The genus Hirsutella includes about 50 entomopathogenic species that attack a wide range of insects.

Genus Hirsutella mencakup sekitar 50 spesies entomopathogenic yang menyerang berbagai serangga. The mononematous hyphomycete fungi, Hirsutella thompsonii var. The mononematous hyphomycete jamur, Hirsutella thompsonii var. thompsonii, H. thompsonii var. thompsonii, H. thompsonii var. vinacea and H. thompsonii synnematosa, all produce hirsutellin A with similar toxicogenic activity. vinacea dan H. thompsonii synnematosa, semua menghasilkan hirsutellin J toxicogenic dengan kegiatan serupa. In vivo, Hirsutella thompsonii and related species produce infective conidia on phialides arising from external mycelia growing from the host. Dalam vivo, Hirsutella thompsonii terkait dan jenis produksi infective conidia pada phialides timbul dari eksternal mycelia berkembang dari tuan rumah. Hyphae can emerge from cadavers through oral and anal openings, appendages, genital opening and at times through the body wall. Hyphae dapat muncul dari cadavers melalui lisan dan anal bukaan, appendages, genital dan sewaktu-waktu melalui badan dinding. Within the host, hyphae usually develop initially in the central area of the hemocoel as oval bodies and then become chain-like as they grow anteriorly or posteriorly along the inner body wall. Di tuan rumah, biasanya hyphae awalnya berkembang di tengah kawasan yang hemocoel sebagai badan lonjong dan kemudian menjadi rantai seperti karena tumbuh anteriorly atau posteriorly sepanjang dinding inner tubuh. In nature, these internal hyphae may break up and form multinucleate spherical chlamydospores. Di alam, ini internal hyphae Mei bubar dan formulir multinucleate bulat chlamydospores. Host mortality appears to result from invasion of tissue by the fungal hyphae, while no toxicogenic activity has been reported. Host nampaknya kematian akibat invasi dari jaringan oleh fungal hyphae, saat tidak ada aktivitas toxicogenic telah dilaporkan. Although different naturally occurring biopesticides have been developed, none has been produced from the metabolites of Hirsutella spp. Walaupun berbeda terjadi secara alami biopesticides telah berkembang, none telah dihasilkan dari metabolites dari Hirsutella spp. This invention addresses this need by providing a safe, naturally occurring biopesticide from Hirsutella spp. Temuan ini perlu alamat ini dengan memberikan yang aman, alami terjadi biopesticide dari Hirsutella spp. which has a broad spectrum insecticidal effect. yang memiliki berbagai insecticidal efek. SUMMARY OF THE INVENTION RANGKUMAN DARI Experiment Recognizing the need to control invertebrate pests which avoids the use of traditional pesticides and their toxic side effects, the inventors searched for a novel biopesticide among the various invertebrate fungal pathogens. Menyadari kebutuhan untuk hewan tdk bertulang punggung pengendalian hama yang menghindari penggunaan pestisida tradisional dan beracun efek samping, yang penemu mencari sebuah novel biopesticide di antara berbagai hewan tdk bertulang punggung fungal pathogens. These efforts have culminated in the substantial purification and isolation of a novel toxin from the genus Hirsutella denoted hirsutellin A. Upaya-upaya telah culminated substansial dalam pemurnian dan isolasi dari novel toksin dari genus Hirsutella denoted hirsutellin A. The hirsutellin A molecule has been characterized and found to possess a broad spectrum of activity with various invertebrate pests. J hirsutellin molekul yang telah ditemukan karakteristik dan memiliki berbagai kegiatan dengan berbagai hewan tdk bertulang punggung hama. These findings advantageously render routine the cloning of the hirsutellin A toxin polynucleotide

thereby enabling the development of novel host systems for use as pest-controlling compositions and as sources of hirsutellin A polypeptide. Temuan-temuan rutin yang menyebabkan advantageously kloning dari hirsutellin J toksin polynucleotide sehingga memungkinkan pengembangan sistem host novel untuk digunakan sebagai pengendalian hama-Komposisi dan sebagai sumber hirsutellin J polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS DESKRIPSI SINGKAT gambar-gambar FIG. Fig. 1 shows a 34 amino acid sequence which comprises a hirsutellin A polypeptide. 1 menunjukkan urutan 34 asam amino yang terdiri dari sebuah hirsutellin J polypeptide. FIG. Fig. 2 shows toxicity of hirsutellin A via Contact and Residual exposure to the citrus rust mite. 2 menunjukkan kebisaan dari hirsutellin J melalui Hubungi dan sisa eksposur ke tungau karat jeruk. FIG. Fig. 2A shows residual toxicity of hirsutellin A to adult citrus rust mite at 24, 48, and 72 hours at 0, 10, 32, 56, and 100 g/ml. 2A menunjukkan sisa racun dari hirsutellin A untuk orang dewasa tungau karat jeruk di 24, 48, dan 72 hari pada 0, 10, 32, 56, dan 100 g / ml. FIG. Fig. 2B shows egg production on the leaves after 72 hours following residual application of hirsutellin A. 2B menunjukkan produksi telur pada daun setelah 72 jam berikut sisa dari aplikasi hirsutellin A. FIG. Fig. 2C shows contact and residual toxicity of hirsutellin A to adult citrus rust mite. 2C menunjukkan kontak dan sisa racun dari hirsutellin A untuk orang dewasa tungau karat jeruk. FIG. Fig. 2D shows egg production after 72 hours following contact and residual application of hirsutellin A. 2D menunjukkan produksi telur setelah 72 jam berikut kontak dan sisa dari aplikasi hirsutellin A. DETAILED DESCRIPTION OF THE INVENTION Detil PENJELASAN ATAS Experiment The present invention discloses the novel insecticidal mycotoxin protein hirsutellin A. This fungal metabolite is toxic to invertebrate pests. Sekarang invensi yang mengungkapkan novel insecticidal mycotoxin protein hirsutellin A. fungal metabolite ini adalah racun untuk hama hewan tdk bertulang punggung. Treatment of adult insects or larvae with hirsutellin A protein is lethal. Perawatan dewasa atau serangga larvae dengan hirsutellin J protein adalah letal. An advantage to the use of hirsutellin A is that pests can be controlled without the environmental and public safety hazards previously presented by chemical control agents. Sebuah keuntungan dengan penggunaan hirsutellin J adalah hama yang dapat dikendalikan tanpa lingkungan dan keselamatan publik bahaya sebelumnya disajikan oleh agen kontrol kimia. The hirsutellin A of this invention is characterized by a monomeric basic protein with a molecular weight of 15 KD and a pl of 10.5., with heat sensitivity at 60 C. or greater after 15 minutes. A of the hirsutellin penemuan ini adalah ditandai dengan monomeric dasar protein dengan berat molekul 15 KD dan pl dari 10,5., Dengan sensitivitas panas pada 60 C. atau lebih

setelah 15 menit. Crude broth extracts of Hirsutella sp. Bulyon crude ekstrak dari Hirsutella sp. grown in vitro in shake cultures, as well as purified hirsutellin A, exhibit toxicogenic activity in various insect species. berkembang di dalam vitro guncangkan budaya, serta suci hirsutellin A, toxicogenic kegiatan pameran di berbagai jenis serangga. The exact mechanism of action for hirsutellin A toxicity is unknown. Mekanisme yang tepat untuk tindakan hirsutellin J kebisaan tidak diketahui. Injection of hirsutellin A into Galleria mellonella larvae results in alterations of the digestive tract, particularly the midgut and the malpighian tubules. Suntikan dari hirsutellin J menjadi Galleria mellonella larvae hasil dari perubahan dalam sistem pencernaan, terutama midgut dan malpighian tubules. Ultrastructural studies of organs and cells show strong cellular changes in the malpighian tubules. Ultrastructural studi organ dan sel selular kuat menunjukkan perubahan dalam malpighian tubules. The pycnotic evolution of the nucleus, which displays large and dense aggregates of chromatin, is intense and many alterations are visible at the cytoplasmic level. The pycnotic evolusi inti, yang akan menampilkan besar dan padat aggregates dari chromatin, adalah intens dan banyak perubahan yang terlihat pada tingkat cytoplasmic. The hyaloplasm shows a reduced electron density and mitochondria show obvious alterations. Hyaloplasm yang menunjukkan electron mengurangi kepadatan mitochondria dan jelas menunjukkan perubahan. The present invention provides substantially pure hirsutellin A polypeptide and method for producing. Temuan ini menyediakan substansial murni hirsutellin J polypeptide dan metode produksi. In addition, polynucleotide sequences encoding hirsutellin A or functional fragments of the polypeptide are included. Selain itu, polynucleotide sequences encoding hirsutellin J fungsional atau fragmen dari polypeptide dimasukkan. Also provided is a method for controlling an invertebrate pest using a pest-controlling amount of hirsutellin A polypeptide. Juga disediakan adalah sebuah metode untuk mengendalikan hama hewan tdk bertulang punggung menggunakan hama-mengendalikan jumlah hirsutellin J polypeptide. The term "pest" refers to any agricultural invertebrate including, but not limited to, arthropods such as ticks, mites, lepidoptera, hemiptera, diptera (mosquitos), homoptera and coleopera (beetles). Istilah "hama" merujuk kepada hewan tdk bertulang punggung pertanian termasuk, namun tidak terbatas pada, seperti arthropods ticks, mites, lepidoptera, hemiptera, diptera (mosquitos), homoptera dan coleopera (beetles). Other pests may include Plutella, Aedes, Galleria, Drosophila, Bombyx, Aphis and Eutetranychus. Lainnya mungkin termasuk hama Plutella, Aedes, Galleria, Drosophila, Bombyx, Aphis dan Eutetranychus. The term "pest-controlling" or "pest controlling activity", used throughout the specification and in the claims include any pesticidal or pest inhibiting activities of a composition of the invention against a particular pest. Istilah "pengendalian hama-" atau "kegiatan pengendalian hama", digunakan di seluruh spesifikasi dan di klaim apapun termasuk pesticidal atau hama inhibiting kegiatan dari komposisi dari penemuan terhadap hama tertentu. These terms include killing as well as activities which cause such injury to the pest so as to inhibit the production or development of future progeny. Istilah tersebut termasuk pembunuhan serta kegiatan-kegiatan seperti itu yang menyebabkan cedera pada hama sehingga menghalangi produksi atau perkembangan masa depan anak.

Amino acids referred to herein may be identified according to the following three-letter or oneletter abbreviations: Asam amino yang disebut di sini dapat diidentifikasi menurut berikut tiga huruf atau satu huruf singkatan: ______________________________________ ______________________________________ Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Tiga Banding Satu Banding amino acid Abbreviation Abbreviation ______________________________________ ______________________________________ L-Alanine Ala A L-Alanine Ala J L-Arginine Arg R L-Arginine Arg R L-Asparagine Asn N L-Asparagine ASN N L-Aspartic Acid L-Aspartic Acid Asp D Asp D L-Cysteine Cys C L-Cysteine Cys C L-Glutamine Gln Q L-Glutamine GLN T L-Glutamic Acid L-Glutamic Acid Glu E ALA E L-Glycine Gly G L-Glycine GLY G L-Histidine His H L-Histidine Nya H L-Isoleucine Ile I L-Isoleucine Ile saya L-Leucine Leu L L-Leucine Leu L L-Lysine Lys K L-Lysine Lys K L-Methionine Met M L-Methionine Met M L-Phenylalanine L-Phenylalanine Phe F TYR M L-Proline Pro P L-Proline Pro P L-Serine Ser S L-Serine Ser S L-Threonine Thr T L-Threonine ASN T L-Tryptophan Trp W L-Tryptophan TRP W L-Tyrosine Tyr Y L-Tyrosine GLU Y L-Valine Val V L-Valine Val V ______________________________________ ______________________________________ The term "substantially pure" as used herein refers to hirsutellin A which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is normally associated. Istilah "substansial murni" seperti yang digunakan di sini merujuk kepada hirsutellin J substansial yang bebas dari protein lainnya, lipids, karbohidrat atau dengan bahan lainnya yang biasanya terkait. The substantially pure hirsutellin A polypeptide yields a single major band of 15 kD on a non-reducing polyacrylamide gel. Substansial yang murni hirsutellin J polypeptide hasil satu band besar dari 15 kd di non-mengurangi polyacrylamide gel. The purity of hirsutellin A can also be determined by amino-terminal amino acid sequence analysis. Yang suci dari hirsutellin J juga dapat ditentukan oleh amino-terminal analisis urutan asam amino. Hirsutellin A polypeptide includes functional fragments of the polypeptide, so long as hirsutellin A activity is retained. Hirsutellin J polypeptide fungsional meliputi fragmen dari polypeptide, selama kegiatan

hirsutellin J diperoleh. Therefore, smaller peptides containing the biological activity of hirsutellin A are included in the invention. Karena itu, lebih kecil yang berisi peptides biologi kegiatan hirsutellin J disertakan dalam ciptaan. Minor modifications of the hirsutellin A primary amino acid sequence may result in proteins which have substantially equivalent activity compared with hirsutellin A described in the invention. Modifikasi kecil dari hirsutellin J utama urutan asam amino dapat menyebabkan protein yang ada secara substansial setara dengan kegiatan dibandingkan hirsutellin J dijelaskan di adakan. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. Modifikasi seperti itu mungkin disengaja, seperti yang diarahkan oleh situs-mutagenesis, atau mungkin spontan. For example, more effective hirsutellin A molecules that may be produced by these modifications are included herein, as long as hirsutellin A activity still exists. Misalnya, lebih efektif hirsutellin J molekul yang dapat dihasilkan oleh modifikasi ini disertakan di sini, selama kegiatan hirsutellin J masih ada. The present invention provides a method for producing substantially pure hirsultellin A. Procedures include those commonly used for the separation of protein substances including, for example, treatment of a sample containing hirsutellin A activity with common precipitants for proteins, followed by fractionation techniques such as ion exchange chromatography, affinity chromatography, molecular sieve chromatography, adsorption chromatography, ultrafiltration and various combinations thereof. Temuan ini memberikan sebuah metode untuk memproduksi substansial murni hirsultellin A. Prosedur termasuk yang umum digunakan untuk pemisahan zat protein termasuk, misalnya, perlakuan terhadap sampel berisi hirsutellin J dengan kegiatan Common precipitants untuk protein, diikuti oleh fractionation teknik seperti ion tukar kromatografi, kromatografi Affinity, saringan molekular kromatografi, kromatografi adsorption, ultrafiltration dan berbagai kombinasi itu. Preferably, the method of the invention includes, a) a salt precipitation step, b) a step for removal of neutral and acidic proteins, such as an ion exchange step, and c) exclusion chromatography. Preferably, metode penemuan yang meliputi, a) garam langkah hujan, b) langkah untuk dihapus dari netral dan acidic protein, seperti pertukaran ion langkah, dan c) pengecualian kromatografi. Preferably, the sample containing toxin is produced by growth of a fungus which produces hirsutellin A such as Hirsutella thompsonii, in yeast extract containing medium such as CzapekDox broth at 25 C. The sample containing hirsutellin A activity is salt precipitated, preferably with ammonium sulfate. Preferably, sampel yang mengandung toksin yang dihasilkan oleh pertumbuhan dari jamur yang memproduksi J hirsutellin seperti Hirsutella thompsonii, dalam ekstrak ragi yang mengandung media seperti Czapek-dox bulyon pada 25 C. sampel berisi hirsutellin J kegiatan precipitated adalah garam, sebaiknya dengan ammonium sulfate. After partial precipitation, the material is desalted using gel filtration, for example, with Sephadex G25, followed by ion exchange chromatography. Setelah hujan sebagian, bahan desalted adalah menggunakan gel penyaringan, misalnya, dengan Sephadex G-25, diikuti oleh pertukaran ion kromatografi. The material is treated with an anion exchange resin with a diethylaminomethyl (DEAE) or a diethyl-(2-hydroxypropyl)aminoethyl (QAE) functional group, preferably a diethylaminomethyl group. Materi yang dianggap dengan resin pertukaran anion dengan diethylaminomethyl (DEAE) atau diethyl-(2-hydroxypropyl) aminoethyl (QAE) fungsional grup, sebaiknya satu grup diethylaminomethyl. DEAE is a weak anion exchange resin and is preferably to stronger anion exchange resins such as QAE agarose for the present invention.

DEAE adalah resin pertukaran anion lemah dan lebih kuat untuk pertukaran anion Resins seperti QAE agarose untuk sementara ini. The treatment conditions, eg, a solution of about pH 8.0 and a linear salt gradient of about 0 to 0.3M, are such that the hirsutellin A activity is not adsorbed to the support. Perawatan kondisi, misalnya, sebuah solusi dari sekitar pH 8,0 dan garam linear lereng sekitar 0 sampai 0.3M, adalah seperti yang hirsutellin J aktivitas tidak adsorbed dengan dukungan. Hirsutellin A activity can be determined by its insecticidal effect. J Hirsutellin aktivitas dapat ditentukan oleh insecticidal efek. One suitable assay for hirsutellin A activity includes that described in EXAMPLE 3, which involves the injection of the hirsutellin A containing solution into Lepidopteran larvae. Kadar logam yang cocok untuk hirsutellin J termasuk aktivitas yang dijelaskan dalam EXAMPLE 3, yang melibatkan suntikan yang berisi J hirsutellin menjadi solusi Lepidopteran larvae. In a preferred embodiment, the pooled fractions containing partially purified hirsutellin A are treated next with a cation exchange material with a carboxymethyl or sulphopropyl functional group, preferably a carboxymethyl group. Penjelmaan dalam penawaran, yang berisi sebagian pecahannya pooled suci hirsutellin J diperlakukan berikutnya dengan cation pertukaran materi dengan carboxymethyl atau sulphopropyl kelompok fungsional, sebaiknya yang carboxymethyl grup. Carboxymethyl resin is preferably to the stronger cation ion exchange resins, such as those with sulphopropyl groups. Carboxymethyl damar adalah lebih baik untuk semakin kuat cation ion pertukaran Resins, seperti mereka yang sulphopropyl kelompok. The treatment conditions, eg, a solution of about pH 5.0 and a salt concentration of about 0.05M to 0.5M, are such that the hirsutellin A activity initially adsorbed to the support, is then eluted at a salt concentration of about 0.3M. Perawatan kondisi, misalnya, sebuah solusi dari sekitar pH 5,0 dan konsentrasi garam tentang 0.05M ke 0.5M, adalah seperti yang hirsutellin J adsorbed pada awalnya kegiatan untuk dukungan, kemudian eluted pada konsentrasi garam sekitar 0.3M. The resulting material is tested for hirsutellin A activity as described above and as described in EXAMPLE 3. Hasilnya adalah bahan untuk diuji hirsutellin J kegiatan seperti yang dijelaskan di atas dan seperti yang dijelaskan dalam EXAMPLE 3. The unbound fractions containing hirsutellin A activity can be further purified by gel filtration using, for example, any commerically available sizing gel such as biogel P10 (Biorad) which has a molecular weight fractionation range of about 1,500 to 20,000 daltons. Yg tdk dijilid pecahannya yang berisi hirsutellin J kegiatan dapat lebih suci oleh penyaringan menggunakan gel, misalnya, setiap komersial tersedia gel perekat seperti biogel P10 (Biorad) yang memiliki berat molekul fractionation berbagai tentang 1500-20000 daltons. The hirsutellin A containing fractions after treatment with the gel are then subjected to SDS PAGE under suitable conditions and the gel slice containing hirsutellin A activity is recovered. Yang berisi hirsutellin J pecahannya setelah pengobatan dengan gel kemudian subyek SDS PAGE bawah sesuai kondisi dan gel slice berisi hirsutellin J kegiatan tersebut dipulihkan. SDS PAGE is performed according to the method of Laemmli, et al., (Nature, 227:680, 1970) and is a technique well known to those in the art. SDS PAGE dilakukan sesuai dengan metode Laemmli, dkk. (Nature, 227:680, 1970) dan merupakan teknik yang dikenal dengan baik kepada mereka dalam seni. Variations in conditions which are within a suitable range are understood to be encompassed within the

