Fragment of recombinant dna (options) and recombinant baculovirus vector for expression neuroparalytic toxin (options)

 

(57) Abstract:

The invention relates to biotechnology. The proposed fragments of recombinant DNA, in which neurotoxicology sequence placed under the control of heterologous promoters. The proposed genetically modified baculoviruses. The invention allows the development of agents for biological control of insects that produce toxic to insects levels of the neurotoxin and have increased toxic effects on insects. 6 c.p. f-crystals, 9 tab., 8 Il.

This invention was made partially with funds from the National institutes of health (grant N NS 26109). The U.S. government may have certain rights in this invention.

The present invention relates to methods and compositions for improved biological control of insect pests. More specifically, the present invention relates to the use and manipulation of genes, coding specific to insects paralytic neurotoxins, for the development of improved agents for biological control of insects. The present invention specifically relates to baculoviruses obtained by the methods of the gene is bream, parasitic insects.

Pyemotes tritici, mite-bellied, is one of the thirteen known species of mites of the genus Pyemotes, all of which are predatory and have poisons that cause moderate to extreme toxicity of insect targets. Thirteen known species can be divided into two morphological groups that differ in host range, methods of dispersion and toxicity in relation to their hosts, as well as the effects that have their toxins in insects and humans. Group scolyti and ventricosus are given in table. 1. The majority of the members of the group ventricosus has toxins, highly toxic to insects. Clamp group scolyti are all foretical and usually found on bark; they may produce paralytic toxins.

The life cycle of ticks lasts only 7-14 days, with 100-300 newborns sexually Mature ticks you receive from the mother. When you are a female, she immediately mate and finds a new host. Time of paralysis of the insect host ranges, as I believe, depending on the type, size, stage of development and the number of attackers mites. All growth stages of the insect host can attract mites, but adults usually me who are overcome with mouthparts of ticks.

The actual poisons mites, apparently, are not specific to certain insects, as the poisons are toxic to a wide range of home and nehoosai insect species. Toxin(s) causing irreversible paralysis without destroying respiratory mechanisms (Weiser and Slama (1964), Ann. Ent. Soc. Am. 57:479).

The toxins that are specific to insects that are present in the venom of P. tritici, purified and characterized (Tomalski et al. (1988), Toxicon 26:127-132; Tomalski et al. (1989), Toxicon 27:1151-1167). These toxins are produced by the female mites and inherits in the victim's body as components of the venom that causes paralysis of the insect. Paralysis allows the female mite be fully pregnant, to provide sufficient nutrients for reproduction. Components of the toxin with a low molecular weight cause rapid paralysis-twitch muscles, while the high molecular weight fraction of the toxin causes flaccid paralysis of muscles.

One component of the toxin designated Dr-I, purified to apparent homogeneity; it has an apparent molecular weight of 27000 defined by polyacrylamide gel electrophoresis using sodium dodecyl sulfate. Analysis of amino acid composition thr-I presented in the work Tomalski et al. (1989) supra. Relatively high is the sequence thr-I published and has the following form: N-asp-asn-gly-asn-val-glu-ser-val-arg-ala-val-val-ile-asp-tyr-[X] -asp-ile-arg-his-pro-. Found that the N-terminal amino acid sequence is homologous to any protein sequence (Resources identification of proteins, the release of the National biomedical Foundation, No. 13 dated June 30, 1987).

Patinirovanie two other components, which exhibit a molecular mass equal to 28000 and 29000; these two components are thr-II. Based on peptide mapping and Western blot turns postulated that the protein components thr-I and Dr-II are isoprotein (Tomalski et al. (1989) supra). The mixture thr-I and Dr-II contains TxP-III.

Drugs toxins P. tritici in fact are not toxic to mammals, as evidenced by the check in mice using and intraperitoneal, and intracerebral introduction. Doses that cause paralysis in 50% of the test insects (PD50), for kr-I, Dr-II and thr-III form 330, 550 and 500 µg/kg, respectively, when tested on the larvae of the moth large wax (Galleria mellonella). Thr-I and Dr-II cause rapid mystically paralysis.

