence according to SEQ ID NO: 1.
  • Figure 1 shows the result of the electrophysiological measurement after injection of helicokinin receptor DNA into Xenopus oocytes and the addition of helicokinin peptide (Helicokinin III, 100nM, Blackburn et al, 1995) in comparison to the application of peptide to control oocytes in which no helicokinin receptor DNA was previously injected.
  • helicokinin peptide Helicokinin III, 100nM, Blackburn et al, 1995
  • Polynucleotides were manipulated using standard recombinant DNA technology methods (Sambro ⁇ k et al., 1989). Bioinformatic processing of nucleotide and amino acid sequences was carried out with the GCG Version 9.1 program package (GCG Genetics Computer Group, Inc., Madison Wisconsin, USA).
  • sequence region of SEQ ID NO: 1 was amplified by means of polymerase chain reaction (PCR) and cloned into the vector pCMV Script EX (Stratagene, La Jolla, USA).
  • G-protein-activatable potassium channels (GIRK1 and GIRK4) are partially co-expressed in order to measure the activation of the receptors (White et al., 1998).
  • the oocytes come from an adult female Xenopus laevis frog (Horst Kahler, Hamburg, Germany).
  • the frogs are kept in large tanks with circulating water at a water temperature of 18-20 ° C. Parts of the Frog ovars are removed under complete anesthesia through a my incision in the abdomen (approx. 1 cm).
  • Injection electrodes with a diameter of 10 - 15 ⁇ m are manufactured with a pipette puller (type L / M-3P-A, List-electronic, Darmstadt-Eberstadt, Germany). Before the injection, aliquots are thawed with the receptor DNA or GIRK1 / 4 DNA and diluted with water to a final concentration of 10ng / ⁇ l. The DNA samples are centrifuged at 3200 g for 120 s (type Biofuge 13, Heraeus Instruments GmbH, Hanau, Germany). A pulled-out PE tube is then used as the transfer tube to fill the pipettes from behind.
  • a pipette puller type L / M-3P-A, List-electronic, Darmstadt-Eberstadt, Germany.
  • the injection electrodes are attached to an X, Y, Z travel unit (machining center EP 1090, isel-automation, Eiterfeld, Germany). With the help of a Macintosh computer, the oocytes in the wells of the microtiter plates are approached and about 50nl of the DNA solution is injected into the oocyte by short pressure application (0.5-3 bar, 3-6s). 3. Electrophysiological measurements
  • a two-electrode voltage clamp with a TURBO TEC-10CD (npi electronic GmbH, Tamm, Germany) amplifier is used for the electrophysiological measurements.
  • Current and voltage electrodes have a diameter of 1 - 3 ⁇ m and are filled with 1.5M KCl and 1.5M potassium acteate.
  • the pipettes have a capacitive resistance of 0.2 - 0.5 MW.
  • the oocytes are transferred to a small chamber which is continuously rinsed with normal Rimland solution (in mM: KCl 90, MgCl 2 3, HEPES 5, pH 7.2).
  • Rimland solution in mM: KCl 90, MgCl 2 3, HEPES 5, pH 7.2.
  • the perfusion solution is replaced by a substance solution with the same composition and additionally the desired substance concentration.
  • a clamping potential of -60mV the successful expression of the receptor DNA is checked after one week. Unresponsive oocytes are discarded. All others are used for substance testing.
  • the data is documented using a YT recorder (YT recorder, model BD 111, Kipp & Zonen Delft BV, AM Delft, The Netherlands).
  • Origin evaluation software Microcal Origin, Microcal Software, Inc., Northampton, MA 01060-4410 USA (Additive GmbH, Friedrichsdorf / Ts, Germany). Average values, standard deviation, IC 5 o values and IC 5 n- Curves are calculated using Origin, and these measurements were performed at least twice.
  • Insect myotropic peptides isolation, structural characterization and biological properties.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Insects & Arthropods (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne des polypeptides, qui exercent l'activité biologique d'un récepteur de l'hélicokinine, ainsi que des polynucléotides codant pour ces polypeptides et leur utilisation dans le repérage de principes actifs phytoprotecteurs.
EP03746285A 2002-04-16 2003-04-04 Recepteur de l'helicokinine issu d'insectes Withdrawn EP1497427A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10216738A DE10216738A1 (de) 2002-04-16 2002-04-16 Helicokinin-Rezeptor aus Insekten
DE10216738 2002-04-16
PCT/EP2003/003519 WO2003087356A2 (fr) 2002-04-16 2003-04-04 Recepteur de l'helicokinine issu d'insectes

Publications (1)

Publication Number Publication Date
EP1497427A2 true EP1497427A2 (fr) 2005-01-19

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP03746285A Withdrawn EP1497427A2 (fr) 2002-04-16 2003-04-04 Recepteur de l'helicokinine issu d'insectes

Country Status (6)

Country Link
US (1) US20050235365A1 (fr)
EP (1) EP1497427A2 (fr)
JP (1) JP2005523003A (fr)
AU (1) AU2003232196A1 (fr)
DE (1) DE10216738A1 (fr)
WO (1) WO2003087356A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10013618A1 (de) * 2000-03-18 2001-09-20 Bayer Ag Rezeptoren für Peptide aus Insekten
WO2001070980A2 (fr) * 2000-03-23 2001-09-27 Pe Corporation (Ny) Recepteurs isoles couples a la proteine g, molecules d'acides nucleiques codant des proteines gpcr et leurs utilisations en tant que cibles insecticides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03087356A2 *

Also Published As

Publication number Publication date
AU2003232196A1 (en) 2003-10-27
US20050235365A1 (en) 2005-10-20
WO2003087356A2 (fr) 2003-10-23
AU2003232196A8 (en) 2003-10-27
WO2003087356A3 (fr) 2003-12-04
JP2005523003A (ja) 2005-08-04
DE10216738A1 (de) 2003-10-30

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