purification procedure. Variasi dalam kondisi yang cocok dalam rentang yang dapat dipahami mencakup pemurnian dengan prosedur. The hirsutellin A activity-containing fraction from the SDS PAGE is subjected to reverse phase HPLC and eluted with acetonitrile. J hirsutellin yang berisi kegiatan-pecahan dari SDS PAGE sasaran adalah membalikkan phase HPLC dan eluted dengan acetonitrile. The hirsutellin A which is obtained is substantially pure to permit N-terminal amino acid sequencing. The J hirsutellin yang diperoleh adalah murni substansial untuk mengizinkan N-terminal amino acid sequencing. The toxic solution is dried under vacuum and redissolved in a small volume of acetonitrile 95% +TFA (0.08%). Beracun adalah solusi yang kering di bawah dan redissolved kekosongan dalam volume kecil acetonitrile 95% + TFA (0,08%). The concentrated sample is then introduced in a sequencer connected to a phenylthiohydantoine (PTH) analyzer. Terkonsentrasi pada contoh ini kemudian diperkenalkan di sequencer terhubung ke phenylthiohydantoine (PTH) analyzer. A preferred embodiment of the invention comprises the polypeptide, APKVTSRPKLDGNEKPFKVDV (SEQ. ID No. 1) and conservative variations of this peptide. J penawaran perwujudan invensi yang terdiri atas polypeptide, APKVTSRPKLDGNEKPFKVDV (ID SEQ. No 1) dan konservatif variasi peptide ini. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar, residue. Istilah "konservatif variasi" seperti yang digunakan di sini menandakan penggantian residu asam amino yang lain, mirip biologis, residu. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. Contoh variasi konservatif termasuk substitution satu residu hydrophobic seperti isoleucine, valine, leucine atau untuk methionine lain, atau substitusi satu kutub sisa untuk yang lain, seperti dari arginine substitution untuk lysine, glutamic untuk aspartic asam, atau untuk glutamine asparagine, dan lain-lain. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that the substituted polypeptide retain the pest controlling activity of the unsubstituted polypeptide. Istilah "konservatif variasi" juga mencakup penggunaan asam amino yang digantikan di tempat yang unsubstituted induk asam amino asalkan yang digantikan polypeptide mempertahankan kegiatan pengendalian hama yang unsubstituted polypeptide. Antibodies provided in the present invention are immunoreactive with hirsutellin A polypeptide or fragments thereof. Antibodies yang hadir dalam ciptaan yang immunoreactive dengan hirsutellin J polypeptide atau fragmen itu. Polyclonal antibodies are prepared by immunization of an animal, eg, rabbit, with an immunogenic sample of hirsutellin A followed by purification of the antibody by methods well known in the art. Polyclonal antibodies imunisasi yang disiapkan oleh suatu hewan, misalnya, kelinci, dengan immunogenic sampel hirsutellin J diikuti oleh pemurnian dari antibodi oleh metode terkenal dalam seni. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations, are provided. Antibodi yang pada dasarnya terdiri dari pooled monoclonal antibodies berbeda dengan epitopic specificities, serta berbeda monoclonal antibodi persiapan, disediakan. Monoclonal antibodies are made from antigen containing fragments of the protein by

methods well known in the art (Kohler, et al., Nature, 256:495, 1975). Monoclonal antibodies antigen yang dibuat dari potongan-potongan yang mengandung protein yang dikenal dengan baik oleh metode dalam seni (Kohler, dkk., Alam, 256:495, 1975). The term "antibody" as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab') 2 , which are capable of binding the epitopic determinant. Istilah "antibodi" seperti yang digunakan dalam penemuan ini dimaksudkan untuk menyertakan molekul utuh serta fragmen itu, seperti Fab dan F (ab ') 2, yang mampu mengikat epitopic yang menentukan. The antibodies of the invention can be used in immunoaffinity chromatography for the isolation of sequences containing the hirsutellin A activity of the present invention. The antibodies invensi yang dapat digunakan dalam kromatografi immunoaffinity untuk isolasi dari sequence yang berisi hirsutellin J kegiatan masa kini invensi. One way by which such immunoaffinity chromatography can be utilized is by the binding of the antibodies of the invention to CNBrSepharose-4B or Tresyl activated Sepharose (Pharmacia). Salah satu cara yang seperti immunoaffinity kromatografi dapat dimanfaatkan oleh yang mengikat dari antibodies dari invensi ke CNBr-4B-Sepharose atau Tresyl diaktifkan Sepharose (Pharmacia). These solid phase-bound antibodies can then be used to specifically bind sequences containing hirsutellin A activity from mixtures of other proteins to enable isolation and purification thereof. Solid-tahap ini terikat antibodies kemudian dapat digunakan untuk mengikat secara khusus yang berisi sequence hirsutellin J kegiatan dari campuran lain untuk mengaktifkan protein isolasi dan pemurnian itu. Bound hirsutellin A sequences can be eluted from the affinity chromatographic material using techniques known to those of ordinary skill in the art such as, for example, chaotropic agents, low pH, or urea. Terikat hirsutellin J sequence dapat eluted dari bahan Affinity chromatographic menggunakan teknik yang dikenal orang-orang biasa dari keahlian dalam seni seperti, misalnya, chaotropic agen, pH rendah, atau urea. The invention provides polynucleotides encoding the hirsutellin A protein. Invensi yang menyediakan polynucleotides encoding yang hirsutellin J protein. These polynucleotides include DNA, cDNA and RNA sequences which encode hirsutellin A. It is understood that all polynucleotides encoding all or a portion of hirsutellin A are also included herein, so long as they encode a polypeptide with hirsutellin A activity. Ini termasuk polynucleotides DNA, RNA dan cDNA sequence yang menyandi hirsutellin A. Penyalahgunaan dipahami bahwa semua polynucleotides Encoding semua atau sebagian hirsutellin J juga dimasukkan di sini, sepanjang mereka yang menyandi polypeptide dengan hirsutellin J kegiatan. Such polynucleotides include both naturally occurring and intentionally manipulated polynucleotides. Seperti polynucleotides termasuk terjadi baik secara alami dan sengaja dimanipulasi polynucleotides. For example, hirsutellin A may be subjected to site-directed mutagenesis. Misalnya, hirsutellin J mungkin terkena diarahkan ke situs mutagenesis. The polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. The polynucleotides dari ciptaan termasuk sequence yang memburuk sebagai akibat dari kode genetik. There are only 20 natural amino acids, most of which are specified by more than one codon. Hanya ada 20 asam amino alam, sebagian besar dari yang ditentukan oleh lebih dari satu codon. Therefore, as long as the amino acid sequence of hirsutellin A is unchanged, or although changed retains hirsutellin A activity, all degenerate nucleotide sequences are included in the invention. Oleh karena itu, selama urutan asam amino hirsutellin A berubah, walaupun berubah atau tetap hirsutellin J kegiatan, semua merosot nucleotide sequence disertakan dalam ciptaan.

DNA sequences of the invention can be isolated by several techniques known in the art. DNA sequence dari invensi dapat terpencil oleh beberapa teknik yang dikenal dalam seni. These include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences, and 2) antibody screening of expression libraries to detect shared structural features. Termasuk, tetapi tidak terbatas pada: 1) hibridisasi dari probes ke genomic atau cDNA perpustakaan untuk mendeteksi nucleotide sequence bersama, dan 2) antibodi skrining ekspresi dari perpustakaan untuk mendeteksi bersama fitur struktural. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Prosedur pemeriksaan yang mengandalkan nucleic asam hibridisasi memungkinkan untuk mengisolasi gene urutan apapun dari organisme, yang diberikan yang sesuai probe tersedia. For example, oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. Misalnya, oligonucleotide probes, yang sesuai dengan bagian yang urutan encoding protein tersebut dapat synthesized kimia. This requires that short, oligopeptide stretches of amino acid sequence must be known. Ini membutuhkan singkat, oligopeptide stretches urutan asam amino yang harus diketahui. Preferably, the oligonucleotide probe of the invention encodes a consecutive sequence from about four to about thirty-four amino acids of the amino acid sequence APKVTSRPKLDGREKPFKVDVATAQAQARKAGLT (SEQ. ID No. 2) including any conservative variations. Preferably, yang oligonucleotide penyelidikan dari penemuan encode yang berturut-turut urutan dari sekitar empat hingga sekitar tiga puluh empat asam amino dari asam amino urutan APKVTSRPKLDGREKPFKVDVATAQAQARKAGLT (ID SEQ. No 2) termasuk konservatif variasi. A preferred oligonucleotide probe encodes a consecutive sequence of the amino acids EKPFKV, including any conservative variations of this peptide. J penawaran oligonucleotide probe encode yang berturut-turut dari urutan asam amino EKPFKV, termasuk konservatif variasi peptide ini. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. DNA encoding urutan protein yang dapat deduced dari kode genetik, namun degeneracy kode yang harus diambil ke dalam rekening. It is possible to perform a mixed addition reaction when the sequence is degenerate. Kemungkinan untuk melakukan penambahan dicampur reaksi adalah ketika urutan merosot. This includes a heterogenous mixture of denatured double-stranded DNA. Ini termasuk heterogenous ganda campuran denatured DNA-terlantar. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Untuk pemutaran film seperti itu, adalah hibridisasi sebaiknya dilakukan pada salah satu-terlantar atau denatured DNA ganda terlantar DNA. This is especially useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. Hal ini sangat berguna dalam deteksi cDNA clones yang berasal dari sumber yang sangat rendah di mana jumlah mRNA sequence polypeptide yang berkaitan dengan kepentingan yang hadir. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by hybridization of the target DNA to the single probe in the mixture which is its complement (Wallace, et al., Nucleic Acid Research, 9:879, 1981). Dengan kata lain, dengan menggunakan hibridisasi kondisi ketat diarahkan untuk menghindari hal-spesifik mengikat, kemungkinan, misalnya, untuk membolehkan autoradiographic visualisasi tertentu cDNA clone oleh hibridisasi dari target DNA tunggal ke

dalam penyelidikan yang adalah campuran melengkapi (Wallace, et al., Nucleic Acid Research, 9:879, 1981). A cDNA expression library, such as gtll, can be screened indirectly for hirsutellin A peptides having at least one antigenic epitope, using antibodies specific for hirsutellin A. Such antibodies can be either monoclonal or polyclonal and used to detect an expression product indicative of the presence of hirsutellin A cDNA. J ekspresi cDNA perpustakaan, seperti gtll, dapat langsung screened untuk hirsutellin J peptides memiliki setidaknya satu antigenic epitope, menggunakan antibodies khusus untuk hirsutellin A. semacam antibodies dapat berupa monoclonal atau polyclonal dan digunakan untuk mendeteksi sebuah produk ekspresi menunjukkan keberadaan dari hirsutellin J cDNA. A hirsutellin A cDNA library can also be screened by injecting different cDNAs into oocytes. J hirsutellin J cDNA perpustakaan juga dapat screened oleh injecting berbeda cDNAs ke oocytes. After expression of the cDNA gene products occurs, the presence of the specific cDNA gene product can be identified by antibody screening with antibody specifically immunoreactive with hirsutellin A polypeptide, for example. Setelah ekspresi dari cDNA gene produk terjadi, keberadaan spesifik cDNA gene produk dapat diidentifikasi oleh skrining antibodi dengan antibodi spesifik immunoreactive dengan hirsutellin J polypeptide, misalnya. Alternatively, functional assays for hirsutellin A toxicogenic activity could be performed to identify Hirsutellin A producing oocytes. Atau, untuk hirsutellin fungsional assays J toxicogenic kegiatan dapat dilakukan untuk mengidentifikasi Hirsutellin J oocytes produksi. Specific DNA sequences encoding hirsutellin A can also be obtained by: (1) isolation of doublestranded DNA sequences from genomic DNA; (2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and (3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell, resulting in a cDNA, or complimentary DNA. Spesifik DNA sequences encoding hirsutellin J juga dapat diperoleh oleh: (1) isolasi dari dua terjalin DNA sequence dari genomic DNA; (2) kimia manufaktur dari urutan DNA yang diperlukan untuk menyediakan codons untuk polypeptide kepentingan; dan (3) vitro di sintesis dari dua terdampar urutan DNA oleh reverse transcription dari mRNA terpencil dari eukaryotic donor sel, sehingga dalam cDNA, atau gratis DNA. Synthesis of DNA sequences is frequently the method chosen when the entire sequence of amino acid residues of the desired polypeptide product is known. Sintesis dari DNA sequence sering metode dipilih bila seluruh urutan residu asam amino yang dikehendaki polypeptide produk dikenal. When the entire sequence of amino acid residues of the desired polypeptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the formation of cDNA sequences. Bila seluruh urutan residu asam amino yang dikehendaki polypeptide tidak diketahui, langsung dari sintesis DNA sequence tidak mungkin dan metode pilihan adalah pembentukan cDNA sequence. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid or bacteriophage based cDNA libraries in which mRNA is reverse transcribed from donor cells with a high level of genetic expression. Di antara standar prosedur untuk isolating cDNA sequence yang menarik adalah pembentukan plasmid atau bacteriophage berbasis cDNA perpustakaan di mana mRNA adalah reverse

transcribed dari sel donor yang tinggi dengan tingkat ekspresi genetik. When used in combination with polymerase chain reaction (PCR) technology, less common mRNA species (cDNA) can be cloned as well. Bila digunakan bersama dengan polymerase chain reaction (PCR) teknologi, kurang umum mRNA spesies (cDNA) dapat clone juga. When significant portions of the amino acid sequence of a polypeptide are known, labeled single or double-stranded DNA or RNA probes which represent a sequence present in the target cDNA, may be used in DNA/DNA hybridization procedures which are performed on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al., Nucleic Acid Research, 11:2325, 1983). Ketika porsi signifikan dari urutan asam amino yang dikenal polypeptide, berlabel satu atau dua kali terdampar DNA atau RNA probes yang mewakili urutan hadir dalam target cDNA, dapat digunakan dalam DNA / DNA hibridisasi prosedur yang dilaksanakan di clone salinan yang cDNA yang telah denatured dalam satu bentuk-terlantar (Jay, et al., Nucleic Acid Research, 11:2325, 1983). Since the novel DNA sequences of the invention encode a unique sequence of hirsutellin A, it is now a routine matter to prepare, subclone, and express smaller polypeptide fragments of DNA from this or corresponding DNA sequences. Sejak novel DNA sequence dari ciptaan yang unik menyandi urutan hirsutellin A, sekarang adalah hal yang rutin untuk mempersiapkan, subclone, dan menyatakan polypeptide kecil fragmen dari DNA dari DNA atau sesuai urutan. Alternatively, by utilizing the DNA fragments disclosed herein which define the unique hirsutellin A polypeptide of the invention, it is possible, in conjunction with known techniques, to determine the DNA sequences encoding the entire hirsutellin A toxin. Atau, dengan memanfaatkan DNA fragmen diungkapkan di sini yang menentukan yang unik hirsutellin J polypeptide dari penemuan itu, dimungkinkan, dalam hubungannya dengan teknik yang dikenal, untuk menentukan DNA sequences encoding seluruh hirsutellin J toksin. Such techniques are described in US Pat. Teknik seperti yang dijelaskan dalam US Pat. Nos. 4,394,443 and 4,446,235 which are incorporated herein by reference. Nos 4394443 dan 4446235 yang dimasukkan di sini oleh referensi. The polypeptide resulting from expression of a DNA sequence of the invention can be further characterized as being free from association with other eukaryotic polypeptides or other contaminants which might otherwise be associated with the polypeptide in its natural cellular environment. Polypeptide yang dihasilkan dari ekspresi dari urutan DNA dari invensi dapat lebih karakteristik sebagai bebas dari asosiasi dengan lainnya eukaryotic polypeptides atau contaminants lainnya yang mungkin dapat dikaitkan dengan alam polypeptide dalam lingkungan selular. Isolation and purification of microbially expressed polypeptides provided by invention may be by conventional means including, preparative chromatographic separations and immunological separations involving monoclonal and/or polyclonal antibody preparation. Isolasi dan pemurnian dari microbially dinyatakan polypeptides diberikan oleh penemuan mungkin oleh konvensional berarti termasuk, chromatographic separations persiapan dan imunologi separations melibatkan monoclonal dan / atau antibodi polyclonal persiapan. For purposes of the present invention, hirsutellin A polypeptides which are homologous to those of the invention can be identified by structural as well as functional similarity. Untuk tujuan

penemuan hadir, hirsutellin J polypeptides yang sesuai untuk penemuan mereka yang dapat diidentifikasi oleh struktural maupun fungsional kesamaan. Structural similarity can be determined, for example, by assessing polynucleotide strand hybridization or by screening with antibody, especially a monoclonal antibody, which recognizes a unique epitope present on the hirsutellin A polypeptides of the invention. Struktural kesamaan dapat ditetapkan, misalnya, menilai polynucleotide oleh helai hibridisasi atau dengan pemeriksaan antibodi, khususnya yang monoclonal antibodi, yang mengakui epitope yang unik hadir di hirsutellin J polypeptides dari ciptaan. When hybridization is used as criteria to establish structural similarity, those polynucleotide sequences which hybridize under stringent conditions to the polynucleotides of the invention are considered to be essentially the same as the polynucleotide sequences of the invention. Hibridisasi bila digunakan sebagai kriteria untuk menentukan kesamaan struktural, yang polynucleotide sequence silang yang ketat di bawah kondisi yang polynucleotides dari invensi dianggap dasarnya sama dengan polynucleotide sequence dari ciptaan. A wide variety of ways are available for introducing a polynucleotide expressing a hirsutellin A toxin into the microorganism host under conditions which allow for stable maintenance and expression of the gene. J berbagai cara yang tersedia untuk memperkenalkan polynucleotide expressing J toksin yang hirsutellin mahluk yg kecil yang menjadi tuan rumah dalam kondisi yang stabil memungkinkan pemeliharaan dan ekspresi dari gene. DNA constructs are available which include the transcriptional and translational regulatory signals for expression of the hirsutellin A polynucleotide; the toxin gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur; and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur. DNA constructs yang tersedia termasuk transcriptional dan peraturan translational sinyal untuk ekspresi dari hirsutellin J polynucleotide; toksin yang gene peraturan mereka di bawah kontrol dan DNA urutan sesuai dengan urutan dalam host organisme, dimana akan terjadi integrasi, dan / atau peniruan fungsional sistem yang di-host, dimana integrasi atau pemeliharaan stabil akan terjadi. The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. Inisiasi yang transcriptional sinyal akan termasuk promotor dan transcriptional inisiasi mulai situs. In some instances, it may be desirable to provide for regulative expression of the hirsutellin A polynucleotide, where expression of the toxin will only occur after release into the environment. Dalam beberapa kasus, mungkin keinginan untuk menyediakan regulative ekspresi dari hirsutellin J polynucleotide, di mana ekspresi dari toksin hanya akan terjadi setelah rilis ke dalam lingkungan. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the host. Hal ini dapat dicapai dengan operator atau ke suatu daerah yang mengikat activator atau enhancers, yang mampu induksi suatu perubahan fisik atau kimia lingkungan host. For example, a temperature sensitive regulatory region may be employed where the organisms may be grown up in the laboratory without expression of a toxin, but upon change in the growth conditions or environment, expression would begin. Misalnya, suhu sensitif peraturan daerah dapat bekerja di mana organisme dapat tumbuh di laboratorium tanpa ekspresi dari toksin, tetapi setelah perubahan dalam kondisi pertumbuhan atau lingkungan, akan mulai ekspresi. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where the nutrient medium in the later environment would

allow for expression of the toxin. Teknik lainnya Mei mempekerjakan gizi media tertentu dalam laboratorium, yang inhibits ekspresi dari toksin, dimana gizi media di lingkungan nantinya akan memungkinkan untuk ekspresi dari toksin. For translational initiation, a ribosomal binding site and an initiation codon will be present. Untuk translational inisiasi, sebuah situs dan ribosomal mengikat codon inisiasi yang akan hadir. Various manipulations may be employed for enhancing the expression of the mRNA, particularly by using an active promoter, as well as by employing sequences, which enhance the stability of the mRNA. Berbagai manipulasi dapat digunakan untuk meningkatkan ekspresi dari mRNA, terutama dengan menggunakan promotor yang aktif, serta Mempekerjakan sequence, yang meningkatkan stabilitas dari mRNA. The initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal. Inisiasi yang translational dan penghentian daerah akan melibatkan menghentikan codon (s), sebuah terminator daerah, dan opsional, yang polyadenylation sinyal. In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. Dalam arah transcription, yaitu di 5 'ke 3' searah dengan coding atau rasa urutan, dengan membangun akan melibatkan transcriptional peraturan daerah, jika ada, dan promotor, di mana peraturan daerah mungkin baik 5 'atau 3' dari promotor, yang mengikat ribosomal situs, inisiasi codon, yang memiliki struktural gene buka membaca bingkai dalam tahap inisiasi dengan codon, yang berhenti codon (s), maka urutan polyadenylation sinyal, jika ada, dan terminator daerah. This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the toxin expression construct during introduction of the DNA into the host. Urutan ini sebagai dua helai dapat digunakan oleh dirinya sendiri untuk transformasi dari mahluk yg kecil host, tapi biasanya akan disertakan dengan DNA melibatkan urutan tanda, di mana urutan kedua DNA mungkin toksin yang tergabung dalam membangun ekspresi selama pengenalan DNA ke dalam tuan rumah. A marker structural gene may be present which provides for selection of those hosts which have been modified or transformed. J tanda gene Mei struktural yang hadir untuk memberikan pilihan yang host yang telah dimodifikasi atau diwujudkan. The marker will normally provide for selective advantage, for example, providing for biocide resistance, for example, resistance to antibiotics or heavy metals; complementation, so as to provide prototropy to an auxotrophic host, or the like. Tanda tersebut biasanya akan memberikan keuntungan untuk selektif, misalnya, untuk menyediakan biocide perlawanan, misalnya, perlawanan terhadap antibiotik atau logam berat; complementation, sehingga memberikan prototropy ke auxotrophic tuan rumah, atau sejenisnya. Preferably, complementation is employed, so that the modified host may not only be selected, but may also be competitive in the field. Preferably, complementation yang bekerja, sehingga tidak dapat diubah host hanya dapat dipilih, tetapi juga dapat bersaing di lapangan. One or more markers may be employed in the development of the constructs, as well as for modifying the host. Satu atau lebih tanda mungkin bekerja dalam pembangunan constructs, serta untuk