The obtained polyclonal antibody using purified thr-I as an antigen. This antibody is reactive against and thr-I, and thr-II, and the antibody will neutralize paralite the protein neurotoxins found in the venom of other arthropods, including Scorpions, wasps and spiders (Zlotkin (1985) Comprehensive Insect Prosiology, Biochemistry and Pharmacology. I. Insects, I. Kervut and L. I. Gilbert (eds.) Pergamon Press, Oxford, U. R., p. 499-546). Several peptide toxins from Scorpions have a specific insects neurotoxic effects, and they are sequenced. These toxins Scorpions have a relatively low molecular weight that is from about 3000 to about 8000 daltons, which is significantly different from the performance of toxins mites. Between toxins ticks and Scorpions, there is no visible relationship in the sequence, however, and toxins mites, and Scorpion toxins have a high content of cysteine. The compact structure of proteins toxins stabilized by disulfide bonds.

Interest in biological control of insect pests has arisen as a result of the inefficiency of traditional chemical pesticides. Chemical pesticides are usually striking and useful and harmful species. Insect pests are becoming resistant to these chemicals, so that new populations of insect pests can get rapid development with acquired resistance to these pesticides. In addition, chemical residues pose a threat to the environment and vozmojnoe pests, which can reduce dependence on chemical pesticides.

Strategies of biological control include the involvement of naturally occurring microorganisms that are pathogenic to insects (entomopathogens), and the development of crops that are more resistant to insect pests. Approaches include the identification and characterization of genes or gene products insects, which can serve as suitable targets for agents for combating insects, identification and use of previously not found the use of microorganisms (including modification of naturally occurring non-pathogenic microorganisms in order to give them the pathogenic nature in regards to insects, modification and processing of the currently used entomopathogens and development on the genetic level crops that exhibit higher resistance to insect pests.

Among entomopathogens developed as biological pesticides, are viruses that cause natural epizootic diseases. Entomopathogenic viruses include baculoviruses, entomopoxvirus, reovirus (viruses citpl is (still classified) and probably some retroviruses.

Baculoviruses are a large group of evolutionarily related viruses that infect only arthropods (Miller, L. K. (1981) Genetic Engineering in the Plant Sciences N. Panopoulous (ed.), Praeger Publ. , New York, p. 203-224; Carstens (1980) Trends in Biochemical Science 52: 107-110; ish and Payne (1979) in Advances in Virus Research, vol. 25, Lawfer et al. (eds.), Academic Press, New York, p. 273-355, Granados, R. R. and Federici, B. A. eds. (1986) The Biology of Baciloviruses, vol. 1, Biological Properties and Molecular Biology, CRC Press Inc., Boca Raton, Florida). Some baculoviruses infect only insects that are pests of commercially important agricultural and forest crops. Known to other baculoviruses that infect other insect pests, such as mosquitoes and fleas. These baculoviruses potentially valuable as agents for biological control. A potential advantage of baculoviruses as biological pesticides is their specificity to the owners. Baculoviruses as a group infect only arthropods, and some strains of baculoviruses normally infect only one or a few insect species. Therefore, they have little or no adverse effect on humans or the environment and can be used without any the rose (NPV), viruses granulata (GV) and "neutropenia" baculoviruses. In the 'embedded' forms of baculovirus virions (nucleocapsid in the shell) immersed in a crystalline protein matrix. This structure, referred to as the body on or off, is a form that is detectable outside the body in nature and responsible for the spread of infection among organisms. Hallmark group NPV is that many virions are immersed in every body off, which are relatively large (up to 5 microns). Body off group GV is smaller and each contain a single virion. Crystalline protein matrix bodies off of both forms is mainly composed of a single polypeptide with molecular mass 25000-38000 daltons, which is known as poliakin or granulin. Neutropenia baculoviruses do not produce protein polyhedrin and do not form a body off (Groner et al. in The Biology of Baculoviruses, vol. 1, supra, and this work is given as a reference, in Chapter 9, table. 2 and 7 give an extensive list of owners of NPV and GV owners as an example).

In nature, the infection begins when the insect ingests food contaminated baculovirus particles, usually in the form of bodies off. Body off dissociable cells, oblative intestine. Within the host cell baculovirus migrates to the nucleus, where replication occurs. Initially, specific viral proteins are produced within the infected cell by transcription and translation of the so-called "early genes". Among other functions, these proteins are required for replication of viral DNA, which begins in 4-6 h after the virus has entered the cell. The viral DNA replication continues for approximately 24 h after infection (PZ). Approximately 8-24 h PZ infecting cells Express a "late genes" at high levels. They include components of the nucleocapsid, which surrounds the viral DNA during the formation of progeny virus particles. Production of progeny virus particles begins approximately 12 h PZ. First, the descendant of the virus migrates to the cell membrane, where it acquires the shell as soon as he otokoyama from the cell surface. Neutropenia viral particles can then infect other cells within the insect. The polyhedrin synthesis begins after approximately 18 h after infection and reaches very high levels for 24-48 h PZ. Approximately 24 h PZ, there is a decrease in the rate of production neutropenia virus, Algaida until death or lysis of the cells. Some baculoviruses infect virtually every tissue in the insect host so that by the end of the process of infection insect fully diluted, liberating extremely large number of bodies off, which can then spread the infection to other insects (The Biology of Baculoviruses, vol. I and II, Granados and Federici (eds.), CRC Press, Boca Raton, Florida, 1986).

Baculoviruses derived from AcMNPV, which are suitable as expressing vectors described in application for U.S. patent N 07/353847, registered on may 17, 1989; international patent application PCT/US 90/02814, registered on may 17, 1990; Rankin et al. (1988) Gene 70: 39-49; Ooi et al. (1989), J. Mol. Biol. 210:221-736, Thiem and Miller (1990), Gene 91:87-95, all of these works are given as references. Extremely strong late and very late promoters described in the literature, and they include a modified polyhedrin promoter LSXIV, hybrid Cap/Polh promoter and a synthetic promoter Syn.

Described baculoviruses, which are improved insecticidal properties. For example, AcMNPV, in which the egt gene (Addison-glucosyltransferase) inactivated, causing early termination of power and the earlier death of the larvae compared with larvae infected with AcMNPV the bathroom on June 29, 1989).

Egt AcMBPV, also genetically modified to expression of the protein, striking a moult, can lead to further improvements insecticidal properties (international patent application PCT/US 90/03758 registered 29 June 1990, entered into this description by reference). Derivatives AcMNPV Egt, which Express the juvenile hormone esterase, hormone hatching or prothoracicotropic hormone, also constructed in this area knowledge. Feeding infected larvae reduced, and the death occurs earlier than in larvae infected with AcMNPV wild-type or AcMNPV egt.

Maeda (1989) Biochem. Biophys. Res. Commun. 165:1177-1183 also described obtained by the methods of recombinant DNA baculovirus with improved pesticidal properties. BmNPV that infects the mulberry silkworm Bombyx mori, modified with the purpose of expression of a synthetic gene encoding diuretic hormone hawk moth Manduca sexta. Fluid balance infected insects was broken, and the killing was approximately 20% faster than when using the wild-type virus.

Dee and co-authors (1990), Bio/Technology 8:339-342 cloned and expressed insecticidal toxin from the Scorpion Androctonus australis in fibroblast cells of the mouse. Coin-2, and the synthesis was controlled by the promoter sequences in the long terminal repeat of the virus murine sarcoma of Malone. Recombinant protein, which was secretarian in the extracellular environment, as noted, is toxic to mosquito larvae, but not to murine cells in culture or mice.