memodifikasi host. The organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field. Organisme yang dapat diubah oleh lebih memberikan keuntungan yang kompetitif terhadap lainnya liar-jenis mikroorganisme di lapangan. For example, genes expressing metal chelating agents, for example, siderophores, may be introduced into the host along with the structural gene expressing the toxin. Misalnya, gen expressing metal chelating agen, misalnya, siderophores, mungkin diperkenalkan ke tuan rumah bersama dengan struktural gene menyatakan toksin. In this manner, the enhanced expression of a siderophore may provide for a competitive advantage for the toxin-producing host, so that it may effectively compete with the wild-type microorganisms and stably occupy a niche in the environment. Dengan cara ini, dengan peningkatan ekspresi dari siderophore untuk dapat memberikan keuntungan kompetitif untuk memproduksi toksin-host, sehingga dapat bersaing secara efektif dengan liar-jenis mikroorganisme stably dan menempati sebuah relung di lingkungan. Where no functional replication system is present, the construct will also include a sequence of at least 50 basepairs (bp), preferably at least about 100 bp, and usually not more than about 1000 bp of a sequence homologous with a sequence in the host. Fungsional, di mana tidak ada sahutan sistem ini, yang juga akan membangun termasuk urutan ke-50 setidaknya basepairs (bp), sebaiknya minimal sekitar 100 bp, dan biasanya tidak lebih dari sekitar 1000 bp dari urutan sesuai dengan urutan dalam host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Dengan cara ini, kemungkinan yang sah recombination ditingkatkan agar generasi akan diintegrasikan ke dalam host dan dikelola oleh stably host. Desirably, the toxin gene will be in close proximity to the gene providing for complementation as well as the gene providing for the competitive advantage. Desirably, gene toksin yang akan di dekat ke generasi untuk memberikan complementation serta gene memberikan keuntungan yang kompetitif. Therefore, in the event that a toxin gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct. Karena itu, dalam acara yang toksin gene terputus, sehingga organisme akan kemungkinan juga kehilangan gene melengkapi dan / atau gene memberikan keuntungan yang kompetitif, sehingga tidak akan dapat bersaing di lingkungan dengan tetap mempertahankan gene membangun yang utuh. A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like. Banyaknya jumlah transcriptional peraturan daerah yang tersedia dari berbagai jenis mahluk yg kecil host, seperti bakteri, bacteriophage, cyanobacteria, algae, jamur, dan lain-lain. Various transcriptional regulatory regions include the regions associated with the trp gene, lac gene, gal gene, the lambda left and right promoters, the Tac promoter, the naturally-occurring promoters associated with the toxin gene, where functional in the host. Transcriptional berbagai daerah termasuk peraturan daerah yang terkait dengan TRP gene, lac gene, gene gal, di kiri dan kanan lambda promoters, maka TAC promotor, yang terjadi secara alami-promoters yang terkait dengan toksin gene, dimana fungsional dalam host. See, for example, US Pat. Lihat, misalnya, US Pat. Nos. 4,332,898, 4,352,832 and 4,356,270. Nos 4332898, 4352832 dan 4356270. The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are

compatible and functional in the host. Penghentian wilayah yang mungkin merupakan wilayah penghentian biasanya terkait dengan transcriptional inisiasi atau wilayah yang berbeda transcriptional inisiasi daerah, sehingga selama dua daerah tersebut kompatibel dan fungsional dalam host. Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system which is functional in the host. Dimana episomal stabil atau pemeliharaan integrasi adalah diinginkan, yang akan digunakan plasmid yang memiliki replikasi sistem yang berfungsi dalam host. The replication system may be derived from the chromosome, an episomal element normally present in the host or a different host, or a replication system from a virus which is stable in the host. Replikasi sistem mungkin berasal dari kromosom, episomal elemen yang biasanya hadir di-host atau host yang berbeda, atau sebuah sistem replikasi dari virus yang stabil pada host. A large number of plasmids are available, such as pBR322, pACYC184, RSFIO1O, pR01614, and the like. Banyaknya jumlah plasmids tersedia, seperti pBR322, pACYC184, RSFIO1O, pR01614, dan lain-lain. (See, for example Olson et al., J. Bacteriol. 150:6069, 1982, and Bagdasarian et al., Gene 16:237, 1981, and US Pat. Nos. 4,356,270, 4,362,817, and 4,371,625.) (Lihat, misalnya Olson et al., J. Bacteriol. 150:6069, 1982, dan Bagdasarian dkk., Gene 16:237, 1981, dan US Pat. Nos 4356270, 4362817, dan 4371625.) The hirsutellin A polynucleotide can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This construct will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, as described above. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome. The transformants can be isolated in accordance with conventional techniques usually employing selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be screened for pesticidal activity. Suitable host cells, where the pesticide-containing cells will be treated to prolong the activity of the toxin in the cell where the treated cell is applied to the environment of target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to limit any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Ervinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and

Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like. Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the hirsutellin A polynucleotide into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like. Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., and the like. Specific organisms include Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like. In general, expression vectors containing promotor sequences which facilitate the efficient transcription of the inserted genetic sequence are used in connection with the host. As described above, biologically functional viral or plasmid DNA vectors caable fo expression and replication in a host are known in the art. Such vectors are used to incorporate hirsutellin A encoding DNA sequences of the invention. Expression vectors typically contain an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. Transformation of the host cell with the recombinant DNA may be carried out by conventional techniques well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by the CaCl 2 method using procedures well known in the art. Alternatively, MgCl 2 or RbCl could be used. Where the host used is a eukaryote, various methods of DNA transfer can be used. These include transfection of DNA by calcium phosphate-precipitates, conventional mechanical procedures such as microinjection or electroporation, insertion of a plasmid encased in liposomes, or the use of viral vectors. Eukaryotic host cells may also include yeast. For example, DNA can be expressed in yeast by inserting the DNA into appropriate expression vectors and introducing the product into the host cells. Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, J. et al., Nature, 340:205, 1989; Rose, M. et al., Gene, 60:237, 1987). Isolation and purification of microbially expressed protein, or fragments thereof provided by the invention, may be carried out by conventional means including preparative chromatography and

immunological separations involving monoclonal or polyclonal antibodies. Antibodies provided in the present invention are immunoreactive with the hirsutellin A polypeptides of the invention. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989). Minor modifications of the hirsutellin A primary amino acid sequence may result in polypeptides which have substantially equivalent activity compared to the hirsutellin A polypeptides described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All proteins produced by these modifications are included herein as long as hirsutellin A pest controlling activity is present. Compositions of the invention include those in which an organism is genetically modified to contain polynucleotide encoding hirsutellin A polypeptides. Treatment of the organism, for example, a microbe, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability in protecting the toxin. Examples of chemical reagents are halogenating agents. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, WH Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host animal. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Where the pesticide is in a proform, the method of inactivation should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink protein and could inhibit processing of the proform of a polypeptide pesticide. The method of inactivation or killing retains at least a substantial portion of the bio-availability or bioactivity of the toxin. The cellular host containing the hirsutellin A polypeptide insecticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain hirsutellin A gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting. The pest-controlling agent of the present invention may be prepared as dusts, water dispersions, emulsions and solutions. They may comprise accessory agents such as dust carriers, solvents,

emulsifiers, wetting and dispersing agents, stickers, deodorants, and masking agents (see, for example, Encyclopedia of Chemical Technology, Vol. 13, pp. 416, et seq.). Dusts generally will contain low concentrations, 0.1-20% of hirsutellin A, although ground preparations may be used and diluted. Carriers commonly include sulfur, silcon oxides, lime gypsum, talc, pyrophylite, bentonite, kaolins, attapulgite, and volcanic ash. Selection of the carrier may be made on the basis of compatibility with the desired pest control composition (including pH, moisture content, and stability), particle size, abrasiveness, absorbability, density, wettability and cost. The agent of the invention, alone or in combination, and eluent is made by a variety of simple operations such as milling, solvent impregnations, fusing, and grinding. Particle sizes usually range from 0.5-4.0 microns in diameter. Wettable powders may be prepared by blending the hirsutellin A of the invention in high concentrations, usually from 15-80% with a dust carrier such as bentonite which wets and suspends properly in water. Twenty-two percent of a surface-active agent is usually added to improve the wetting and suspendability of the powder. Hirsutellin A may also be used in granules, which are pelleted mixtures of the agents, usually at 2.5-20%, and a dust carrier, eg, adsorptive clay, bentonite or diatomaceous earth, and common within particle sizes of 250-590 microns. Granules may be prepared by impregnations of the carrier with a solution or slurry of the organism and may be used principally for soil applications or for mosquito larvae treatment The hirsutellin A may also be applied in the form of an emulsion, which comprises a solution of hirsutellin A in water-immisicible organic solvents, commonly at 15-50%, with a few percent of surface active agent to promote emulsification, wetting, and spreading. The choice of solvent is predicated upon solubility, safety to plants and animals, volatility, flammability, safety to plants and animals, compatibility, odor and cost. The hirsutellin A of the present invention may also be applied depending on the properties of the particular pest-controlling compound, the habits of the pest to be controlled, and the site of the application to be made. It may be applied by spraying, dusting or fumigation. Sprays are a common means of application and generally will involve the use of water as the principal carrier, although volatile oils may also be used, The pest-controlling agents of the invention may be used in dilute sprays or in concentrate sprays, and the amount of carrier to be applied is reduced. The use of concentrate and ultra low volume sprays will allow the use of atomizing nozzles producing droplets of 30-80 microns in diameter. Aerosols may also be used to apply hirsutellin A. Aerosols are applied by atomizing amounts of liquified gas dispersion or bomb, but can be generated on a larger scale by rotary atomizers or twin-fluid atomizers. Carriers used as aerosols or liquified gas may include mineral spirits, ethanol, isopropanol, deionized water, and hydrocarbon propellants, such as isobutane, n-butane, and propane or nonflammable fluorinated hydrocarbon propellants, or compressed gases, such as nitrogen, carbon dioxide, or nitrous oxide. The aerosol composition will typically comprise about

0.001-10% hirsutellin A of the invention, and the remainder being aerosol or liquified gas ingredients (The Science and Technology of Aerosol Packaging, John Wiley & Sons, 1974). The following examples are intended to illustrate but not limit the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used. EXAMPLE 1 PURIFICATION OF HIRSUTELLIN A A. CRUDE FILTRATE PRODUCTION Solid cultures of H. thompsonii var. thompsonii (ATCC 24874) were grown on soil fungus medium (SFM) for 5 days in 90 mm petri plates at 28 C. An inoculum was prepared by washing the conidia from the surface of a sporulating aerial mycelial mat of a given number of petri plates with sterile distilled water and then concentrating the conidial suspension by centrifugation. Broth culture containing Czapek-Dox medium plus yeast extract at 10 g/l were prepared in Erlenmeyer flasks. Each flask was inoculated with a conidial suspension at 410 6 conidia per 35 ml of broth. Flasks were then placed randomly on a gyrator water bath shaker maintained at 25 C. Cultures were agitated at 100 RPM for 10 days. After incubation, the mycelial biomass was separated from the supernatant via filtration through cheesecloth and filter paper. Each crude broth was then filtered again through a 0.45 m filter to ensure sterility and stored at 4 C. or lyophilized for extended storage. B. INITIAL PRECIPITATION AND CONCENTRATION The following strategy was developed to separate the different toxic metabolites of Hirsutell thompsonii var. thompsonii as pure compounds. This purification procedure was developed by initially adding ammonium sulfate to the crude filtrate to precipitate and concentrate proteins, desalting by gel filtration on Sephadex G-25 and applying different steps of ion exchange and exclusion chromatography. The different fractions collected were analyzed for their absorption at 280 nm. These fractions were screened for biological activity by injection of 8 l of each solution in larvae (L6) of the experimental host, Galleria mellonella. The contents of each fraction were also analyzed by SDS PAGE electrophoresis to monitor the evolution of the purification process. The proteins of the crude filtrate were precipitated by addition of ammonium sulfate, at a concentration of 0.76 mg/ml (90% of saturation). After a storage of 15 hr at 4 C., the pellet of precipitated proteins was collected by centrifugation for 1 hr at 8500 rpm, and the supernatant was discarded. The pellet was dissolved in a small amount of distilled water (1/30 to 1/40 of the volume of filtrate) to obtain an increase of concentration. C. GEL FILTRATION ON SEPHADEX G-25

In a next step, exclusion liquid chromatography with Sephadex G-25 was performed for desalting the precipitate. The concentrated solution prepared above was applied oa 2.525 cm column and eluted with 0.05M Tris-HCl buffer, pH 8. Fractions of 7.5 ml were collected, analyzed for absorption at 280 nm, and tested for biological activity by injection of 0.8 l of each of these solutions to L6 larvae of the lepidopteran insect Galleria mellonella (200 mg of mean body weight). Among these fractions (F) only F1, 2 and 3, collected immediately after the void volume and containing the macromolecules excluded from the gel (MW cut-off=5000D), showed an insecticidal effect. A pool of F1, 2 and 3, with a total concentration in proteins of 500 g/ml (Biorad method of dosage) induced a rapid rate of mortality in 100% of the injected larvae. D. ION EXCHANGE CHROMATOGRAPHY WITH DEAE Ion exchange chromatography was performed by DEAE-Trisacryl, a weak anion exchanger. Fractions 1-3 eluted from Sephadex G-25 were filtrated on membranes of a porosity of 0.2, and then applied to DEAE-Trisacryl column (116 cm). The column was equilibrated with 0.05M Tris-HCl buffer, pH 8. This column was washed with the same buffer, and the bound material was eluted with a 60 ml 0 to 0.3M linear NaCl gradient in the same buffer, at a flow rate of 50 ml/hr. Fractions of 3.5 ml were collected, and the absorbance at 280 nm was recorded. A first peak, corresponding to non-adsorbed material, was obtained during washing, at the level of fractions F2, 3 and 4. A pool of these fractions (200 g/ml of proteins) induced (by injection) a rate of mortality of 85-95% in the G. mellonella larvae. The intoxicated larvae showed a characteristic aspect; they were swollen and displayed small dispersed melanized spots on their cuticle. At the beginning of the elution of the NaCl gradient a second peak of compounds previously bound to DEAE appeared, but detached quickly. Fraction F15, whose concentration in proteins was 200 g/ml, also caused a rate of lethality of 90%. The larvae were not swollen, and the brownish color of the cuticle was more apparent. E. ION EXCHANGE CHROMATOGRAPHY WITH A CATION EXCHANGER Fractions 1-3 from DEAE were applied to a carboxymethyl-Trisacryl column (116 cm) equilibrated with a 0.05M sodium acetate buffer, pH 5. The column was washed with this buffer, and the bound material was eluted in a first step with 0.1M NaCl in the same buffer, and in a second step by elution with a 0.2 to 0.4M NaCl gradient. During elution of the gradient, a peak was obtained at a NaCl concentration of 0.3M, at the level of fraction 50. Among the fractions tested for their biological activity, only those corresponding to this peak were toxic, causing a mortality at 100% when injected into Galleria larvae. Hirsutellin A was therefore present in these fractions. However, this material was not pure as SDS-PAGE electrophoresis of these solutions revealed two bands after staining of gels with Coomassie brilliant blue.

F. EXCLUSION CHROMATOGRAPHY The native fractions collected by ion exchange chromatography on carboxymethyl-Trisacryl were dialyzed against distilled water and then freeze-dried. The freeze-dried powder was dissolved in 0.05M Tris-HCl buffer, pH 8, in conditions to increase the concentration to 40 for exclusion chromatography. Gel permeation runs were then performed with Biogel P 10, quality medium (Biorad) (fractionation range: 1500-20000 D). During these experiments, the concentrated solution was applied to a biogel column (1100 cm), and eluted with 0.05M TrisHCl buffer, pH 8, at a flow rate of 15 ml/hr. Fractions of 1.5 ml were collected. When these samples were analyzed, only one small peak was observed which eluted approximately the same as Cytochrome C (MW=12300). This fraction was very active as injection caused 100% mortality in G. mellonella larvae (see EXAMPLE 2B). SDS-PAGE revealed only one band, even after silver staining (Biorad), and comparison with the MW standards showed that the MW of the components(s) of this band was about 15D. When isoelectrofocusing was performed with a flat bed apparatus and Servalyt Precotes (Serva) 3-11 it also appeared as only one band. The pl was 10.5 according to the comparison with markers (Serva). When the same sample was submitted successively to isoelectrofocusing and to SDSPAGE electrophoresis, there was again only one spot on the gel, thereby showing that the hirsutellin A was purified. EXAMPLE 2 CHARACTERIZATION AND CLONING OF HIRSUTELLIN A Column chromatography, electrophoresis, and isoelectrofocusing studies described above showed that hirsutellin A is a basic macromolecular monomeric protein with a molecular weight of 15 kD, an isoelectric point (pl) of 10.5. The purity of hirsutellin A was confirmed via HPLC and the composition of its amino acids determined. Determination of the sequence of amino acids of the NH-terminal portion of the hirsutellin A molecule was based on the Phenylthiohydantoine (PTH) method of Edman, et al. (Europ. J. Biochem., 1:80, 1967). It was performed with a gaseous phase sequencer connected to a PTH analyzer. The sequence of the first 34 amino acids of the NH-terminal part of the hirsutellin A molecule obtained by this method has been defined in FIG. 1. Search via a computer data base (CITI2) did not reveal a significant degree of homology between hirsutellin A and other known proteins. In addition, research designed to identify a glycosylated fraction in hirsutellin A has proven negative. EXAMPLE 3 TOXICITY OF HIRSUTELLIN A A. TOXICITY OF HIRSUTELLIN A TO Galleria mellonella LARVAE To test the toxicity of hirsutellin A to larvae (L6) of G. mellonella, three concentrations of toxin, 25, 50 and 100 g/ml, corresponding to final doses of 1,2 and 4 g/g of body weight, respectively, were prepared and 8 l of each treatment injected into the proleg of 20

larvae/replicate. A Tris buffer solution injection was performed as a control. Each treatment was replicated three times. Larval mortality was recorded daily. All concentrations of hirsutellin A killed 100% of the larvae; however, the LT 50 increased with a decrease in dose. The LT 50 was 8.81.4, 17.11.8 and 24.22.0 at 100, 50 and 25 g/ml, respectively. There was no mortality in the control (TABLE 1). TABLE 1 ______________________________________ MEAN PERCENT MORTALITY TO Galleria mellonella LARVAE (L6) FOLLOWING INJECTION OF DIFFERENT CONCENTRATIONS OF HIRSUTELLIN A Dosage (g/ Mean % Mortality; Days Post-Treatment ml) 4 5 10 15 20 25 30 LT 50 ______________________________________ 25 0.0 -- 0.0 6.7 40.0 73.3 100.0 24.2 2.0 50 0.0 7.5 35.0 -- 80.0 100.0 -- 17.1 1.8 100 18.0 45.0 63.6 100.0 -- -- -- 8.8 1.4 Con- 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -trol ______________________________________ B. TOXICITY OF HIRSUTELLIN A TO NEONATAL Aedas aegypti LARVAE To test the toxicity of hirsutellin A to neonatal mosquito larvae of Aedes aegypti, different concentrations of toxin, 0, 5, 10, 15 and 20 g/ml, in 200 l of water were placed into microtitration plates. A control containing only water was included. Each treatment was replicated three times. Twenty-four vigorous neonatal larvae were placed into each well under continuous exposure. Larval mortality increased with an increase in toxin dosage. At 5 and 10 g/ml, larval mortality reached 90% after 48 hr and 100% by 72 hr post-inoculation. Virtually no mortality was recorded from the control. C. TOXICITY OF HIRSUTELLIN A TO Plutella xylostella CELL LINE To test the toxicity of hirsutellin A on Plutella xylostella cells produced in tissue culture, different concentrations of toxin (0, 0.5, 1.0, 2.5, 5.0 and 10.0 g/ml) were added via dilution to achieve a given test concentration in 2 ml of tissue culture medium containing 210 5 cells. A control containing cells in tissue culture media only was included. Living and dead cells per day were determined by staining with Trypan blue and the number of live cells at 1, 2, 3, 4 and 5 days was recorded (TABLE 2).

The growth rate of P. xylostella cells is tissue culture declined with an increase in concentration of hirsutellin A. At 10 g/ml, all cells were killed after 2 days. TABLE 2 ______________________________________ Toxin Conc. No. Live Cells/ml; Days Post-Treatment a (g/ml) 0 1 2 3 4 5 ______________________________________ 0.0 2.0 5.5 6.4 8.6 9.0 12.0 0.5 2.0 4.0 6.9 8.3 9.8 9.1 1.0 2.0 4.7 7.3 7.8 9.4 9.7 2.5 2.0 2.6 3.3 3.1 3.8 3.4 5.0 2.0 2.1 2.2 2.2 2.2 2.5 10.0 2.0 1.7 3.2 0.0 0.0 0.0 ______________________________________
a

Live cells counted in hemocytometer after staining with Trypan blu 10 5

D. TOXICITY OF HIRSUTELLIN A VIA CONTACT AND RESIDUAL EXPOSURE TO THE CITRUS RUST MITE Citrus rust mite (CRM), Phyllocoptruta oleivora, were reared on Sunburst mandarin citrus seedlings kept under plexiglass cages. Residual+contact (R+C) and residual (R) activity of hirsutellin A was tested at 0, 10, 32, 56 and 100 g of toxin/ml. For determining R+C activity, citrus leaves were first placed on a wet cotton pad in an open Petri dish. Tanglefoot was then placed around the perimeter of leaves and 30 adult CRM mites were transferred to each leaf. About 2 ml of hirsutellin A at five concentrations were sprayed on each of four leaves using Potter tower. For determining R activity, leaves were sprayed with different concentrations of hirsutellin A before transferring the mites. Dishes were confined to a closed plastic container and held in a temperature cabinet at 25 C. +1 C. and photoperiod of 15 hr. Mortality was evaluated 24, 48 and 72 hr after infestation. The number of eggs found on each leaf surface was recorded at 72 hr (FIG. 2). Good biological activity against CRM was observed 48 hr after infestation at 100 g/ml. Interestingly, hirsutellin A also showed some effect on mite physiology because, at lower concentrations where low mortality was detected, oviposition was significantly reduced in comparison to the control. The higher number of eggs recorded in the R+C bioassay compared to the R bioassay was probably because some eggs were laid on the leaf before the application of the toxin. Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

________________________________________________________ __________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 2 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AlaProLysValThrSerArgProLysLeuAspGlyArgGluLysPro 151015 PheLysValAspVal 20 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..34 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: A laProLysValThrSerArgProLysLeuAspGlyArgGluLysPro 151015 PheLysValAspValAlaThrAlaGlnAlaGlnAlaArgLysAlaGly 202530 LeuThr

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US Patent 5338675 - Toxic metabolic product of Hirsutella sp. US Patent 5338675 - Toxic metabolis produk Hirsutella sp.
US Patent Issued on August 16, 1994 US Patent Issued pada 16 Agustus 1994 Estimated Patent Expiration Date: Perkiraan Paten Tanggal Kadaluarsa: June 23, 2012 Estimated Expiration Date is calculated based on simple USPTO term provisions. 23 Jun 2012 Perkiraan Tanggal Kadaluarsa dihitung berdasarkan ketentuan istilah USPTO sederhana. It does not account for terminal disclaimers, term adjustments, failure to pay maintenance fees, or other

factors which might affect the term of a patent. Tidak account Penyangkalan terminal, istilah penyesuaian, kegagalan untuk membayar biaya pemeliharaan, atau faktor lainnya yang mungkin mempengaruhi jangka waktu paten. Abstract Claims Description Full Text Abstrak Claims Description Full Text loading... loading ...