The gene encoding an insect toxin from Buthus eupeus (Central Asian subspecies of Scorpions), synthesized, and cloned into the genome of AcMNPV (nuclear polyhedrosis virus from Autographa californica) and expressed under the control of the polyhedrin promoter. Also carried out the design, in which the Scorpion toxin expressed from a synthetic gene, including the toxin-coding sequence, fused to the sequence encoding the signal peptide, or peptidoglicanova protein with 58 amino acids polyhedrin at N-end. In all cases, there is a certain expression, as indicated [35S] -methionine-radioactive torment, gel-electrophoresis in polyacrylamide using sodium dodecyl sulfate and autoradiography, however, not observed any paralytic activity against insects by observing the products of expression. I believe that this is partly due to nestabil the sensitivity in the system analysis or due to inability of the recombinant protein to form a functional three-dimensional structure (Carbonell et al. (1988), Gene 73:409-418).

Hammock et al. (1990) Nature 344:458-461 describe baculovirus-mediated expression in insect gene encoding the juvenile hormone esterase (JHE), the enzyme that inactivates evolutionary hormone.

Merryweather et al. (1990), J. Gen. Virology 71:1535-1544 lead the construction of baculovirus containing the Delta-endotoxin HD-73 subspecies kurstaki species of Bacillus thuringiensis. Gene endotoxin HD-73 BTk was placed under the control of poliakin-promoter.

The aim of the present invention to provide genes encoding neurotoxins that cause paralysis in insects, for example, from mites, parasitic insects, such as belong to the genus Pyemotes, in particular those that belong to the group ventricoccus of the genus Pyemotes. In a specific embodiment, the gene neurotoxins, agents against insects, is a gene Toh Pyemotes tritici, which is identified by the nucleotide sequence shown in the table. 2; the second specific option paralytic to insects of the neurotoxin and the gene encoding it, shown in the nucleotide and amino acid sequences Taha also kind of Pyemotes tritici, which are listed in the table. 4. It should be understood that it can be isolated and identified by homology nucleation in experiments on hybridization (see, for example, Hanes and Higgins (1985), Nucleic Acid Hybridization, IRL Press, Washington, D. C.), using for this purpose the information here about the sequence.

Genes specific to insects paralytic neurotoxins, having at least about 70 nukleinovokisly homology relative to the coding Toh or Toha sequences can be easily isolated using well-known hybridization assays or screening. Such techniques are particularly suitable for the identification of such genes neurotoxins from mites, parasitic insects, and are most suitable for selection of mites of the genus Pyemotes. Functional equivalents of the specific insect paralytic neurotoxins in accordance with the present invention, for example Toh and Taha, include proteins having the biological activity Toh and/or Toga, and are basically similar in structure, that is, amino acid sequence, with Toh and/or Toga, as indicated in the table. 2 and 4, respectively.

In accordance with this present invention includes specific to insects paralytic neurotoxin that has at least 70% sequence identical to the amino acid sequence in the table. 4.

Neurotoxins, basically similar Toh and Taha include those that are at least about 70% identical in amino acid sequence Toh and/or Toga. Basically similar neurotoxins also include those that have at least about 70% amino acid sequence similar to the sequence Toh or Toha that allows substitution of conservative amino acids, amino acids Toh and Taha. The person skilled in the art will understand that the function of the protein may not be affected as a result of minor structural modifications, especially if these structural modifications are substitutions of amino acids, which is similar in chemical and physical properties. Structural modifications including amino acid deletions and insertions may be permissible if they do not affect functionality.

Genes encoding neurotoxins, which is functionally equivalent Toh and/or Toga, can be isolated and identified any of the presented information about the sequences. For example, amino acid homology and/or nucleotidebinding homology measured by the methods of hybridization, it is possible to connect with the methods described in this work regarding the assessment of neurotoxicity for insects, with the aim of identifying functional neurotoxins for insects. Methods PCR (polymerase-cableway reaction), for example, in combination with other well-known techniques described in this paper can be used for isolation of genes encoding the neurotoxins that are functionally equivalent to those provided in the present invention. The information presented herein, in combination with the known methodology relating to the synthesis of proteins and DNA, conservation properties between amino acids and the use of codons that enables a person skilled in the art can easily construct and synthesize neurotoxins insect genes for insect neurotoxins, which is functionally equivalent Toh and Toga.

Accordingly, the invention includes a recombinant DNA molecule containing the gene encoding for specific insects paralytic neurotoxin.

The present invention also includes a recombinant DNA molecule that contains nificence to insects paralytic neurotoxin has at least about 70% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 2.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and the specified coded specific to insects paralytic neurotoxin has at least about 83% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 2.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and the specified coded specific to insects paralytic neurotoxin has at least about 88% amino acid sequence similarity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 2.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and the specified gene includes the nucleotide sequence encoding a specific paralytic to insects, neurotoxi which incorporates both the recombinant DNA molecule, containing the gene encoding specific to insects paralytic neurotoxin, and the specified gene has at least about 70% homology to the nucleotide sequence with a nucleotide sequence that encodes a specific insects paralytic neurotoxin, as shown in the table. 2.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and the specified coded specific to insects paralytic neurotoxin contains the amino acid sequence shown in table. 2, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and indicated specific to insects paralytic neurotoxin has at least about 70% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 4.

The present izobreteniyami neurotoxin, and specified specific to insects paralytic neurotoxin has at least about 83% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 4.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and indicated specific to insects paralytic neurotoxin has at least about 88% amino acid sequence similarity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 4.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and the specified gene has at least about 70% homology to the nucleotide sequence with a nucleotide sequence that encodes a specific insects paralytic neurotoxin, as shown in the table. 4.

The present invention also includes a recombinant DNA molecule containing the gene encoding specificity is eticheski neurotoxin mite of the genus Pyemotes.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and the specified gene encodes specific to insects paralytic neurotoxin mite of the genus Pyemotes, with this mite belongs to the species of Pyemotes tritici.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and the specified gene has a nucleotide sequence encoding specific to insects paralytic neurotoxin, as shown in the table. 4.

The present invention also includes a recombinant DNA molecule containing the gene encoding specific to insects paralytic neurotoxin, and the specified gene encodes specific to insects paralytic neurotoxin having the amino acid sequence shown in table. 4.

Another objective of the present invention is a receiving agent for combating insects, expressing the gene specific to insects paralytic neurotoxin such as baculovirus, such as AcMNPV constructed by methods of recombinant DNA to target the specific customers to insect neurotoxin gene is placed under the regulatory control of appropriate regulatory sequences of the gene, such as the promoter, in order to get the amount of neurotoxin, effective to create toxic effects such as paralysis, in the sought insect. Specific variants of genetically modified AcMNPV include VETL-Toh, vCap/Polh-Tox34, vEV-Toh and vSp-Toh, where gene Toh Express under the control of the early promoter, a strong of late and/or very late promoter; and especially preferred variants of genetically modified disseminated AcMNPV include vSp-Tox34, representing disseminated the virus, and vCap/Polh-Tox34, which is nevirapine, but provides improved combat insects earlier than other examples. The specialist will understand how to construct similar to the disseminated form of the virus. The specialist will also understand that the virus can be disseminated through co-infection of cells by viral helper, which carries the polyhedrin gene function.

The person skilled in the art also will understand how to construct recombinant viruses in which the toxin gene insertion in other provisions of the AcMNPV genome. Such viruses would have a toxin gene, synthesized with an appropriate promoter, inserzioni in a nonessential region of the AcMNPV genome. Insignificant is al. (1990), J. Gen. Virology, 71:1029-1037), the area of ETL (Grawford and Miller (1988) J. Virology, 62:2773-2781), region egt (O'reilly and Miller (1990), J. Virology 64:1321-1328), the region of the open reading frame (orf) (Gearing and Possee (1990) J. Gen. Virology 71:251-262), the orf region p94 (Friesen and Miller (1987), J. Virology 61:2264-2272) or other areas that the expert can easily determine. Because there is considerable homology among these various genes of baculoviruses, the specialist will also understand how to incarcerate toxin gene, synthesized with an appropriate promoter, in the genomes of other baculoviruses in a similar insignificant places.