Inventors Inventors

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McCoy, Clayton W. McCoy, Clayton W. Vey, Alain J. Vey, Alain J. Mazet, Isabelle M. Mazet, Isabelle M.

BACKGROUND OF THE INVENTION LATAR BELAKANG TERHADAP Experiment 1. 1. Field of the Invention Lapangan dari Production The present invention relates to a unique fungal toxin and its use in controlling invertebrate pests. Saat ini berkaitan dengan penemuan unik fungal toksin dan penggunaannya dalam pengendalian hama hewan tdk bertulang punggung. 2. 2. Description of Related Art Keterangan terkait Seni Insects and other pests are responsible for billions of dollars in loss of important agricultural crops every year. Serangga dan hama lainnya yang bertanggung jawab atas milyaran dolar hilangnya penting dalam pertanian tanaman pangan setiap tahun. Therefore, it is of critical importance to find suitable protection from such pests for these plants. Karena itu, penting untuk menemukan pentingnya cocok seperti perlindungan dari hama untuk tanaman. Traditionally, broad spectrum chemical pesticides have been used to protect the world's crops. Tradisional, berbagai pestisida kimia telah digunakan untuk melindungi tanaman dari seluruh dunia. Unfortunately, in addition to pests,

Assignee Diberi Tugas

University of Florida University of Florida l'Institut National de la Recherche Agronomi que l'Institut National de la Recherche Agronomi que Le Centre

other beneficial insects are often destroyed as well. Sayangnya, selain hama, serangga bermanfaat lainnya seringkali hancur juga. Chemical pesticides may also have deleterious effects on humans or animals that have been exposed to them. Pestisida kimia juga memiliki efek mengganggu pada manusia atau hewan yang telah dipaparkan kepada mereka. Chemical pesticides can pollute the environment and can create health hazards to both agricultural workers and consumers. Pestisida kimia dapat mengotori lingkungan dan dapat membuat kesehatan untuk pekerja pertanian dan konsumen. Also, because many pests develop resistance to chemical pesticides, these compounds are often rendered ineffective or must be utilized in resistance management strategies only. Selain itu, karena banyak hama mengembangkan perlawanan terhadap pestisida kimia, compounds ini sering tidak efektif atau diberikan harus dimanfaatkan dalam manajemen strategi hanya perlawanan. The use of biological methods of pest control was first suggested in 1895, when a fungal disease was discovered in silkworms. Penggunaan biologi metode pengendalian hama pertama kali diusulkan pada 1895, ketika sebuah penyakit fungal Di silkworms. Later, in 1940, spores of Bacillus popilliae were used to control the Japanese beetle. Kemudian, pada tahun 1940, spores of Bacillus popilliae digunakan untuk mengontrol Jepang kelam. This was the first known successful use of biological pesticides. Ini adalah yang pertama dikenal berhasil penggunaan pestisida biologi.

National de la Recherche Scientifiqu e Le Centre National de la Recherche Scientifiqu e

Applicatio n Lamaran
No. 903005 filed on 06/23/1992 No. 903005 filed pada 06/23/1992

In more recent years, considerable research has been done trying to develop biopesticides against agriculturally and medically important pests. Lebih dalam beberapa tahun terakhir, banyak penelitian telah dilakukan untuk mencoba mengembangkan biopesticides terhadap agriculturally dan medis 435/183 , penting hama. In large part, these studies have focused on strains and toxins ENZYME (EG, of Bacillus thuringensis (BT). Dalam sebagian besar, studi ini telah terfokus LIGASES (6. ), pada jenis dan toxins dari Bacillus thuringensis (BT). However, in recent ETC.), years reports have appeared that pests can develop resistance to various PROENZYME; strains of BT and their toxins and other naturally occurring microbes, thereby COMPOSITIONS demonstrating that pest control in the future must be diverse, such as by THEREOF; utilizing both chemical and biological methods in an integrated manner. PROCESS FOR Namun, dalam beberapa tahun terakhir telah muncul laporan bahwa hama PREPARING, dapat mengembangkan perlawanan terhadap berbagai jenis dari BT dan toxins ACTIVATING, dan mikroba lainnya yang muncul secara alami, sehingga menunjukkan INHIBITING, bahwa pengendalian hama di masa mendatang harus beragam, seperti dengan SEPARATING, memanfaatkan kedua metode kimia dan biologi di secara terpadu. OR PURIFYING ENZYMES There is a need for more naturally occurring "biopesticides" which are safe to 435/71.1 , Using a humans and animals yet toxic to agricultural pests. Ada kebutuhan untuk micro-organism lebih terjadi secara alami "biopesticides" yang aman untuk manusia dan to make a protein hewan namun beracun ke pertanian hama. Biopesticides are often safer and or polypeptide ecologically more acceptable than chemical pesticides and there is a lower 435/71.3 , chance for the pests to develop resistance to naturally occurring biopesticides. Antibiotic or

US Classes: US Kelas:

Biopesticides biasanya lebih aman dan lebih dapat diterima daripada ekologis pestisida kimia dan ada yang lebih rendah untuk hama kesempatan untuk mengembangkan perlawanan terhadap biopesticides terjadi secara alami. This invention provides a novel, broad spectrum, naturally occurring mycoinsecticide. Temuan ini memberikan sebuah novel, spektrum luas, mycoinsecticide terjadi secara alami. The genus Hirsutella includes about 50 entomopathogenic species that attack a wide range of insects. Genus Hirsutella mencakup sekitar 50 spesies entomopathogenic yang menyerang berbagai serangga. The mononematous hyphomycete fungi, Hirsutella thompsonii var. The mononematous hyphomycete jamur, Hirsutella thompsonii var. thompsonii, H. thompsonii var. thompsonii, H. thompsonii var. vinacea and H. thompsonii synnematosa, all produce hirsutellin A with similar toxicogenic activity. vinacea dan H. thompsonii synnematosa, semua menghasilkan hirsutellin J toxicogenic dengan kegiatan serupa.

In vivo, Hirsutella thompsonii and related species produce infective conidia on phialides arising from external mycelia growing from the host. Dalam vivo, Hirsutella thompsonii terkait dan jenis produksi infective conidia pada phialides timbul dari eksternal mycelia berkembang dari tuan rumah. Hyphae can emerge from cadavers through oral and anal openings, appendages, genital opening and at times through the body wall. Hyphae dapat muncul dari cadavers melalui lisan dan anal bukaan, appendages, genital dan sewaktu-waktu melalui badan dinding. Within the host, hyphae usually develop initially in the central area of the hemocoel as oval bodies and then become chain-like as they grow anteriorly or posteriorly along the inner body wall. Di tuan rumah, biasanya hyphae awalnya berkembang di tengah kawasan yang hemocoel sebagai badan lonjong dan kemudian menjadi rantai seperti karena tumbuh anteriorly atau posteriorly sepanjang dinding inner tubuh. In nature, these internal hyphae may break up and form multinucleate spherical chlamydospores. Di alam, ini internal hyphae Mei bubar dan formulir multinucleate bulat chlamydospores. Host mortality appears to result from invasion of tissue by the fungal hyphae, while no toxicogenic activity has been reported. Host nampaknya kematian akibat invasi dari jaringan oleh fungal hyphae, saat tidak ada aktivitas toxicogenic telah dilaporkan. Although different naturally occurring biopesticides have been developed, none has been produced from the metabolites of Hirsutella spp. Walaupun berbeda terjadi secara alami biopesticides telah berkembang, none telah dihasilkan dari metabolites dari Hirsutella spp. This invention addresses this need by providing a safe, naturally occurring biopesticide from Hirsutella spp. Temuan ini perlu alamat ini dengan memberikan yang aman, alami terjadi biopesticide dari Hirsutella spp. which has a broad spectrum insecticidal effect. yang memiliki berbagai insecticidal efek.

toxin 536/22.1 , N-glycosides, polymers thereof, metal derivatives (eg, nucleic acids, oligonucleotides, etc.) 536/23.1 , DNA or RNA fragments or modified forms thereof (eg, genes, etc.) 536/23.2 , Encodes an enzyme 536/23.7 Encodes a microbial polypeptide 435/183, enzim (EG, LIGASES (6.), ETC.), PROENZYME; Komposisi Thereof; PROSES UNTUK Mempersiapkan, mengaktifkannya, INHIBITING, memisahkan, ATAU pemurnian ENZYMES 435/71.1, Menggunakan mikro organisme untuk membuat protein atau polypeptide 435/71.3, antibiotik atau toksin 536/22.1, N-glycosides, Polimer itu, logam derivatif (misalnya, nucleic acids, oligonucleotides, dll) 536/23.1,

SUMMARY OF THE INVENTION RANGKUMAN DARI Experiment

DNA atau RNA fragmen atau Recognizing the need to control invertebrate pests which avoids the use of diubah bentuk itu traditional pesticides and their toxic side effects, the inventors searched for a (misalnya, gen, novel biopesticide among the various invertebrate fungal pathogens. dll .) 536/23.2, Menyadari kebutuhan untuk hewan tdk bertulang punggung pengendalian encode sebuah hama yang menghindari penggunaan pestisida tradisional dan beracun efek enzim 536/23.7 samping, yang penemu mencari sebuah novel biopesticide di antara berbagai encode yang hewan tdk bertulang punggung fungal pathogens. These efforts have Microbial culminated in the substantial purification and isolation of a novel toxin from polypeptide the genus Hirsutella denoted hirsutellin A. Upaya-upaya telah culminated substansial dalam pemurnian dan isolasi dari novel toksin dari genus Field of Hirsutella denoted hirsutellin A. The hirsutellin A molecule has been characterized and found to possess a broad spectrum of activity with various invertebrate pests. J hirsutellin molekul yang telah ditemukan karakteristik dan memiliki berbagai kegiatan dengan berbagai hewan tdk bertulang punggung hama. These findings advantageously render routine the cloning of the hirsutellin A toxin polynucleotide thereby enabling the development of novel host systems for use as pest-controlling compositions and as sources of hirsutellin A polypeptide. Temuan-temuan rutin yang menyebabkan advantageously kloning dari hirsutellin J toksin polynucleotide sehingga memungkinkan pengembangan sistem host novel untuk digunakan sebagai pengendalian hama-Komposisi dan sebagai sumber hirsutellin J polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS DESKRIPSI SINGKAT gambar-gambar FIG. Fig. 1 shows a 34 amino acid sequence which comprises a hirsutellin A polypeptide. 1 menunjukkan urutan 34 asam amino yang terdiri dari sebuah hirsutellin J polypeptide. FIG. Fig. 2 shows toxicity of hirsutellin A via Contact and Residual exposure to the citrus rust mite. 2 menunjukkan kebisaan dari hirsutellin J melalui Hubungi dan sisa eksposur ke tungau karat jeruk. FIG. Fig. 2A shows residual toxicity of hirsutellin A to adult citrus rust mite at 24, 48, and 72 hours at 0, 10, 32, 56, and 100 g/ml. 2A menunjukkan sisa racun dari hirsutellin A untuk orang dewasa tungau karat jeruk di 24, 48, dan 72 hari pada 0, 10, 32, 56, dan 100 g / ml. FIG. Fig. 2B shows egg production on the leaves after 72 hours following residual application of hirsutellin A. 2B menunjukkan produksi telur pada daun setelah 72 jam berikut sisa dari aplikasi hirsutellin A.

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435/71.1 , Using a micro-organism to make a protein or polypeptide 435/71.3 , Antibiotic or toxin 435/183 , ENZYME (EG, LIGASES (6. ), ETC.), PROENZYME; COMPOSITIONS THEREOF; PROCESS FOR PREPARING, ACTIVATING, INHIBITING, SEPARATING, OR PURIFYING ENZYMES 530/350 , PROTEINS, IE, MORE THAN 100 AMINO ACID RESIDUES 530/412 , Separation or

FIG. Fig. 2C shows contact and residual toxicity of hirsutellin A to adult citrus rust mite. 2C menunjukkan kontak dan sisa racun dari hirsutellin A untuk orang dewasa tungau karat jeruk. FIG. Fig. 2D shows egg production after 72 hours following contact and residual application of hirsutellin A. Menunjukkan produksi telur 2D setelah 72 jam berikut kontak dan sisa dari aplikasi hirsutellin A. DETAILED DESCRIPTION OF THE INVENTION Detil PENJELASAN ATAS Experiment The present invention discloses the novel insecticidal mycotoxin protein hirsutellin A. This fungal metabolite is toxic to invertebrate pests. Sekarang invensi yang mengungkapkan novel insecticidal mycotoxin protein hirsutellin A. fungal metabolite ini adalah racun untuk hama hewan tdk bertulang punggung. Treatment of adult insects or larvae with hirsutellin A protein is lethal. Perawatan dewasa atau serangga larvae dengan hirsutellin J protein adalah letal. An advantage to the use of hirsutellin A is that pests can be controlled without the environmental and public safety hazards previously presented by chemical control agents. Sebuah keuntungan dengan penggunaan hirsutellin J adalah hama yang dapat dikendalikan tanpa lingkungan dan keselamatan publik bahaya sebelumnya disajikan oleh agen kontrol kimia. The hirsutellin A of this invention is characterized by a monomeric basic protein with a molecular weight of 15 KD and a pl of 10.5., with heat sensitivity at 60 C. or greater after 15 minutes. A of the hirsutellin penemuan ini adalah ditandai dengan monomeric dasar protein dengan berat molekul 15 KD dan pl dari 10,5., Dengan sensitivitas panas pada 60 C. atau lebih setelah 15 menit. Crude broth extracts of Hirsutella sp. Bulyon crude ekstrak dari Hirsutella sp. grown in vitro in shake cultures, as well as purified hirsutellin A, exhibit toxicogenic activity in various insect species. berkembang di dalam vitro guncangkan budaya, serta suci hirsutellin A, toxicogenic kegiatan pameran di berbagai jenis serangga. The exact mechanism of action for hirsutellin A toxicity is unknown. Mekanisme yang tepat untuk tindakan hirsutellin J kebisaan tidak diketahui. Injection of hirsutellin A into Galleria mellonella larvae results in alterations of the digestive tract, particularly the midgut and the malpighian tubules. Suntikan dari hirsutellin J menjadi Galleria mellonella larvae hasil dari perubahan dalam sistem pencernaan, terutama midgut dan malpighian tubules. Ultrastructural studies of organs and cells show strong cellular changes in the malpighian tubules. Ultrastructural studi organ dan sel selular kuat menunjukkan perubahan dalam malpighian tubules. The pycnotic evolution of the nucleus, which displays large and dense aggregates of chromatin, is intense and many alterations are visible at the cytoplasmic level.

purification 530/413 , Immunological separation or affinity chromatography 530/414 , Ultra filtration or osmosis 530/416 , Ion exchange 530/417 , Chromatography or by septum selective as to material, eg, gel filtration, molecular sieve dialysis, etc. 536/22.1 , Nglycosides, polymers thereof, metal derivatives (eg, nucleic acids, oligonucleotides, etc.) 536/23.1 , DNA or RNA fragments or modified forms thereof (eg, genes, etc.) 536/23.2 , Encodes an enzyme 536/23.7 Encodes a microbial polypeptide 435/71.1, Menggunakan mikro organisme untuk membuat protein atau polypeptide 435/71.3, antibiotik atau toksin 435/183, enzim (EG, LIGASES (6.),

The pycnotic evolusi inti, yang akan menampilkan besar dan padat aggregates dari chromatin, adalah intens dan banyak perubahan yang terlihat pada tingkat cytoplasmic. The hyaloplasm shows a reduced electron density and mitochondria show obvious alterations. Hyaloplasm yang menunjukkan electron mengurangi kepadatan mitochondria dan jelas menunjukkan perubahan. The present invention provides substantially pure hirsutellin A polypeptide and method for producing. Temuan ini menyediakan substansial murni hirsutellin J polypeptide dan metode produksi. In addition, polynucleotide sequences encoding hirsutellin A or functional fragments of the polypeptide are included. Selain itu, polynucleotide sequences encoding hirsutellin J fungsional atau fragmen dari polypeptide dimasukkan. Also provided is a method for controlling an invertebrate pest using a pest-controlling amount of hirsutellin A polypeptide. Juga disediakan adalah sebuah metode untuk mengendalikan hama hewan tdk bertulang punggung menggunakan hamamengendalikan jumlah hirsutellin J polypeptide. The term "pest" refers to any agricultural invertebrate including, but not limited to, arthropods such as ticks, mites, lepidoptera, hemiptera, diptera (mosquitos), homoptera and coleopera (beetles). Istilah "hama" merujuk kepada hewan tdk bertulang punggung pertanian termasuk, namun tidak terbatas pada, seperti arthropods ticks, mites, lepidoptera, hemiptera, diptera (mosquitos), homoptera dan coleopera (beetles). Other pests may include Plutella, Aedes, Galleria, Drosophila, Bombyx, Aphis and Eutetranychus. Lainnya mungkin termasuk hama Plutella, Aedes, Galleria, Drosophila, Bombyx, Aphis dan Eutetranychus. The term "pest-controlling" or "pest controlling activity", used throughout the specification and in the claims include any pesticidal or pest inhibiting activities of a composition of the invention against a particular pest. Istilah "pengendalian hama-" atau "kegiatan pengendalian hama", digunakan di seluruh spesifikasi dan di klaim apapun termasuk pesticidal atau hama inhibiting kegiatan dari komposisi dari penemuan terhadap hama tertentu. These terms include killing as well as activities which cause such injury to the pest so as to inhibit the production or development of future progeny. Istilah tersebut termasuk pembunuhan serta kegiatan-kegiatan seperti itu yang menyebabkan cedera pada hama sehingga menghalangi produksi atau perkembangan masa depan anak. Amino acids referred to herein may be identified according to the following three-letter or one-letter abbreviations: Asam amino yang disebut di sini dapat diidentifikasi menurut berikut tiga huruf atau satu huruf singkatan: ______________________________________ Three-Letter One-Letter Amino Acid Abbreviation Abbreviation

ETC.), PROENZYME; Komposisi Thereof; PROSES UNTUK Mempersiapkan, mengaktifkan , INHIBITING, memisahkan, ATAU pemurnian ENZYMES 530/350, protein, IE, LEBIH DARI 100 amino acid residu 530/412, Pemisahan atau pemurnian 530/413, imunologi atau pemisahan kromatografi Affinity 530/414, Ultra penyaringan atau osmosa 530/416, pertukaran ion 530/417, kromatografi atau septum selektif sebagai bahan untuk, misalnya, gel penyaringan, saringan molekular dialysis, dll 536/22.1, Nglycosides, Polimer itu, logam derivatif (misalnya, nucleic acids, oligonucleotides, dll) 536/23.1, DNA atau RNA fragmen atau diubah bentuk itu

______________________________________ L-Alanine Ala A L-Arginine Arg R L-Asparagine Asn N L-Aspartic Acid Asp D L-Cysteine Cys C LGlutamine Gln Q L-Glutamic Acid Glu E L-Glycine Gly G L-Histidine His H L-Isoleucine Ile I L-Leucine Leu L L-Lysine Lys K L-Methionine Met M LPhenylalanine Phe F L-Proline Pro P L-Serine Ser S L-Threonine Thr T LTryptophan Trp W L-Tyrosine Tyr Y L-Valine Val V ______________________________________ ______________________________________-Tiga Banding Satu Banding amino acid Abbreviation Abbreviation ______________________________________ L-Alanine Ala J L-Arginine L-Arg R Asparagine ASN N L-Aspartic Acid Asp D L-Cysteine Cys C LGlutamine GLN T L-Glutamic Acid ALA E L G-Glycine GLY Histidine LNya H L-Isoleucine Ile saya L-Leucine Leu L L-Lysine Lys K L-Methionine Met M L-Phenylalanine TYR M L-Proline Pro P L-Serine Ser S L-Threonine ASN T L TRP W-Tryptophan L-Tyrosine GLU Y L-Valine Val V ______________________________________ The term "substantially pure" as used herein refers to hirsutellin A which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is normally associated. Istilah "substansial murni" seperti yang digunakan di sini merujuk kepada hirsutellin J substansial yang bebas dari protein lainnya, lipids, karbohidrat atau dengan bahan lainnya yang biasanya terkait. The substantially pure hirsutellin A polypeptide yields a single major band of 15 kD on a non-reducing polyacrylamide gel. Substansial yang murni hirsutellin J polypeptide hasil satu band besar dari 15 kd di non-mengurangi polyacrylamide gel. The purity of hirsutellin A can also be determined by amino-terminal amino acid sequence analysis. Yang suci dari hirsutellin J juga dapat ditentukan oleh amino-terminal analisis urutan asam amino. Hirsutellin A polypeptide includes functional fragments of the polypeptide, so long as hirsutellin A activity is retained. Hirsutellin J polypeptide fungsional meliputi fragmen dari polypeptide, selama kegiatan hirsutellin J diperoleh. Therefore, smaller peptides containing the biological activity of hirsutellin A are included in the invention. Karena itu, lebih kecil yang berisi peptides biologi kegiatan hirsutellin J disertakan dalam ciptaan. Minor modifications of the hirsutellin A primary amino acid sequence may result in proteins which have substantially equivalent activity compared with hirsutellin A described in the invention. Modifikasi kecil dari hirsutellin J utama urutan asam amino dapat menyebabkan protein yang ada secara substansial setara dengan kegiatan dibandingkan hirsutellin J dijelaskan di adakan. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. Modifikasi seperti itu mungkin disengaja, seperti yang diarahkan oleh situsmutagenesis, atau mungkin spontan. For example, more effective hirsutellin A molecules that may be produced by these modifications are included herein, as long as hirsutellin A activity still exists. Misalnya, lebih efektif hirsutellin J molekul yang dapat dihasilkan oleh modifikasi ini disertakan di sini, selama kegiatan hirsutellin J masih ada.