Therefore, the present invention includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified gene encodes specific to insects paralytic neurotoxin mite of the genus Pyemotes.

The present invention also includes an agent bio is Yong, encoding specific to insects paralytic neurotoxin, with the specified gene encodes specific to insects paralytic neurotoxin mite of the genus Pyemotes, and this mite belongs to the species of Pyemotes tritici.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified specific to insects paralytic neurotoxin has at least about 70% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 2, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified specific to insects paralytic neurotoxin has at least about 83% Ident paralytic neurotoxin, are given in table. 2, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified specific to insects paralytic neurotoxin has at least about 88% amino acid sequence similarity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 2, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified gene has at least about 70% homology to the nucleotide sequence with a nucleotide sequence that encodes a specific paralytic to insects nanotexture includes an agent for biological control of insects, which is genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified gene contains a nucleotide sequence specific to insects paralytic neurotoxin, are given in table. 2, from about 118 to nucleotide 873 nucleotides.

The present invention also includes an agent for biological control of insects, which is about genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified coded specific to insects paralytic neurotoxin contains the amino acid sequence shown in table. 2, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus.

Now, however, to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus, and it is derived baculovirus is a baculovirus NPV.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus, and it is derived baculovirus is a baculovirus NPV, which in turn is derived from AcMNPV.

The present invention also includes a biological agent is, encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus, and it is derived baculovirus is a baculovirus NPV, which in turn is derived from AcMNPV, the latter expresses a gene specific to insects paralytic neurotoxin under the regulatory control of the promoter, acting very late during infection.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus, and it is derived baculovirus is a baculovirus NPV, which in turn is derived from AcMNPV, the latter expresses a gene specific to insects paralytic neurotoxin under the regulatory control of the promoter, acting very late during infection, and AcMNPV is a vEV-Toh.

The present ISM, to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus, and it is derived baculovirus is a baculovirus NPV, which in turn is derived from AcMNPV, the latter expresses a gene specific to insects paralytic neurotoxin under the regulatory control of the promoter, expressed earlier during viral infection.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus, and it is derived baculovirus is a baculovirus NPV, which in turn is derived from AcMNPV, the latter expresses a gene specific to insects paralytic neurotoxin under the regulatory control of the promoter, expressed earlier in viral infection, ukaoi insect, which is genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus, and it is derived baculovirus is a baculovirus NPV, which in turn is derived from AcMNPV, with the specified specific to insects paralytic neurotoxin is expressed under regulatory control of a synthetic promoter.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus, and it is derived baculovirus is a baculovirus NPV, which in turn is derived from AcMNPV, with the specified specific to insects paralytic neurotoxin is expressed under regulatory control of a synthetic promoter, and AcMNPV is a vS-Toh.

On the Rowan with the to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insects is a virus insect-derived baculovirus, and it is derived baculovirus is a baculovirus NPV, which in turn is derived from AcMNPV, with the specified specific to insects paralytic neurotoxin is expressed under regulatory control of a hybrid promoter.

The present invention also includes an agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified agent against insect is an insect virus, which is derived from baculovirus, and it is derived baculovirus is a baculovirus NPV, which in turn is derived from AcMNPV, with the specified specific to insects paralytic neurotoxin is expressed under regulatory control of the hybrid promoter and agent for combating insects is a vCap/Polh-Tox34.

The biological agent be specific to insects paralytic neurotoxin, while specified coded, specific to insects paralytic neurotoxin has at least about 70% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 4, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

Agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified coded, specific to insects paralytic neurotoxin has at least about 83% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 4, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

Agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotol. 4, has at least about 88% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

Agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified gene contains a nucleotide sequence specific to insects paralytic neurotoxin, are given in table. 4, from about 118 to nucleotide 873 nucleotides.

Agent for biological control of insects, which are genetically modified to contain and Express a gene encoding specific to insects paralytic neurotoxin, with the specified gene has at least about 70% homology to the nucleotide sequence with a nucleotide sequence specific to insects paralytic neurotoxin, as shown in the table. 4.