(misalnya, gen, dll) 536/23.2, encode sebuah enzim 536/23.7 encode yang Microbial polypeptide

Examiners Examiners
Primary: Wax, Robert A. Utama: Wax, Robert A. Assistant: Kim, Hyosuk Asisten: Kim, Hyosuk

Attorney, Agent or Firm Pengacara, Agen atau Firm

Spensley Horn Jubas & Lubitz Spensley Horn Jubas & Lubitz

Internation al Classes Kelas internasion al

The present invention provides a method for producing substantially pure hirsultellin A. Procedures include those commonly used for the separation of protein substances including, for example, treatment of a sample containing hirsutellin A activity with common precipitants for proteins, followed by fractionation techniques such as ion exchange chromatography, affinity chromatography, molecular sieve chromatography, adsorption chromatography, ultrafiltration and various combinations thereof. Temuan ini memberikan sebuah metode untuk memproduksi substansial murni hirsultellin A. Prosedur termasuk yang umum digunakan untuk pemisahan zat protein termasuk, misalnya, perlakuan terhadap sampel berisi hirsutellin J dengan kegiatan Common precipitants untuk protein, diikuti oleh fractionation teknik seperti ion tukar kromatografi, kromatografi Affinity, saringan molekular kromatografi, kromatografi adsorption, ultrafiltration dan berbagai kombinasi itu. Preferably, the method of the invention includes, a) a salt precipitation step, b) a step for removal of neutral and acidic proteins, such as an ion exchange step, and c) exclusion chromatography. Preferably, metode penemuan yang meliputi, a) garam langkah hujan, b) langkah untuk dihapus dari netral dan acidic protein, seperti pertukaran ion langkah, dan c) pengecualian kromatografi. Preferably, the sample containing toxin is produced by growth of a fungus which produces hirsutellin A such as Hirsutella thompsonii, in yeast extract containing medium such as Czapek-Dox broth at 25 C. The sample containing hirsutellin A activity is salt precipitated, preferably with ammonium sulfate. Preferably, sampel yang mengandung toksin yang dihasilkan oleh pertumbuhan dari jamur yang memproduksi J hirsutellin seperti Hirsutella thompsonii, dalam ekstrak ragi yang mengandung media seperti Czapek-dox bulyon pada 25 C. sampel berisi hirsutellin J kegiatan precipitated adalah garam, sebaiknya dengan ammonium sulfate. After partial precipitation, the material is desalted using gel filtration, for example, with Sephadex G-25, followed by ion exchange chromatography. Setelah hujan sebagian, bahan desalted adalah menggunakan gel penyaringan, misalnya, dengan Sephadex G-25, diikuti oleh pertukaran ion kromatografi. The material is treated with an anion exchange resin with a diethylaminomethyl (DEAE) or a diethyl-(2-hydroxypropyl)aminoethyl (QAE) functional group, preferably a diethylaminomethyl group. Materi yang dianggap dengan resin pertukaran anion dengan diethylaminomethyl (DEAE) atau diethyl-(2hydroxypropyl) aminoethyl (QAE) fungsional grup, sebaiknya satu grup diethylaminomethyl. DEAE is a weak anion exchange resin and is preferably to stronger anion exchange resins such as QAE agarose for the present invention. DEAE adalah resin pertukaran anion lemah dan lebih kuat untuk pertukaran anion Resins seperti QAE agarose untuk sementara ini. The treatment conditions, eg, a solution of about pH 8.0 and a linear salt gradient of about 0 to 0.3M, are such that the hirsutellin A activity is not adsorbed to the support. Perawatan kondisi, misalnya, sebuah solusi dari sekitar pH 8,0

C12N 009/00 C12N 009/00 A01N 063/00 A01N 063/00 C12P 021/04 C12P 021/04 C07H 019/00 C07H 019/00

dan garam linear lereng sekitar 0 sampai 0.3M, adalah seperti yang hirsutellin J aktivitas tidak adsorbed dengan dukungan. Hirsutellin A activity can be determined by its insecticidal effect. J Hirsutellin aktivitas dapat ditentukan oleh insecticidal efek. One suitable assay for hirsutellin A activity includes that described in EXAMPLE 3, which involves the injection of the hirsutellin A containing solution into Lepidopteran larvae. Kadar logam yang cocok untuk hirsutellin J termasuk aktivitas yang dijelaskan dalam EXAMPLE 3, yang melibatkan suntikan yang berisi J hirsutellin menjadi solusi Lepidopteran larvae. In a preferred embodiment, the pooled fractions containing partially purified hirsutellin A are treated next with a cation exchange material with a carboxymethyl or sulphopropyl functional group, preferably a carboxymethyl group. Penjelmaan dalam penawaran, yang berisi sebagian pecahannya pooled suci hirsutellin J diperlakukan berikutnya dengan cation pertukaran materi dengan carboxymethyl atau sulphopropyl kelompok fungsional, sebaiknya yang carboxymethyl grup. Carboxymethyl resin is preferably to the stronger cation ion exchange resins, such as those with sulphopropyl groups. Carboxymethyl damar adalah lebih baik untuk semakin kuat cation ion pertukaran Resins, seperti mereka yang sulphopropyl kelompok. The treatment conditions, eg, a solution of about pH 5.0 and a salt concentration of about 0.05M to 0.5M, are such that the hirsutellin A activity initially adsorbed to the support, is then eluted at a salt concentration of about 0.3M. Perawatan kondisi, misalnya, sebuah solusi dari sekitar pH 5,0 dan konsentrasi garam tentang 0.05M ke 0.5M, adalah seperti yang hirsutellin J adsorbed pada awalnya kegiatan untuk dukungan, kemudian eluted pada konsentrasi garam sekitar 0.3M. The resulting material is tested for hirsutellin A activity as described above and as described in EXAMPLE 3. Hasilnya adalah bahan untuk diuji hirsutellin J kegiatan seperti yang dijelaskan di atas dan seperti yang dijelaskan dalam EXAMPLE 3. The unbound fractions containing hirsutellin A activity can be further purified by gel filtration using, for example, any commerically available sizing gel such as biogel P10 (Biorad) which has a molecular weight fractionation range of about 1,500 to 20,000 daltons. Yg tdk dijilid pecahannya yang berisi hirsutellin J kegiatan dapat lebih suci oleh penyaringan menggunakan gel, misalnya, setiap komersial tersedia gel perekat seperti biogel P10 (Biorad) yang memiliki berat molekul fractionation berbagai tentang 1500-20000 daltons. The hirsutellin A containing fractions after treatment with the gel are then subjected to SDS PAGE under suitable conditions and the gel slice containing hirsutellin A activity is recovered. Yang berisi hirsutellin J pecahannya setelah pengobatan dengan gel kemudian subyek SDS PAGE bawah sesuai kondisi dan gel slice berisi hirsutellin J kegiatan tersebut dipulihkan. SDS PAGE is performed according to the method of Laemmli, et al., (Nature, 227:680, 1970) and is a technique well known to those in the art.

SDS PAGE dilakukan sesuai dengan metode Laemmli, dkk. (Nature, 227:680, 1970) dan merupakan teknik yang dikenal dengan baik kepada mereka dalam seni. Variations in conditions which are within a suitable range are understood to be encompassed within the purification procedure. Variasi dalam kondisi yang cocok dalam rentang yang dapat dipahami mencakup pemurnian dengan prosedur. The hirsutellin A activity-containing fraction from the SDS PAGE is subjected to reverse phase HPLC and eluted with acetonitrile. J hirsutellin yang berisi kegiatan-pecahan dari SDS PAGE sasaran adalah membalikkan phase HPLC dan eluted dengan acetonitrile. The hirsutellin A which is obtained is substantially pure to permit N-terminal amino acid sequencing. The J hirsutellin yang diperoleh adalah murni substansial untuk mengizinkan N-terminal amino acid sequencing. The toxic solution is dried under vacuum and redissolved in a small volume of acetonitrile 95% +TFA (0.08%). Beracun adalah solusi yang kering di bawah dan redissolved kekosongan dalam volume kecil acetonitrile 95% + TFA (0,08%). The concentrated sample is then introduced in a sequencer connected to a phenylthiohydantoine (PTH) analyzer. Terkonsentrasi pada contoh ini kemudian diperkenalkan di sequencer terhubung ke phenylthiohydantoine (PTH) analyzer. A preferred embodiment of the invention comprises the polypeptide, APKVTSRPKLDGNEKPFKVDV (SEQ. ID No. 1) and conservative variations of this peptide. J penawaran perwujudan invensi yang terdiri atas polypeptide, APKVTSRPKLDGNEKPFKVDV (ID SEQ. No 1) dan konservatif variasi peptide ini. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar, residue. Istilah "konservatif variasi" seperti yang digunakan di sini menandakan penggantian residu asam amino yang lain, mirip biologis, residu. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. Contoh variasi konservatif termasuk substitution satu residu hydrophobic seperti isoleucine, valine, leucine atau untuk methionine lain, atau substitusi satu kutub sisa untuk yang lain, seperti dari arginine substitution untuk lysine, glutamic untuk aspartic asam, atau untuk glutamine asparagine, dan lain-lain. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that the substituted polypeptide retain the pest controlling activity of the unsubstituted polypeptide. Istilah "konservatif variasi" juga mencakup penggunaan asam amino yang digantikan di tempat yang unsubstituted induk asam amino asalkan yang digantikan polypeptide mempertahankan kegiatan pengendalian hama yang unsubstituted polypeptide.

Antibodies provided in the present invention are immunoreactive with hirsutellin A polypeptide or fragments thereof. Antibodies yang hadir dalam ciptaan yang immunoreactive dengan hirsutellin J polypeptide atau fragmen itu. Polyclonal antibodies are prepared by immunization of an animal, eg, rabbit, with an immunogenic sample of hirsutellin A followed by purification of the antibody by methods well known in the art. Polyclonal antibodies imunisasi yang disiapkan oleh suatu hewan, misalnya, kelinci, dengan immunogenic sampel hirsutellin J diikuti oleh pemurnian dari antibodi oleh metode terkenal dalam seni. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations, are provided. Antibodi yang pada dasarnya terdiri dari pooled monoclonal antibodies berbeda dengan epitopic specificities, serta berbeda monoclonal antibodi persiapan, disediakan. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975). Monoclonal antibodies antigen yang dibuat dari potongan-potongan yang mengandung protein yang dikenal dengan baik oleh metode dalam seni (Kohler, dkk., Alam, 256:495, 1975). The term "antibody" as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab') 2 , which are capable of binding the epitopic determinant. Istilah "antibodi" seperti yang digunakan dalam penemuan ini dimaksudkan untuk menyertakan molekul utuh serta fragmen itu, seperti Fab dan F (ab ') 2, yang mampu mengikat epitopic yang menentukan. The antibodies of the invention can be used in immunoaffinity chromatography for the isolation of sequences containing the hirsutellin A activity of the present invention. The antibodies invensi yang dapat digunakan dalam kromatografi immunoaffinity untuk isolasi dari sequence yang berisi hirsutellin J kegiatan masa kini invensi. One way by which such immunoaffinity chromatography can be utilized is by the binding of the antibodies of the invention to CNBr-Sepharose-4B or Tresyl activated Sepharose (Pharmacia). Salah satu cara yang seperti immunoaffinity kromatografi dapat dimanfaatkan oleh yang mengikat dari antibodies dari invensi ke CNBr-4B-Sepharose atau Tresyl diaktifkan Sepharose (Pharmacia). These solid phase-bound antibodies can then be used to specifically bind sequences containing hirsutellin A activity from mixtures of other proteins to enable isolation and purification thereof. Solid-tahap ini terikat antibodies kemudian dapat digunakan untuk mengikat secara khusus yang berisi sequence hirsutellin J kegiatan dari campuran lain untuk mengaktifkan protein isolasi dan pemurnian itu. Bound hirsutellin A sequences can be eluted from the affinity chromatographic material using techniques known to those of ordinary skill in the art such as, for example, chaotropic agents, low pH, or urea. Terikat hirsutellin J sequence dapat eluted dari bahan Affinity chromatographic menggunakan teknik yang dikenal orang-orang biasa dari keahlian dalam seni seperti, misalnya, chaotropic agen, pH rendah, atau urea.

The invention provides polynucleotides encoding the hirsutellin A protein. Invensi yang menyediakan polynucleotides encoding yang hirsutellin J protein. These polynucleotides include DNA, cDNA and RNA sequences which encode hirsutellin A. It is understood that all polynucleotides encoding all or a portion of hirsutellin A are also included herein, so long as they encode a polypeptide with hirsutellin A activity. Ini termasuk polynucleotides DNA, RNA dan cDNA sequence yang menyandi hirsutellin A. Penyalahgunaan dipahami bahwa semua polynucleotides Encoding semua atau sebagian hirsutellin J juga dimasukkan di sini, sepanjang mereka yang menyandi polypeptide dengan hirsutellin J kegiatan. Such polynucleotides include both naturally occurring and intentionally manipulated polynucleotides. Seperti polynucleotides termasuk terjadi baik secara alami dan sengaja dimanipulasi polynucleotides. For example, hirsutellin A may be subjected to site-directed mutagenesis. Misalnya, hirsutellin J mungkin terkena diarahkan ke situs mutagenesis. The polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. The polynucleotides dari ciptaan termasuk sequence yang memburuk sebagai akibat dari kode genetik. There are only 20 natural amino acids, most of which are specified by more than one codon. Hanya ada 20 asam amino alam, sebagian besar dari yang ditentukan oleh lebih dari satu codon. Therefore, as long as the amino acid sequence of hirsutellin A is unchanged, or although changed retains hirsutellin A activity, all degenerate nucleotide sequences are included in the invention. Oleh karena itu, selama urutan asam amino hirsutellin A berubah, walaupun berubah atau tetap hirsutellin J kegiatan, semua merosot nucleotide sequence disertakan dalam ciptaan. DNA sequences of the invention can be isolated by several techniques known in the art. DNA sequence dari invensi dapat terpencil oleh beberapa teknik yang dikenal dalam seni. These include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences, and 2) antibody screening of expression libraries to detect shared structural features. Termasuk, tetapi tidak terbatas pada: 1) hibridisasi dari probes ke genomic atau cDNA perpustakaan untuk mendeteksi nucleotide sequence bersama, dan 2) antibodi skrining ekspresi dari perpustakaan untuk mendeteksi bersama fitur struktural. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Prosedur pemeriksaan yang mengandalkan nucleic asam hibridisasi memungkinkan untuk mengisolasi gene urutan apapun dari organisme, yang diberikan yang sesuai probe tersedia. For example, oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. Misalnya, oligonucleotide probes, yang sesuai dengan bagian yang urutan encoding protein tersebut dapat synthesized kimia. This requires that short, oligopeptide

stretches of amino acid sequence must be known. Ini membutuhkan singkat, oligopeptide stretches urutan asam amino yang harus diketahui. Preferably, the oligonucleotide probe of the invention encodes a consecutive sequence from about four to about thirty-four amino acids of the amino acid sequence APKVTSRPKLDGREKPFKVDVATAQAQARKAGLT (SEQ. ID No. 2) including any conservative variations. Preferably, yang oligonucleotide penyelidikan dari penemuan encode yang berturut-turut urutan dari sekitar empat hingga sekitar tiga puluh empat asam amino dari asam amino urutan APKVTSRPKLDGREKPFKVDVATAQAQARKAGLT (ID SEQ. No 2) termasuk konservatif variasi. A preferred oligonucleotide probe encodes a consecutive sequence of the amino acids EKPFKV, including any conservative variations of this peptide. J penawaran oligonucleotide probe encode yang berturut-turut dari urutan asam amino EKPFKV, termasuk konservatif variasi peptide ini. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. DNA encoding urutan protein yang dapat deduced dari kode genetik, namun degeneracy kode yang harus diambil ke dalam rekening. It is possible to perform a mixed addition reaction when the sequence is degenerate. Kemungkinan untuk melakukan penambahan dicampur reaksi adalah ketika urutan merosot. This includes a heterogenous mixture of denatured double-stranded DNA. Ini termasuk heterogenous ganda campuran denatured DNA-terlantar. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Untuk pemutaran film seperti itu, adalah hibridisasi sebaiknya dilakukan pada salah satu-terlantar atau denatured DNA ganda terlantar DNA. This is especially useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. Hal ini sangat berguna dalam deteksi cDNA clones yang berasal dari sumber yang sangat rendah di mana jumlah mRNA sequence polypeptide yang berkaitan dengan kepentingan yang hadir. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by hybridization of the target DNA to the single probe in the mixture which is its complement (Wallace, et al., Nucleic Acid Research, 9:879, 1981). Dengan kata lain, dengan menggunakan hibridisasi kondisi ketat diarahkan untuk menghindari hal-spesifik mengikat, kemungkinan, misalnya, untuk membolehkan autoradiographic visualisasi tertentu cDNA clone oleh hibridisasi dari target DNA tunggal ke dalam penyelidikan yang adalah campuran melengkapi (Wallace, et al., Nucleic Acid Research, 9:879, 1981). A cDNA expression library, such as gtll, can be screened indirectly for hirsutellin A peptides having at least one antigenic epitope, using antibodies specific for hirsutellin A. Such antibodies can be either monoclonal or polyclonal and used to detect an expression product indicative of the presence of hirsutellin A cDNA. J ekspresi cDNA perpustakaan, seperti gtll, dapat

langsung screened untuk hirsutellin J peptides memiliki setidaknya satu antigenic epitope, menggunakan antibodies khusus untuk hirsutellin A. semacam antibodies dapat berupa monoclonal atau polyclonal dan digunakan untuk mendeteksi sebuah produk ekspresi menunjukkan keberadaan dari hirsutellin J cDNA. A hirsutellin A cDNA library can also be screened by injecting different cDNAs into oocytes. J hirsutellin J cDNA perpustakaan juga dapat screened oleh injecting berbeda cDNAs ke oocytes. After expression of the cDNA gene products occurs, the presence of the specific cDNA gene product can be identified by antibody screening with antibody specifically immunoreactive with hirsutellin A polypeptide, for example. Setelah ekspresi dari cDNA gene produk terjadi, keberadaan spesifik cDNA gene produk dapat diidentifikasi oleh skrining antibodi dengan antibodi spesifik immunoreactive dengan hirsutellin J polypeptide, misalnya. Alternatively, functional assays for hirsutellin A toxicogenic activity could be performed to identify Hirsutellin A producing oocytes. Atau, untuk hirsutellin fungsional assays J toxicogenic kegiatan dapat dilakukan untuk mengidentifikasi Hirsutellin J oocytes produksi. Specific DNA sequences encoding hirsutellin A can also be obtained by: (1) isolation of double-stranded DNA sequences from genomic DNA; (2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and (3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell, resulting in a cDNA, or complimentary DNA. Spesifik DNA sequences encoding hirsutellin J juga dapat diperoleh oleh: (1) isolasi dari dua terjalin DNA sequence dari genomic DNA; (2) kimia manufaktur dari urutan DNA yang diperlukan untuk menyediakan codons untuk polypeptide kepentingan; dan (3) vitro di sintesis dari dua terdampar urutan DNA oleh reverse transcription dari mRNA terpencil dari eukaryotic donor sel, sehingga dalam cDNA, atau gratis DNA. Synthesis of DNA sequences is frequently the method chosen when the entire sequence of amino acid residues of the desired polypeptide product is known. Sintesis dari DNA sequence sering metode dipilih bila seluruh urutan residu asam amino yang dikehendaki polypeptide produk dikenal. When the entire sequence of amino acid residues of the desired polypeptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the formation of cDNA sequences. Bila seluruh urutan residu asam amino yang dikehendaki polypeptide tidak diketahui, langsung dari sintesis DNA sequence tidak mungkin dan metode pilihan adalah pembentukan cDNA sequence. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid or bacteriophage based cDNA libraries in which mRNA is reverse transcribed from donor cells with a high level of genetic expression. Di antara standar prosedur untuk isolating cDNA

sequence yang menarik adalah pembentukan plasmid atau bacteriophage berbasis cDNA perpustakaan di mana mRNA adalah reverse transcribed dari sel donor yang tinggi dengan tingkat ekspresi genetik. When used in combination with polymerase chain reaction (PCR) technology, less common mRNA species (cDNA) can be cloned as well. Bila digunakan bersama dengan polymerase chain reaction (PCR) teknologi, kurang umum mRNA spesies (cDNA) dapat clone juga. When significant portions of the amino acid sequence of a polypeptide are known, labeled single or double-stranded DNA or RNA probes which represent a sequence present in the target cDNA, may be used in DNA/DNA hybridization procedures which are performed on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al., Nucleic Acid Research, 11:2325, 1983). Ketika porsi signifikan dari urutan asam amino yang dikenal polypeptide, berlabel satu atau dua kali terdampar DNA atau RNA probes yang mewakili urutan hadir dalam target cDNA, dapat digunakan dalam DNA / DNA hibridisasi prosedur yang dilaksanakan di clone salinan yang cDNA yang telah denatured dalam satu bentuk-terlantar (Jay, et al., Nucleic Acid Research, 11:2325, 1983). Since the novel DNA sequences of the invention encode a unique sequence of hirsutellin A, it is now a routine matter to prepare, subclone, and express smaller polypeptide fragments of DNA from this or corresponding DNA sequences. Sejak novel DNA sequence dari ciptaan yang unik menyandi urutan hirsutellin A, sekarang adalah hal yang rutin untuk mempersiapkan, subclone, dan menyatakan polypeptide kecil fragmen dari DNA dari DNA atau sesuai urutan. Alternatively, by utilizing the DNA fragments disclosed herein which define the unique hirsutellin A polypeptide of the invention, it is possible, in conjunction with known techniques, to determine the DNA sequences encoding the entire hirsutellin A toxin. Atau, dengan memanfaatkan DNA fragmen diungkapkan di sini yang menentukan yang unik hirsutellin J polypeptide dari penemuan itu, dimungkinkan, dalam hubungannya dengan teknik yang dikenal, untuk menentukan DNA sequences encoding seluruh hirsutellin J toksin. Such techniques are described in US Pat. Teknik seperti yang dijelaskan dalam US Pat. Nos. 4,394,443 and 4,446,235 which are incorporated herein by reference. Nos 4394443 dan 4446235 yang dimasukkan di sini oleh referensi. The polypeptide resulting from expression of a DNA sequence of the invention can be further characterized as being free from association with other eukaryotic polypeptides or other contaminants which might otherwise be associated with the polypeptide in its natural cellular environment. Polypeptide yang dihasilkan dari ekspresi dari urutan DNA dari invensi dapat lebih karakteristik sebagai bebas dari asosiasi dengan lainnya eukaryotic polypeptides atau contaminants lainnya yang mungkin dapat dikaitkan dengan alam polypeptide dalam lingkungan selular. Isolation and purification of microbially expressed polypeptides provided by

invention may be by conventional means including, preparative chromatographic separations and immunological separations involving monoclonal and/or polyclonal antibody preparation. Isolasi dan pemurnian dari microbially dinyatakan polypeptides diberikan oleh penemuan mungkin oleh konvensional berarti termasuk, chromatographic separations persiapan dan imunologi separations melibatkan monoclonal dan / atau antibodi polyclonal persiapan. For purposes of the present invention, hirsutellin A polypeptides which are homologous to those of the invention can be identified by structural as well as functional similarity. Untuk tujuan penemuan hadir, hirsutellin J polypeptides yang sesuai untuk penemuan mereka yang dapat diidentifikasi oleh struktural maupun fungsional kesamaan. Structural similarity can be determined, for example, by assessing polynucleotide strand hybridization or by screening with antibody, especially a monoclonal antibody, which recognizes a unique epitope present on the hirsutellin A polypeptides of the invention. Struktural kesamaan dapat ditetapkan, misalnya, menilai polynucleotide oleh helai hibridisasi atau dengan pemeriksaan antibodi, khususnya yang monoclonal antibodi, yang mengakui epitope yang unik hadir di hirsutellin J polypeptides dari ciptaan. When hybridization is used as criteria to establish structural similarity, those polynucleotide sequences which hybridize under stringent conditions to the polynucleotides of the invention are considered to be essentially the same as the polynucleotide sequences of the invention. Hibridisasi bila digunakan sebagai kriteria untuk menentukan kesamaan struktural, yang polynucleotide sequence silang yang ketat di bawah kondisi yang polynucleotides dari invensi dianggap dasarnya sama dengan polynucleotide sequence dari ciptaan. A wide variety of ways are available for introducing a polynucleotide expressing a hirsutellin A toxin into the microorganism host under conditions which allow for stable maintenance and expression of the gene. J berbagai cara yang tersedia untuk memperkenalkan polynucleotide expressing J toksin yang hirsutellin mahluk yg kecil yang menjadi tuan rumah dalam kondisi yang stabil memungkinkan pemeliharaan dan ekspresi dari gene. DNA constructs are available which include the transcriptional and translational regulatory signals for expression of the hirsutellin A polynucleotide; the toxin gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur; and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur. DNA constructs yang tersedia termasuk transcriptional dan peraturan translational sinyal untuk ekspresi dari hirsutellin J polynucleotide; toksin yang gene peraturan mereka di bawah kontrol dan DNA urutan sesuai dengan urutan dalam host organisme, dimana akan terjadi integrasi, dan / atau peniruan fungsional sistem yang di-host, dimana integrasi atau pemeliharaan stabil akan terjadi.

The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. Inisiasi yang transcriptional sinyal akan termasuk promotor dan transcriptional inisiasi mulai situs. In some instances, it may be desirable to provide for regulative expression of the hirsutellin A polynucleotide, where expression of the toxin will only occur after release into the environment. Dalam beberapa kasus, mungkin keinginan untuk menyediakan regulative ekspresi dari hirsutellin J polynucleotide, di mana ekspresi dari toksin hanya akan terjadi setelah rilis ke dalam lingkungan. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the host. Hal ini dapat dicapai dengan operator atau ke suatu daerah yang mengikat activator atau enhancers, yang mampu induksi suatu perubahan fisik atau kimia lingkungan host. For example, a temperature sensitive regulatory region may be employed where the organisms may be grown up in the laboratory without expression of a toxin, but upon change in the growth conditions or environment, expression would begin. Misalnya, suhu sensitif peraturan daerah dapat bekerja di mana organisme dapat tumbuh di laboratorium tanpa ekspresi dari toksin, tetapi setelah perubahan dalam kondisi pertumbuhan atau lingkungan, akan mulai ekspresi. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where the nutrient medium in the later environment would allow for expression of the toxin. Teknik lainnya Mei mempekerjakan gizi media tertentu dalam laboratorium, yang inhibits ekspresi dari toksin, dimana gizi media di lingkungan nantinya akan memungkinkan untuk ekspresi dari toksin. For translational initiation, a ribosomal binding site and an initiation codon will be present. Untuk translational inisiasi, sebuah situs dan ribosomal mengikat codon inisiasi yang akan hadir. Various manipulations may be employed for enhancing the expression of the mRNA, particularly by using an active promoter, as well as by employing sequences, which enhance the stability of the mRNA. Berbagai manipulasi dapat digunakan untuk meningkatkan ekspresi dari mRNA, terutama dengan menggunakan promotor yang aktif, serta Mempekerjakan sequence, yang meningkatkan stabilitas dari mRNA. The initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal. Inisiasi yang translational dan penghentian daerah akan melibatkan menghentikan codon (s), sebuah terminator daerah, dan opsional, yang polyadenylation sinyal. In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. Dalam arah transcription, yaitu di 5 'ke 3' searah dengan

coding atau rasa urutan, dengan membangun akan melibatkan transcriptional peraturan daerah, jika ada, dan promotor, di mana peraturan daerah mungkin baik 5 'atau 3' dari promotor, yang mengikat ribosomal situs, inisiasi codon, yang memiliki struktural gene buka membaca bingkai dalam tahap inisiasi dengan codon, yang berhenti codon (s), maka urutan polyadenylation sinyal, jika ada, dan terminator daerah. This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the toxin expression construct during introduction of the DNA into the host. Urutan ini sebagai dua helai dapat digunakan oleh dirinya sendiri untuk transformasi dari mahluk yg kecil host, tapi biasanya akan disertakan dengan DNA melibatkan urutan tanda, di mana urutan kedua DNA mungkin toksin yang tergabung dalam membangun ekspresi selama pengenalan DNA ke dalam tuan rumah. A marker structural gene may be present which provides for selection of those hosts which have been modified or transformed. J tanda gene Mei struktural yang hadir untuk memberikan pilihan yang host yang telah dimodifikasi atau diwujudkan. The marker will normally provide for selective advantage, for example, providing for biocide resistance, for example, resistance to antibiotics or heavy metals; complementation, so as to provide prototropy to an auxotrophic host, or the like. Tanda tersebut biasanya akan memberikan keuntungan untuk selektif, misalnya, untuk menyediakan biocide perlawanan, misalnya, perlawanan terhadap antibiotik atau logam berat; complementation, sehingga memberikan prototropy ke auxotrophic tuan rumah, atau sejenisnya. Preferably, complementation is employed, so that the modified host may not only be selected, but may also be competitive in the field. Preferably, complementation yang bekerja, sehingga tidak dapat diubah host hanya dapat dipilih, tetapi juga dapat bersaing di lapangan. One or more markers may be employed in the development of the constructs, as well as for modifying the host. Satu atau lebih tanda mungkin bekerja dalam pembangunan constructs, serta untuk memodifikasi host. The organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field. Organisme yang dapat diubah oleh lebih memberikan keuntungan yang kompetitif terhadap lainnya liar-jenis mikroorganisme di lapangan. For example, genes expressing metal chelating agents, for example, siderophores, may be introduced into the host along with the structural gene expressing the toxin. Misalnya, gen expressing metal chelating agen, misalnya, siderophores, mungkin diperkenalkan ke tuan rumah bersama dengan struktural gene menyatakan toksin. In this manner, the enhanced expression of a siderophore may provide for a competitive advantage for the toxin-producing host, so that it may effectively compete with the wild-type microorganisms and stably occupy a niche in the environment. Dengan cara ini, dengan peningkatan ekspresi dari siderophore untuk dapat memberikan keuntungan kompetitif untuk memproduksi toksinhost, sehingga dapat bersaing secara efektif dengan liar-jenis mikroorganisme

stably dan menempati sebuah relung di lingkungan. Where no functional replication system is present, the construct will also include a sequence of at least 50 basepairs (bp), preferably at least about 100 bp, and usually not more than about 1000 bp of a sequence homologous with a sequence in the host. Fungsional, di mana tidak ada sahutan sistem ini, yang juga akan membangun termasuk urutan ke-50 setidaknya basepairs (bp), sebaiknya minimal sekitar 100 bp, dan tidak lebih dari biasanya sekitar 1000 bp dari urutan sesuai dengan urutan dalam host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Dengan cara ini, kemungkinan yang sah recombination ditingkatkan agar generasi akan diintegrasikan ke dalam host dan dikelola oleh stably host. Desirably, the toxin gene will be in close proximity to the gene providing for complementation as well as the gene providing for the competitive advantage. Desirably, gene toksin yang akan di dekat ke generasi untuk memberikan complementation serta gene memberikan keuntungan yang kompetitif. Therefore, in the event that a toxin gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct. Karena itu, dalam acara yang toksin gene terputus, sehingga organisme akan kemungkinan juga kehilangan gene melengkapi dan / atau gene memberikan keuntungan yang kompetitif, sehingga tidak akan dapat bersaing di lingkungan dengan tetap mempertahankan gene membangun yang utuh. A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like. Banyaknya jumlah transcriptional peraturan daerah yang tersedia dari berbagai jenis mahluk yg kecil host, seperti bakteri, bacteriophage, cyanobacteria, algae, jamur, dan lain-lain. Various transcriptional regulatory regions include the regions associated with the trp gene, lac gene, gal gene, the lambda left and right promoters, the Tac promoter, the naturally-occurring promoters associated with the toxin gene, where functional in the host. Transcriptional berbagai daerah termasuk peraturan daerah yang terkait dengan TRP gene, lac gene, gene gal, di kiri dan kanan lambda promoters, maka TAC promotor, yang terjadi secara alamipromoters yang terkait dengan toksin gene, dimana fungsional dalam host. See, for example, US Pat. Lihat, misalnya, US Pat. Nos. 4,332,898, 4,352,832 and 4,356,270. Nos 4332898, 4352832 dan 4356270. The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host. Penghentian wilayah yang mungkin merupakan wilayah penghentian biasanya terkait dengan transcriptional inisiasi atau wilayah yang berbeda transcriptional inisiasi daerah, sehingga selama dua daerah tersebut kompatibel dan fungsional

dalam host. Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system which is functional in the host. Dimana episomal stabil atau pemeliharaan integrasi adalah diinginkan, yang akan digunakan plasmid yang memiliki replikasi sistem yang berfungsi dalam host. The replication system may be derived from the chromosome, an episomal element normally present in the host or a different host, or a replication system from a virus which is stable in the host. Replikasi sistem mungkin berasal dari kromosom, episomal elemen yang biasanya hadir dihost atau host yang berbeda, atau sebuah sistem replikasi dari virus yang stabil pada host. A large number of plasmids are available, such as pBR322, pACYC184, RSFIO1O, pR01614, and the like. Banyaknya jumlah plasmids tersedia, seperti pBR322, pACYC184, RSFIO1O, pR01614, dan lain-lain. (See, for example Olson et al., J. Bacteriol. 150:6069, 1982, and Bagdasarian et al., Gene 16:237, 1981, and US Pat. Nos. 4,356,270, 4,362,817, and 4,371,625.) (Lihat, misalnya Olson et al., J. Bacteriol. 150:6069, 1982, dan Bagdasarian dkk., Gene 16:237, 1981, dan US Pat. Nos 4356270, 4362817, dan 4371625.) The hirsutellin A polynucleotide can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. The hirsutellin J polynucleotide dapat diperkenalkan antara transcriptional dan inisiasi translational wilayah dan transcriptional dan translational penghentian daerah, sehingga peraturan di bawah kontrol dari inisiasi daerah. This construct will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. Membangun ini akan dimasukkan dalam plasmid, yang akan berisi minimal satu replikasi sistem, tetapi dapat mencakup lebih dari satu, di mana satu replikasi sistem yang digunakan untuk kloning selama pembangunan plasmid dan yang kedua adalah replikasi sistem yang diperlukan untuk berfungsi di ultimate host. In addition, one or more markers may be present, as described above. Selain itu, satu atau lebih tanda-tanda akan hadir, seperti dijelaskan di atas. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome. Dimana integrasi yang diinginkan, maka plasmid akan desirably termasuk urutan sesuai dengan host genome. The transformants can be isolated in accordance with conventional techniques usually employing selection of the desired organism as against unmodified organisms or transferring organisms, when present. Transformants yang dapat terpencil sesuai dengan teknik konvensional biasanya Mempekerjakan pilihan yang dikehendaki terhadap organisme sebagai unmodified organisme atau

mentransfer organisme, saat ini. The transformants then can be screened for pesticidal activity. Transformants yang kemudian dapat screened untuk pesticidal kegiatan. Suitable host cells, where the pesticide-containing cells will be treated to prolong the activity of the toxin in the cell where the treated cell is applied to the environment of target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. Sesuai host sel, di mana pestisida yang mengandung sel-akan dirawat untuk memperpanjang kegiatan yang toksin dalam sel di mana sel dirawat diterapkan kepada lingkungan sasaran hama (s), mungkin termasuk salah satu prokaryotes atau eukaryotes, yang biasanya terbatas pada sel-sel mereka yang tidak memproduksi bahan beracun untuk organisme yang lebih tinggi, misalnya mamalia. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to limit any possibility of toxicity to a mammalian host. Namun, organisme yang memproduksi bahan beracun untuk organisme yang lebih tinggi dapat digunakan, dimana toksin yang tidak stabil atau tingkat aplikasi cukup rendah untuk setiap batas kemungkinan racun yang berhubung dgn binatang menyusui host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Sebagai tuan rumah, yang tertentu akan menjadi prokaryotes dan eukaryotes rendah, seperti jamur. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Ervinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Ilustrasi prokaryotes, baik Gram-positif dan negatif, termasuk Enterobacteriaceae, seperti Escherichia, Ervinia, Shigella, Salmonella, dan Proteus; Bacillaceae; Rhizobiceae, seperti Rhizobium; Spirillaceae, seperti photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio , Spirillum; Lactobacillaceae; Pseudomonadaceae, seperti Pseudomonas dan Acetobacter; Azotobacteraceae dan Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like. Diantara eukaryotes adalah jamur, seperti Phycomycetes dan Ascomycetes, termasuk ragi, seperti Saccharomyces dan Schizosaccharomyces; Basidiomycetes dan ragi, seperti Rhodotorula, Aureobasidium, Sporobolomyces, dan lain-lain. Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the hirsutellin A polynucleotide into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities.

Karakteristik tertentu dalam memilih host sel untuk keperluan produksi termasuk kemudahan pengenalan hirsutellin J polynucleotide menjadi tuan rumah, ketersediaan sistem ekspresi, ekspresi efisiensi, stabilitas dari pestisida di host, dan adanya kemampuan genetik penolong. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Karakteristik yang menarik untuk digunakan sebagai pestisida microcapsule termasuk kualitas perlindungan untuk pestisida, seperti dinding sel tebal, pewarnaan pigmen, dan intracellular kemasan atau penyertaan pembentukan badan; daun Affinity; kurangnya berhubung dgn binatang menyusui racun; attractiveness ke hama untuk proses menelan; kemudahan membunuh dan pemasangan tanpa kerusakan pada toksin, dan sebagainya. Other considerations include ease of formulation and handling, economics, storage stability, and the like. Pertimbangan lainnya termasuk kemudahan formulasi dan penanganan, ekonomi, stabilitas penyimpanan, dan lain-lain. Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. Host organisme tertentu menarik termasuk ragi, seperti Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., Dan Sporobolomyces sp.; Phylloplane organisme seperti Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., and the like. dan Flavobacterium sp.; atau organisme lain sebagai Escherichia, Lactobacillus sp., Bacillus sp., dan lain-lain. Specific organisms include Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like. Spesifik organisme termasuk Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, dan lain-lain. In general, expression vectors containing promotor sequences which facilitate the efficient transcription of the inserted genetic sequence are used in connection with the host. Secara umum, ekspresi vektor berisi Promotor sequence yang memfasilitasi efisien turunan yang terpasang urutan genetik digunakan dalam kaitannya dengan host. As described above, biologically functional viral or plasmid DNA vectors caable fo expression and replication in a host are known in the art. Seperti dijelaskan di atas, atau virus biologis fungsional DNA plasmid vektor caable untuk berekspresi dan replikasi di host dikenal dalam seni. Such vectors are used to incorporate hirsutellin A encoding DNA sequences of the invention. Vektor seperti itu digunakan untuk menggabungkan hirsutellin J encoding DNA sequences dari ciptaan. Expression vectors typically contain an origin of replication, a promoter, and

a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. Ekspresi vektor biasanya berisi asal jawab, seorang promotor, dan terminator, serta gen khusus yang mampu memberikan pilihan phenotypic dari transformasi sel. Transformation of the host cell with the recombinant DNA may be carried out by conventional techniques well known to those skilled in the art. Transformasi dari sel-host dengan recombinant DNA dapat dilakukan oleh teknik konvensional dikenal dengan yang terampil dalam seni. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by the CaCl 2 method using procedures well known in the art. Dimana host adalah prokaryotic, seperti E. coli, kompeten sel yang mampu DNA uptake dapat disiapkan dari sel eksponen pertumbuhan setelah dipanen dan kemudian dirawat oleh CaCl 2 menggunakan metode prosedur terkenal dalam seni. Alternatively, MgCl 2 or RbCl could be used. Atau, MgCl 2 atau RbCl dapat digunakan. Where the host used is a eukaryote, various methods of DNA transfer can be used. Dimana host yang digunakan adalah eukaryote, berbagai metode transfer DNA dapat digunakan. These include transfection of DNA by calcium phosphate-precipitates, conventional mechanical procedures such as microinjection or electroporation, insertion of a plasmid encased in liposomes, or the use of viral vectors. Termasuk transfection dari DNA oleh kalsium fosfat-precipitates, konvensional mekanis prosedur seperti microinjection atau electroporation, masuknya plasmid yang encased di liposomes, atau penggunaan vektor virus. Eukaryotic host cells may also include yeast. Eukaryotic sel host mungkin juga termasuk ragi. For example, DNA can be expressed in yeast by inserting the DNA into appropriate expression vectors and introducing the product into the host cells. Misalnya, DNA dapat dinyatakan dalam ragi dengan memasukkan ke dalam DNA vektor dan berekspresi sesuai memperkenalkan produk ke dalam sel tuan rumah. Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, J. et al., Nature, 340:205, 1989; Rose, M. et al., Gene, 60:237, 1987). Berbagai shuttle vektor untuk ekspresi gen asing dalam ragi telah dilaporkan (Heinemann, J. dkk., Alam, 340:205, 1989; Rose, M. dkk., Gene, 60:237, 1987). Isolation and purification of microbially expressed protein, or fragments thereof provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies. Isolasi dan pemurnian dari microbially dinyatakan protein, atau potongan-potongan itu diberikan oleh penemuan, dapat dilakukan oleh konvensional berarti termasuk persiapan kromatografi dan imunologi separations melibatkan monoclonal atau

polyclonal antibodies. Antibodies provided in the present invention are immunoreactive with the hirsutellin A polypeptides of the invention. Antibodies yang hadir dalam ciptaan yang immunoreactive dengan hirsutellin J polypeptides dari ciptaan. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Antibodi yang pada dasarnya terdiri dari pooled monoclonal antibodies berbeda dengan epitopic specificities, serta berbeda monoclonal antibodi persiapan disediakan. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989). Monoclonal antibodies antigen yang dibuat dari potongan-potongan yang mengandung protein yang dikenal dengan baik oleh metode dalam seni (Kohler, dkk., Alam, 256:495, 1975; Peristiwa Protokol di Molecular Biology, Ausubel, dkk., Ed., 1989) . Minor modifications of the hirsutellin A primary amino acid sequence may result in polypeptides which have substantially equivalent activity compared to the hirsutellin A polypeptides described herein. Modifikasi kecil dari hirsutellin J utama urutan asam amino dapat menyebabkan polypeptides yang substansial setara aktivitas dibandingkan dengan hirsutellin J polypeptides dijelaskan di sini. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. Modifikasi seperti itu mungkin disengaja, seperti yang diarahkan oleh situs-mutagenesis, atau mungkin spontan. All proteins produced by these modifications are included herein as long as hirsutellin A pest controlling activity is present. Semua protein yang diproduksi oleh modifikasi ini disertakan di sini selama hirsutellin J kegiatan pengendalian hama hadir. Compositions of the invention include those in which an organism is genetically modified to contain polynucleotide encoding hirsutellin A polypeptides. Komposisi dari penemuan termasuk orang-orang yang dalam suatu organisme yang genetically dimodifikasi untuk berisi polynucleotide Encoding hirsutellin J polypeptides. Treatment of the organism, for example, a microbe, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability in protecting the toxin. Perawatan dari organisme, misalnya, mikroba, dapat oleh sarana fisik atau kimia, atau dengan kombinasi kimia dan / atau sarana fisik, asalkan teknik tidak mempengaruhi deleteriously properti dari toksin, atau berkurang selular kemampuan dalam melindungi toksin. Examples of chemical reagents are halogenating agents. Contoh kimia reagents adalah agen halogenating. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Lebih khusus lagi, yodium dapat digunakan di bawah kondisi dan ringan untuk waktu yang cukup untuk mencapai hasil yang diinginkan. Other

suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, WH Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host animal. Lainnya sesuai dengan teknik pengobatan termasuk aldehydes, seperti formaldehida dan glutaraldehyde; anti-infectives, seperti zephiran khlorida dan cetylpyridinium chloride; alcohols, seperti isopropyl dan etanol; berbagai histologic fixatives, seperti yg digambarkan dari Bouin dan Helly dari yg digambarkan (Lihat: Humason, Gretchen L., Animal Tissue Techniques, WH Freeman and Company, 1967), atau kombinasi fisik (panas) dan agen kimia yang melestarikan dan memperpanjang kegiatan toksin yang diproduksi di dalam sel ketika sel diselenggarakan untuk host hewan. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. Contoh fisik yang berarti pendek panjang gelombang radiasi seperti radiasi gamma-X dan radiasi, pembekuan, penyinaran UV, lyophilization, dan lain-lain. The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Sel umumnya akan ditingkatkan stabilitas struktural yang akan meningkatkan perlawanan terhadap kondisi lingkungan. Where the pesticide is in a proform, the method of inactivation should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. Dimana pestisida dalam proform, dengan metode yang inactivation harus dipilih sehingga tidak menghalangi proses yang proform dengan matang bentuk pestisida oleh target hama pathogen. For example, formaldehyde will crosslink protein and could inhibit processing of the proform of a polypeptide pesticide. Misalnya, formaldehida akan crosslink protein dan dapat menghalangi proses yang proform dari polypeptide pestisida. The method of inactivation or killing retains at least a substantial portion of the bio-availability or bioactivity of the toxin. Cara membunuh inactivation atau setidaknya tetap menjadi bagian bioketersediaan atau bioactivity dari toksin. The cellular host containing the hirsutellin A polypeptide insecticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain hirsutellin A gene. Selular host yang berisi hirsutellin J polypeptide gene insecticidal dapat tumbuh di media gizi nyaman, di mana DNA membangun selektif memberikan keuntungan, untuk menyediakan media selektif agar semua substansial atau semua sel tetap hirsutellin J gene. These cells may then be harvested in accordance with conventional ways. Sel-sel ini mungkin akan dipanen sesuai dengan cara

konvensional. Alternatively, the cells can be treated prior to harvesting. Atau, sel dapat diobati sebelum panen. The pest-controlling agent of the present invention may be prepared as dusts, water dispersions, emulsions and solutions. Dengan pengendalian hama-agen yang hadir invensi dapat disiapkan sebagai dusts, air dispersions, emulsions dan solusi. They may comprise accessory agents such as dust carriers, solvents, emulsifiers, wetting and dispersing agents, stickers, deodorants, and masking agents (see, for example, Encyclopedia of Chemical Technology, Vol. 13, pp. 416, et seq.). Mereka terdiri Mei aksesori agen seperti debu operator, larutan, emulsifiers, wetting dan dispersing agen, stiker, deodorants, dan agen masking (lihat, misalnya, Ensiklopedia dari Teknologi Kimia, Vol. 13, hal. 416, et seq.). Dusts generally will contain low concentrations, 0.1-20% of hirsutellin A, although ground preparations may be used and diluted. Dusts umumnya akan konsentrasi rendah, 0,1-20% dari hirsutellin A, walaupun persiapan tanah dapat digunakan dan dicairkan. Carriers commonly include sulfur, silcon oxides, lime gypsum, talc, pyrophylite, bentonite, kaolins, attapulgite, and volcanic ash. Operator umum termasuk belerang, silcon oxides, batu kapur, talek, pyrophylite, bentonite, kaolins, attapulgite, dan abu gunung berapi. Selection of the carrier may be made on the basis of compatibility with the desired pest control composition (including pH, moisture content, and stability), particle size, abrasiveness, absorbability, density, wettability and cost. Pilihan operator yang boleh dibuat berdasarkan kompatibilitas dengan yang diinginkan kontrol hama komposisi (termasuk pH, moisture content, dan stabilitas), ukuran butiran, abrasiveness, absorbability, kepadatan, wettability dan biaya. The agent of the invention, alone or in combination, and eluent is made by a variety of simple operations such as milling, solvent impregnations, fusing, and grinding. Agen dari ciptaan, sendiri atau dalam kombinasi, dan eluent dibuat oleh berbagai operasi sederhana seperti penggilingan, larutan impregnations, fusing, dan digiling. Particle sizes usually range from 0.5-4.0 microns in diameter. Ukuran particle biasanya berkisar antara 0,5-4,0 microns in diameter. Wettable powders may be prepared by blending the hirsutellin A of the invention in high concentrations, usually from 15-80% with a dust carrier such as bentonite which wets and suspends properly in water. Wettable powders mungkin disiapkan oleh campuran yang hirsutellin J dari invensi dalam konsentrasi tinggi, biasanya 15-80% debu dengan operator seperti bentonite yang wets dan suspends baik di dalam air. Twenty-two percent of a surface-active agent is usually added to improve the wetting and suspendability of the powder. Dua puluh dua persen dari permukaan-agen aktif biasanya ditambahkan untuk memperbaiki wetting dan suspendability dari bubuk.

Hirsutellin A may also be used in granules, which are pelleted mixtures of the agents, usually at 2.5-20%, and a dust carrier, eg, adsorptive clay, bentonite or diatomaceous earth, and common within particle sizes of 250-590 microns. J Hirsutellin dapat juga digunakan dalam granules, yang pelleted campuran dari agen, biasanya di 2,5-20%, dan debu operator, misalnya, adsorptive clay, bentonite atau diatomaceous bumi, dan dalam ukuran particle 250-590 microns. Granules may be prepared by impregnations of the carrier with a solution or slurry of the organism and may be used principally for soil applications or for mosquito larvae treatment Granules dapat disiapkan oleh impregnations dari operator dengan solusi atau slurry dari organisme dan dapat digunakan terutama untuk aplikasi atau tanah untuk nyamuk larvae perawatan The hirsutellin A may also be applied in the form of an emulsion, which comprises a solution of hirsutellin A in water-immisicible organic solvents, commonly at 15-50%, with a few percent of surface active agent to promote emulsification, wetting, and spreading. J hirsutellin yang juga dapat diaplikasikan dalam bentuk emulsi, yang terdiri dari solusi hirsutellin Jimmisicible dalam air larutan organik, pada umumnya 15-50%, dengan beberapa persen dari permukaan aktif agen untuk mempromosikan emulsification, wetting, dan penyebarannya. The choice of solvent is predicated upon solubility, safety to plants and animals, volatility, flammability, safety to plants and animals, compatibility, odor and cost. Pilihan larutan adalah kelarutan predicated atas, keamanan untuk tanaman dan hewan, keriangan, flammability, keselamatan untuk tanaman dan hewan, kompatibilitas, bau dan biaya. The hirsutellin A of the present invention may also be applied depending on the properties of the particular pest-controlling compound, the habits of the pest to be controlled, and the site of the application to be made. The hirsutellin J penemuan masa kini juga dapat diterapkan, tergantung pada properti tertentu pengendalian hama-kompleks, kebiasaan dari hama dikontrol, dan situs dari aplikasi yang akan dibuat. It may be applied by spraying, dusting or fumigation. Mungkin diterapkan oleh penyemprotan, pengasapan atau debu. Sprays are a common means of application and generally will involve the use of water as the principal carrier, although volatile oils may also be used, The pest-controlling agents of the invention may be used in dilute sprays or in concentrate sprays, and the amount of carrier to be applied is reduced. Semprot adalah sarana umum dan aplikasi umumnya akan melibatkan penggunaan air sebagai operator sekolah, walaupun minyak volatile dapat juga digunakan, yang mengendalikan hama-agen dari invensi dapat digunakan di dalam atau memperjarang semprot semprot berkonsentrasi, dan jumlah dari operator yang akan diterapkan berkurang. The use of concentrate and ultra low volume sprays will allow the use of atomizing nozzles producing droplets

of 30-80 microns in diameter. Penggunaan berkonsentrasi dan volume semprot ultra rendah akan memungkinkan penggunaan atomizing nozzles memproduksi tetesan dari 30-80 microns in diameter. Aerosols may also be used to apply hirsutellin A. Aerosols are applied by atomizing amounts of liquified gas dispersion or bomb, but can be generated on a larger scale by rotary atomizers or twin-fluid atomizers. Aerosol juga dapat digunakan untuk menerapkan hirsutellin A. aerosol diterapkan oleh atomizing jumlah liquified gas pertebaran atau bom, tapi dapat dihasilkan pada skala yang lebih besar oleh atomizers atau rotary twin-atomizers cairan. Carriers used as aerosols or liquified gas may include mineral spirits, ethanol, isopropanol, deionized water, and hydrocarbon propellants, such as isobutane, n-butane, and propane or nonflammable fluorinated hydrocarbon propellants, or compressed gases, such as nitrogen, carbon dioxide, or nitrous oxide. Operator yang digunakan sebagai aerosol atau liquified gas mungkin termasuk mineral roh, ethanol, isopropanol, deionized air, dan zat air arang propellants, seperti isobutane, n-butana, dan propana atau nonflammable fluorinated zat air arang propellants, dikompresi atau gas, seperti nitrogen, karbon dioksida, atau nitro. The aerosol composition will typically comprise about 0.001-10% hirsutellin A of the invention, and the remainder being aerosol or liquified gas ingredients (The Science and Technology of Aerosol Packaging, John Wiley & Sons, 1974). Erosol komposisi yang biasanya akan terdiri tentang 0,001-10% J hirsutellin dari penemuan, dan sisanya sedang erosol liquified gas atau bahan (The Sains dan Teknologi erosol Packaging, John Wiley & Sons, 1974). The following examples are intended to illustrate but not limit the invention. Contoh berikut dimaksudkan untuk menggambarkan tapi tidak membatasi invensi. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used. Sedangkan mereka adalah orang-orang biasa yang dapat digunakan, prosedur lainnya yang dikenal orang-orang yang terampil dalam seni Mei kalau tidak digunakan. EXAMPLE 1 EXAMPLE 1 PURIFICATION OF HIRSUTELLIN A Pemurnian DARI HIRSUTELLIN J A. CRUDE FILTRATE PRODUCTION A. Crude filtrat PRODUKSI Solid cultures of H. thompsonii var. Solid budaya dari H. thompsonii var. thompsonii (ATCC 24874) were grown on soil fungus medium (SFM) for 5 days in 90 mm petri plates at 28 C. An inoculum was prepared by washing the conidia from the surface of a sporulating aerial mycelial mat of a given number of petri plates with sterile distilled water and then concentrating the conidial suspension by centrifugation. thompsonii (ATCC 24874) yang

tumbuh di tanah jamur menengah (SFM) selama 5 hari dalam 90 mm petri plates pada 28 C. An inoculum telah disiapkan oleh mencuci yang conidia dari permukaan yang sporulating udara mycelial mat suatu jumlah petri piring dengan air steril distilled kemudian berkonsentrasi pada conidial penskorsan oleh centrifugation. Broth culture containing Czapek-Dox medium plus yeast extract at 10 g/l were prepared in Erlenmeyer flasks. Bulyon budaya berisi Czapek-dox media plus yeast extract di 10 g / l telah disiapkan di Erlenmeyer flasks. Each flask was inoculated with a conidial suspension at 410 6 conidia per 35 ml of broth. Setiap botol telah inoculated dengan conidial penskorsan di 4 10 6 conidia per 35 ml kaldu. Flasks were then placed randomly on a gyrator water bath shaker maintained at 25 C. Cultures were agitated at 100 RPM for 10 days. Flasks yang kemudian diletakkan secara acak pada gyrator botol air mandi dipertahankan pada 25 C. Budaya gelisah adalah pada 100 RPM selama 10 hari. After incubation, the mycelial biomass was separated from the supernatant via filtration through cheesecloth and filter paper. Setelah inkubasi, mycelial biomas yang telah dipisahkan dari yg berenang di atas melalui penyaringan melalui penyaring kain katun tipis dan kertas. Each crude broth was then filtered again through a 0.45 m filter to ensure sterility and stored at 4 C. or lyophilized for extended storage. Setiap crude bulyon kemudian disaring lagi melalui 0,45 m filter untuk memastikan kesterilan dan disimpan pada 4 C. diperpanjang atau lyophilized untuk penyimpanan. B. INITIAL PRECIPITATION AND CONCENTRATION B. awal hujan dan konsentrasi The following strategy was developed to separate the different toxic metabolites of Hirsutell thompsonii var. Berikut strategi dikembangkan untuk memisahkan racun metabolites berbeda dari Hirsutell thompsonii var. thompsonii as pure compounds. thompsonii murni sebagai compounds. This purification procedure was developed by initially adding ammonium sulfate to the crude filtrate to precipitate and concentrate proteins, desalting by gel filtration on Sephadex G-25 and applying different steps of ion exchange and exclusion chromatography. Pemurnian prosedur ini dikembangkan oleh awalnya menambahkan ammonium sulfate ke crude filtrat untuk mempercepat dan konsentrat protein, desalting oleh gel penyaringan pada Sephadex G-25 dan menerapkan langkah-langkah yang berbeda dan pertukaran ion kromatografi pengecualian. The different fractions collected were analyzed for their absorption at 280 nm. Berbagai pecahannya telah dikumpulkan dianalisis untuk mereka penyerapan di 280 nm. These fractions were screened for biological activity by injection of 8 l of each solution in larvae (L6) of the experimental host, Galleria mellonella. Ini adalah pecahannya screened untuk aktivitas biologis oleh suntikan dari 8 l setiap solusi larvae (L6) dari percobaan host, Galleria

mellonella. The contents of each fraction were also analyzed by SDS PAGE electrophoresis to monitor the evolution of the purification process. Isi dari setiap pecahan juga dianalisa oleh SDS PAGE electrophoresis untuk memantau perkembangan dari proses pemurnian. The proteins of the crude filtrate were precipitated by addition of ammonium sulfate, at a concentration of 0.76 mg/ml (90% of saturation). The crude protein dari filtrat yang precipitated oleh penambahan ammonium sulfate, pada konsentrasi 0,76 mg / ml (90% dari kejenuhan). After a storage of 15 hr at 4 C., the pellet of precipitated proteins was collected by centrifugation for 1 hr at 8500 rpm, and the supernatant was discarded. Setelah penyimpanan selama 15 jam pada 4 C., peluru yang precipitated protein yang telah dikumpulkan oleh centrifugation selama 1 jam pada 8500 rpm, dan yg berenang di atas telah dibuang. The pellet was dissolved in a small amount of distilled water (1/30 to 1/40 of the volume of filtrate) to obtain an increase of concentration. Peluru yang telah larut dalam jumlah kecil distilled air (1 / 30 sampai 1 / 40 dari volume filtrat) untuk mendapatkan peningkatan konsentrasi. C. GEL FILTRATION ON SEPHADEX G-25 C. GEL penyaringan ON SEPHADEX G-25 In a next step, exclusion liquid chromatography with Sephadex G-25 was performed for desalting the precipitate. Dalam sebuah langkah berikutnya, dengan pengecualian cair kromatografi Sephadex G-25 telah dilakukan untuk desalting yang tergesa-gesa. The concentrated solution prepared above was applied oa 2.525 cm column and eluted with 0.05M Tris-HCl buffer, pH 8. Terkonsentrasi solusi yang telah disiapkan di atas diterapkan oa 2,5 25 cm dan kolom eluted dengan 0.05M Tris-HCl buffer, pH 8. Fractions of 7.5 ml were collected, analyzed for absorption at 280 nm, and tested for biological activity by injection of 0.8 l of each of these solutions to L6 larvae of the lepidopteran insect Galleria mellonella (200 mg of mean body weight). Pecahannya dari 7,5 ml dikumpulkan, dianalisis untuk penyerapan pada 280 nm, dan diuji untuk aktivitas biologis oleh suntikan dari 0,8 l dari masing-masing upaya solusi L6 larvae dari lepidopteran serangga Galleria mellonella (200 mg berarti dari berat badan). Among these fractions (F) only F1, 2 and 3, collected immediately after the void volume and containing the macromolecules excluded from the gel (MW cut-off=5000D), showed an insecticidal effect. Diantara ini pecahannya (M) hanya F1, 2 dan 3, yang dikumpulkan segera setelah void volume dan berisi macromolecules dikecualikan dari gel (MW cut-off = 5000D), menunjukkan sebuah insecticidal efek. A pool of F1, 2 and 3, with a total concentration in proteins of 500 g/ml (Biorad method of dosage) induced a rapid rate of

mortality in 100% of the injected larvae. J renang dari F1, 2 dan 3, dengan total protein dalam konsentrasi 500 g / ml (Biorad metode dosis) dipaksa pesat di tingkat kematian 100% dari menyuntikkan larvae. D. ION EXCHANGE CHROMATOGRAPHY WITH DEAE D. ION EXCHANGE WITH kromatografi DEAE Ion exchange chromatography was performed by DEAE-Trisacryl, a weak anion exchanger. Pertukaran ion kromatografi dilakukan oleh DEAETrisacryl, yang lemah, anion Exchanger. Fractions 1-3 eluted from Sephadex G-25 were filtrated on membranes of a porosity of 0.2, and then applied to DEAE-Trisacryl column (116 cm). Pecahannya 1-3 eluted dari Sephadex G-25 yang filtrated pada membranes dari kerenikan dari 0.2, dan kemudian diterapkan ke kolom DEAE-Trisacryl (1 16 cm). The column was equilibrated with 0.05M Tris-HCl buffer, pH 8. Kolom equilibrated adalah dengan 0.05M Tris-HCl buffer, pH 8. This column was washed with the same buffer, and the bound material was eluted with a 60 ml 0 to 0.3M linear NaCl gradient in the same buffer, at a flow rate of 50 ml/hr. Kolom ini telah dicuci dengan penyangga, dan telah terikat materi eluted dengan 60 ml 0 untuk 0.3M linear NaCl di lereng penyangga yang sama, di alur menilai dari 50 ml / jam. Fractions of 3.5 ml were collected, and the absorbance at 280 nm was recorded. Pecahannya dari 3,5 ml dikumpulkan dan absorbance at 280 nm yang direkam. A first peak, corresponding to non-adsorbed material, was obtained during washing, at the level of fractions F2, 3 and 4. Puncak pertama, berhubungan dengan non-materi adsorbed, telah diperoleh selama mencuci, di tingkat pecahannya F2, 3 dan 4. A pool of these fractions (200 g/ml of proteins) induced (by injection) a rate of mortality of 85-95% in the G. mellonella larvae. J renang ini pecahannya (200 g / ml protein) dipaksa (dengan suntikan) yang tingkat kematian dari 85-95% pada mellonella G. larvae. The intoxicated larvae showed a characteristic aspect; they were swollen and displayed small dispersed melanized spots on their cuticle. Teler larvae yang memperlihatkan karakteristik aspek; mereka bengkak dan muncul bintikbintik kecil melanized cerai pada kulit. At the beginning of the elution of the NaCl gradient a second peak of compounds previously bound to DEAE appeared, but detached quickly. Pada awal elution dari NaCl lerengan kedua puncak compounds sebelumnya terikat DEAE muncul, namun mengelopak cepat. Fraction F15, whose concentration in proteins was 200 g/ml, also caused a rate of lethality of 90%. Pecahan F15, yang dalam konsentrasi protein adalah 200 g / ml, juga menyebabkan tingkat lethality dari 90%. The larvae were not swollen, and the brownish color of the cuticle was more apparent. Larvae yang tidak bengkak, dan coklat warna kulit yang lebih jelas.

E. ION EXCHANGE CHROMATOGRAPHY WITH A CATION EXCHANGER E. ION EXCHANGE kromatografi WITH A CATION Exchanger Fractions 1-3 from DEAE were applied to a carboxymethyl-Trisacryl column (116 cm) equilibrated with a 0.05M sodium acetate buffer, pH 5. Pecahannya 1-3 dari DEAE telah diterapkan ke carboxymethyl-Trisacryl kolom (1 16 cm) equilibrated dengan 0.05M sodium acetate buffer, pH 5. The column was washed with this buffer, and the bound material was eluted in a first step with 0.1M NaCl in the same buffer, and in a second step by elution with a 0.2 to 0.4M NaCl gradient. Kolom ini telah dicuci dengan penyangga, dan telah terikat materi eluted dalam langkah pertama dengan 0.1M NaCl dalam buffer yang sama, dan dalam tahap kedua oleh elution dengan 0.4M NaCl 0,2 ke lereng. During elution of the gradient, a peak was obtained at a NaCl concentration of 0.3M, at the level of fraction 50. Selama elution dari lereng, puncak yang telah diperoleh di NaCl konsentrasi 0.3M, di tingkat pecahan 50. Among the fractions tested for their biological activity, only those corresponding to this peak were toxic, causing a mortality at 100% when injected into Galleria larvae. Di antara pecahan mereka diuji untuk aktivitas biologis, hanya sesuai untuk puncak ini adalah racun, menyebabkan kematian pada 100% bila disuntikkan ke Galleria larvae. Hirsutellin A was therefore present in these fractions. J Hirsutellin itu telah hadir di pecahannya. However, this material was not pure as SDS-PAGE electrophoresis of these solutions revealed two bands after staining of gels with Coomassie brilliant blue. Namun, bahan ini tidak murni sebagai SDS-PAGE electrophoresis solusi ini terungkap setelah dua band staining dari gels dengan brilian Coomassie biru. F. EXCLUSION CHROMATOGRAPHY F. PENGECUALIAN kromatografi The native fractions collected by ion exchange chromatography on carboxymethyl-Trisacryl were dialyzed against distilled water and then freeze-dried. Asli pecahannya dikumpulkan oleh pertukaran ion kromatografi pada carboxymethyl-Trisacryl adalah dialyzed terhadap distilled air dan kemudian beku-kering. The freeze-dried powder was dissolved in 0.05M TrisHCl buffer, pH 8, in conditions to increase the concentration to 40 for exclusion chromatography. Yang beku-kering bubuk dibubarkan di 0.05M Tris-HCl buffer, pH 8, dalam kondisi untuk meningkatkan konsentrasi untuk 40 untuk pengecualian kromatografi. Gel permeation runs were then performed with Biogel P 10, quality medium (Biorad) (fractionation range: 1500-20000 D). Gel peresapan kemudian dilakukan telah berjalan dengan Biogel P 10, kualitas medium (Biorad) (fractionation kisaran: 1500-20000 D). During these experiments, the concentrated solution was applied to a biogel column (1100 cm), and eluted with 0.05M Tris-HCl buffer, pH 8, at a flow rate of 15 ml/hr. Selama percobaan ini, yang dipusatkan solusi yang

diterapkan ke biogel kolom (1 100 cm), dan eluted dengan 0.05M Tris-HCl buffer, pH 8, di tingkat arus 15 ml / jam. Fractions of 1.5 ml were collected. Pecahannya 1,5 ml dikumpulkan. When these samples were analyzed, only one small peak was observed which eluted approximately the same as Cytochrome C (MW=12300). Ketika sampel yang dianalisis, hanya satu puncak kecil yang telah diamati eluted kira-kira sama dengan Cytochrome C (MW = 12.300). This fraction was very active as injection caused 100% mortality in G. mellonella larvae (see EXAMPLE 2B). Pecahan ini sangat aktif sebagai suntikan menyebabkan kematian 100% di G. mellonella larvae (lihat EXAMPLE 2B). SDS-PAGE revealed only one band, even after silver staining (Biorad), and comparison with the MW standards showed that the MW of the components(s) of this band was about 15D. SDS-PAGE diwahyukan hanya satu band, bahkan setelah perak staining (Biorad), dan dibandingkan dengan standar MW menunjukkan bahwa MW dari komponen (s) dari band ini adalah tentang 15D. When isoelectrofocusing was performed with a flat bed apparatus and Servalyt Precotes (Serva) 3-11 it also appeared as only one band. Ketika isoelectrofocusing dilakukan dengan flat tempat tidur dan aparat Servalyt Precotes (Serva) 3-11 juga muncul sebagai satu-satunya band. The pl was 10.5 according to the comparison with markers (Serva). Pl 10,5 yang telah sesuai dengan perbandingan dengan tanda-tanda (Serva). When the same sample was submitted successively to isoelectrofocusing and to SDSPAGE electrophoresis, there was again only one spot on the gel, thereby showing that the hirsutellin A was purified. Apabila sampel yang sama telah disampaikan berturut-turut ke isoelectrofocusing dan SDS-PAGE electrophoresis, ada lagi satu tempat di gel, sehingga menunjukkan bahwa hirsutellin yang telah suci. EXAMPLE 2 EXAMPLE 2 CHARACTERIZATION AND CLONING OF HIRSUTELLIN A Pemeranan Cloning DAN DARI HIRSUTELLIN J Column chromatography, electrophoresis, and isoelectrofocusing studies described above showed that hirsutellin A is a basic macromolecular monomeric protein with a molecular weight of 15 kD, an isoelectric point (pl) of 10.5. Kromatografi kolom, electrophoresis, dan isoelectrofocusing studi di atas menunjukkan bahwa hirsutellin J adalah dasar macromolecular monomeric protein dengan berat molekul 15 kd, sebuah isoelectric point (pl) dari 10,5. The purity of hirsutellin A was confirmed via HPLC and the composition of its amino acids determined. Yang suci dari hirsutellin J telah dikonfirmasi melalui HPLC dan komposisi dari asam amino ditentukan. Determination of the sequence of amino acids of the NH-terminal portion of the hirsutellin A molecule was based on the Phenylthiohydantoine (PTH)

method of Edman, et al. Penentuan urutan asam amino dari dari NH-terminal bagian hirsutellin J molekul berdasarkan Phenylthiohydantoine (PTH) metode Edman, dkk. (Europ. J. Biochem., 1:80, 1967). (Europ. J. Biochem., 1:80, 1967). It was performed with a gaseous phase sequencer connected to a PTH analyzer. Ia dilakukan dengan gas tahap sequencer terhubung ke PTH analyzer. The sequence of the first 34 amino acids of the NH-terminal part of the hirsutellin A molecule obtained by this method has been defined in FIG. Urutan pertama dari 34 asam amino dari NH-terminal bagian dari molekul hirsutellin J diperoleh oleh metode ini telah ditetapkan dalam Fig. 1. 1. Search via a computer data base (CITI2) did not reveal a significant degree of homology between hirsutellin A and other known proteins. Mencari melalui komputer data base (CITI2) tidak signifikan mengungkapkan derajat homology antara hirsutellin A dan protein lainnya dikenal. In addition, research designed to identify a glycosylated fraction in hirsutellin A has proven negative. Selain itu, penelitian yang dirancang untuk mengidentifikasi glycosylated pecahan di hirsutellin J telah membuktikan negatif. EXAMPLE 3 EXAMPLE 3 TOXICITY OF HIRSUTELLIN A Kebisaan DARI HIRSUTELLIN J A. TOXICITY OF HIRSUTELLIN A TO Galleria mellonella LARVAE A. kebisaan DARI HIRSUTELLIN J UNTUK Galleria mellonella LARVAE To test the toxicity of hirsutellin A to larvae (L6) of G. mellonella, three concentrations of toxin, 25, 50 and 100 g/ml, corresponding to final doses of 1,2 and 4 g/g of body weight, respectively, were prepared and 8 l of each treatment injected into the proleg of 20 larvae/replicate. Untuk menguji kebisaan dari hirsutellin A larvae (L6) dari G. mellonella, tiga konsentrasi dari toksin, 25, 50 dan 100 g / ml, sesuai final dari dosis 1,2 dan 4 g / g berat tubuh, masing-masing, telah disiapkan dan 8 l setiap perawatan disuntikkan ke dalam proleg dari 20 larvae / replikasi. A Tris buffer solution injection was performed as a control. J Tris buffer solusi suntikan dilakukan sebagai kontrol. Each treatment was replicated three times. Setiap perawatan direplikasi telah tiga kali. Larval mortality was recorded daily. Larval kematian tercatat harian. All concentrations of hirsutellin A killed 100% of the larvae; however, the LT 50 increased with a decrease in dose. Semua konsentrasi dari hirsutellin J membunuh 100% dari larvae, namun di LT 50 meningkat dengan penurunan dosis. The LT 50 was 8.81.4, 17.11.8 and 24.22.0 at 100, 50 and 25 g/ml, respectively. The LT 50 adalah 8,8 1,4, 17,1 1,8 dan 24,2 2,0 pada 100, 50 dan 25 g / ml, masing-masing. There was no mortality in the control (TABLE 1). Tidak ada kematian di kontrol (TABEL 1). TABLE 1 ______________________________________ MEAN PERCENT MORTALITY TO Galleria mellonella LARVAE (L6) FOLLOWING

INJECTION OF DIFFERENT CONCENTRATIONS OF HIRSUTELLIN A Dosage (g/ Mean % Mortality; Days Post-Treatment ml) 4 5 10 15 20 25 30 LT 50 ______________________________________ 25 0.0 -- 0.0 6.7 40.0 73.3 100.0 24.2 2.0 50 0.0 7.5 35.0 -- 80.0 100.0 -- 17.1 1.8 100 18.0 45.0 63.6 100.0 -- -- -- 8.8 1.4 Con- 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -- trol ______________________________________ TABLE 1 ______________________________________ MEAN PERCENT kematian UNTUK Galleria mellonella LARVAE (L6) Mengikuti INJECTION OF DIFFERENT konsentrasi DARI HIRSUTELLIN J Dosis (g / Mean% Mortality; Hari Pasca-Treatment ml) 4 5 10 15 20 25 30 LT 50 ______________________________________ 25 0,0 - 0,0 6,7 40,0 73,3 100,0 24,2 2,0 50 0,0 7,5 35,0 - 80,0 100,0 - 17,1 1,8 100 18,0 45,0 63,6 100,0 - - - 8,8 1,4 Con-0,0 0,0 0,0 0,0 0,0 0,0 0,0 - trol ______________________________________ B. TOXICITY OF HIRSUTELLIN A TO NEONATAL Aedas aegypti LARVAE B. kebisaan DARI HIRSUTELLIN J UNTUK neonatal Aedas aegypti LARVAE To test the toxicity of hirsutellin A to neonatal mosquito larvae of Aedes aegypti, different concentrations of toxin, 0, 5, 10, 15 and 20 g/ml, in 200 l of water were placed into microtitration plates. Untuk menguji kebisaan dari hirsutellin A neonatal larvae dari nyamuk Aedes aegypti, konsentrasi yang berbeda dari toksin, 0, 5, 10, 15 dan 20 g / ml, dalam 200 l air yang ditempatkan dalam piring microtitration. A control containing only water was included. J kontrol hanya berisi air yang disertakan. Each treatment was replicated three times. Setiap perawatan direplikasi telah tiga kali. Twentyfour vigorous neonatal larvae were placed into each well under continuous exposure. Dua puluh empat cegak neonatal larvae telah ditempatkan ke dalam masing-masing dengan baik di bawah eksposur kontinyu. Larval mortality increased with an increase in toxin dosage. Larval kematian meningkat dengan peningkatan dosis toksin. At 5 and 10 g/ml, larval mortality reached 90% after 48 hr and 100% by 72 hr post-inoculation. Pada 5 dan 10 g / ml, larval kematian mencapai 90% setelah 48 jam dan 100% oleh 72 jam pasca-inokulasi. Virtually no mortality was recorded from the control. Hampir tidak ada kematian tercatat dari kontrol. C. TOXICITY OF HIRSUTELLIN A TO Plutella xylostella CELL LINE C. kebisaan DARI HIRSUTELLIN J UNTUK Plutella xylostella CELL LINE To test the toxicity of hirsutellin A on Plutella xylostella cells produced in tissue culture, different concentrations of toxin (0, 0.5, 1.0, 2.5, 5.0 and 10.0 g/ml) were added via dilution to achieve a given test concentration in 2 ml of tissue culture medium containing 210 5 cells. Untuk menguji kebisaan dari hirsutellin J pada Plutella xylostella sel yang dihasilkan di jaringan

budaya, konsentrasi dari toksin yang berbeda (0, 0.5, 1.0, 2.5, 5,0 dan 10,0 g / ml) telah ditambahkan melalui pengenceran untuk mencapai suatu konsentrasi dalam uji 2 ml jaringan budaya media yang 2 10 5 sel. A control containing cells in tissue culture media only was included. J kontrol yang mengandung sel-sel di jaringan budaya media saja disertakan. Living and dead cells per day were determined by staining with Trypan blue and the number of live cells at 1, 2, 3, 4 and 5 days was recorded (TABLE 2). Hidup dan mati sel per hari yang ditentukan oleh staining dengan Trypan biru dan jumlah tinggal di sel 1, 2, 3, 4 dan 5 hari tercatat (TABEL 2). The growth rate of P. xylostella cells is tissue culture declined with an increase in concentration of hirsutellin A. At 10 g/ml, all cells were killed after 2 days. Tingkat pertumbuhan dari P. xylostella adalah sel jaringan budaya ditolak dengan peningkatan konsentrasi hirsutellin A. Pada 10 g / ml, semua sel dibunuh setelah 2 hari. TABLE 2 ______________________________________ Toxin Conc. TABEL 2 ______________________________________ toksin Conc. No. Live Cells/ml; Days Post-Treatment a (g/ml) 0 1 2 3 4 5 ______________________________________ 0.0 2.0 5.5 6.4 8.6 9.0 12.0 0.5 2.0 4.0 6.9 8.3 9.8 9.1 1.0 2.0 4.7 7.3 7.8 9.4 9.7 2.5 2.0 2.6 3.3 3.1 3.8 3.4 5.0 2.0 2.1 2.2 2.2 2.2 2.5 10.0 2.0 1.7 3.2 0.0 0.0 0.0 ______________________________________ a Live cells counted in hemocytometer after staining with Trypan blu 10 5 Live no. Sel / ml; Hari Perlakuan pasca-a (g / ml) 0 1 2 3 4 5 ______________________________________ 0,0 2,0 5,5 6,4 8,6 9,0 12,0 0,5 2,0 4,0 6,9 8,3 9,8 9,1 1,0 2,0 4,7 7,3 7,8 9,4 9,7 2,5 2,0 2,6 3,3 3,1 3,8 3,4 5,0 2,0 2,1 2,2 2,2 2,2 2,5 10,0 2,0 1,7 3,2 0,0 0,0 0,0 ______________________________________ Live sel yang dihitung dalam hemocytometer setelah staining dengan Trypan blu 10 5 D. TOXICITY OF HIRSUTELLIN A VIA CONTACT AND RESIDUAL EXPOSURE TO THE CITRUS RUST MITE D. DARI HIRSUTELLIN kebisaan J VIA KONTAK DAN sisa Exposure DENGAN Citrus Rust tengu Citrus rust mite (CRM), Phyllocoptruta oleivora, were reared on Sunburst mandarin citrus seedlings kept under plexiglass cages. Tungau karat jeruk (CRM), Phyllocoptruta oleivora, yang pada reared Sunburst jeruk mandarin bibit disimpan di bawah plexiglass cages. Residual+contact (R+C) and residual (R) activity of hirsutellin A was tested at 0, 10, 32, 56 and 100 g of toxin/ml. Sisa kontak + (R + C) dan sisa (R) kegiatan hirsutellin J telah diuji pada 0, 10, 32, 56 dan 100 dari toksin g / ml. For determining R+C activity, citrus leaves were first placed on a wet cotton pad in an open Petri dish. Untuk menentukan aktivitas R + C, daun jeruk pertama yang ditempatkan pada pad kapas basah di buka Petri dish. Tanglefoot was then placed around the perimeter of leaves and 30 adult CRM mites were transferred to each leaf.

Tanglefoot kemudian ditempatkan di sekitar garis daun dan 30 orang dewasa CRM mites telah dipindahkan ke masing-masing daun. About 2 ml of hirsutellin A at five concentrations were sprayed on each of four leaves using Potter tower. Sekitar 2 ml hirsutellin J di lima konsentrasi itu kecipratan pada setiap empat daun menggunakan Potter menara. For determining R activity, leaves were sprayed with different concentrations of hirsutellin A before transferring the mites. Untuk menentukan aktivitas R, daun yang kecipratan dengan konsentrasi yang berbeda dari sebelum hirsutellin J mentransfer mites. Dishes were confined to a closed plastic container and held in a temperature cabinet at 25 C. +1 C. and photoperiod of 15 hr. Piring itu kepada yang tertutup kontainer plastik dan disimpan dalam suhu kabinet pada 25 C. 1 C. dan photoperiod dari 15 jam. Mortality was evaluated 24, 48 and 72 hr after infestation. Kematian telah dievaluasi 24, 48 dan 72 jam setelah kutu. The number of eggs found on each leaf surface was recorded at 72 hr (FIG. 2). Jumlah telur yang ditemukan pada setiap permukaan daun yang direkam pada 72 jam (Fig. 2). Good biological activity against CRM was observed 48 hr after infestation at 100 g/ml. Bagus biologis terhadap kegiatan CRM telah diamati setelah 48 jam kutu pada 100 g / ml. Interestingly, hirsutellin A also showed some effect on mite physiology because, at lower concentrations where low mortality was detected, oviposition was significantly reduced in comparison to the control. Menariknya, hirsutellin J juga menunjukkan beberapa efek pada tungau fisiologi karena konsentrasi rendah di tempat rendah kematian terdeteksi, oviposition telah berkurang secara signifikan dibandingkan dengan kontrol. The higher number of eggs recorded in the R+C bioassay compared to the R bioassay was probably because some eggs were laid on the leaf before the application of the toxin. Semakin tinggi jumlah telur tercatat dalam R + C bioassay dibandingkan dengan R bioassay mungkin karena beberapa telur telah dibebankan pada daun sebelum penerapan toksin. Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Meskipun penemuan tersebut telah dijelaskan dengan merujuk pada saat penawaran penjelmaan, perlu dipahami bahwa berbagai modifikasi dapat dibuat tanpa berangkat dari semangat ini. Accordingly, the invention is limited only by the following claims. Dengan demikian, penemuan itu hanya dibatasi sebagai berikut klaim. ______________________________________________________________ ____________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 2 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Peptide

(B) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AlaProLysValThrSerArgProLysLeuAspGlyArgGluLysPro 151015 PheLysValAspVal 20 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..34 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: A laProLysValThrSerArgProLysLeuAspGlyArgGluLysPro 151015 PheLysValAspValAlaThrAlaGlnAlaGlnAlaArgLysAlaGly 202530 LeuThr ______________________________________________________________ ____________ SEQUENCE LISTING (1) INFORMASI UMUM: (iii) JUMLAH sequence: 2 (2) UNTUK INFORMASI seq ID NO: 1: (i) SEQUENCE KARAKTERISTIK: (A) LENGTH: 21 asam amino (B) TYPE: asam amino ( C) STRANDEDNESS: tunggal (D) topologi: linear (ii) molecule TYPE: peptide (ix) Fitur: (A) NAMA / KEY: Peptide (B) LOKASI: 1 .. 21 (xi) SEQUENCE DESCRIPTION: seq ID NO: 1: AlaProLysValThrSerArgProLysLeuAspGlyArgGluLysPro 151015 PheLysValAspVal 20 (2) UNTUK INFORMASI seq ID NO: 2: (i) SEQUENCE KARAKTERISTIK: (A) LENGTH: 34 asam amino (B) TYPE: asam amino (C) STRANDEDNESS: tunggal (D) topologi: linear (ii) molecule TYPE: peptide (ix) Fitur: (A) NAMA / KEY: Peptide (B) LOKASI: 1 .. 34 (xi) SEQUENCE DESCRIPTION: seq ID NO: 2: J laProLysValThrSerArgProLysLeuAspGlyArgGluLysPro 151015 PheLysValAspValAlaThrAlaGlnAlaGlnAlaArgLysAlaGly 202530 LeuThr ********** Other References Referensi lainnya
Strongman

et al "Hirsutella longicolla new species . . . " J. Invertebrate Pathol 55(1):11-16 1990 Strongman dkk "Hirsutella longicolla spesies baru..." J. hewan tdk bertulang punggung Pathol 55 (1) :11-16 1990 Freifelder "Chromatography", Physical Biochemistry pp. Freifelder "kromatografi", Physical Biochemistry pp. 216-272 198 216-272 198 Add comment Tambahkan komentar

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