Agent for biological control of insects that genetically modificarea the Shin, while specified specific to insects paralytic neurotoxin has the amino acid sequence shown in table. 4.

Another objective of the present invention is to obtain toxic to insects compositions containing toxic to insects the amount of virus insects such as baculovirus, genetically constructed for the expression of specific insects paralytic neurotoxin at a level that results in a toxic effect in relation to the desired insect, and a carrier acceptable for use in agricultural or other environmental conditions. Such compositions can be used to protect plants from insect pests. Preferred agents for combating insects are those that Express a gene specific to the insect paralytic neurotoxin on for parasitic insect, mite and especially mites of the genus Pyemotes. If the insect virus is a baculovirus group GV or NPV, it is preferable that viral particles were present in the occluded form.

Accordingly, the present invention includes toxic to insects composition comprising live in the omogo, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin has at least about 70% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 2, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on askasci to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin has at least about 83% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 2, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin has at least about 88% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 2, from aspartate encoded in approximately 120 nucleotides to cysteine,insect composition, contains the number of agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin has at least about 70% homology to the nucleotide sequence with a nucleotide sequence specific to insects paralytic neurotoxin from about nucleotide 118 to nucleotide 873, as shown in the table. 2.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified gene contains a nucleotide sequence specific to insects paralytic neurotoxin that are listed in the what know for insect composition, contains the number of agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin comprises the amino acid sequence shown in table. 2, from aspartate encoded in approximately 120 nucleotides to cysteine encoded in approximately 873 nucleotides.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin has at least about 70% amino acid sequence identity with the amino acid sequence specification is the train includes toxic to insects composition, contains the number of agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin has at least about 83% amino acid sequence identity with the amino acid sequence specific to insects paralytic neurotoxin, are given in table. 4.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin has at least about 88% amino acid sequence identity with amino acid posledovati the invention also includes toxic to insects composition, contains the number of agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin comprises the nucleotide sequence shown in the table. 4.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified specific to insects paralytic neurotoxin comprises the amino acid sequence shown in table. 4.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain toxiciy gene, coding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified agent against insect is an insect virus.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified agent against insect is an insect virus, representing the derived baculovirus.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified agent how much it is derived baculovirus is derived AcMNPV.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified agent against insect is an insect virus, representing the derived baculovirus, and it is derived baculovirus is derived AcMNPV and the specified AcMNPV expresses this gene is specific to insects paralytic neurotoxin under the regulatory control of a promoter active very late during infection.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier,otvagnoe baculovirus, and it is derived baculovirus is derived AcMNPV and the specified AcMNPV expresses this gene is specific to insects paralytic neurotoxin under the regulatory control of a promoter active very late during infection, and the specified AcMNPV is a vEV-Toh.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified agent against insect is an insect virus, representing the derived baculovirus, and it is derived baculovirus is derived AcMNPV and the specified AcMNPV expresses this gene is specific to insects paralytic neurotoxin under the regulatory control of the promoter, expressed earlier in viral infection.

The present invention also includes toxic to insects composition comprising live in the omogo, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified agent against insect is an insect virus, representing the derived baculovirus, and it is derived baculovirus is derived AcMNPV and the specified AcMNPV, expresses this gene is specific to insects paralytic neurotoxin under the regulatory control of the promoter, expressed earlier in viral infection, and specified AcMNPV is a vETL-Toh.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified agent against insect is an insect virus, representing the derived baculovirus, and it is derived paralytic neurotoxin under the regulatory control of the hybrid promoter.

The present invention also includes toxic to insects composition comprising an amount of an agent for combating insects, effective to obtain a toxic effect on the desired insect, this agent is genetically modified to Express the gene encoding specific to insects paralytic neurotoxin, and containing an agricultural acceptable carrier, with the specified agent against insect is an insect virus, representing the derived baculovirus, and it is derived baculovirus is derived AcMNPV and the specified AcMNPV expresses this gene is specific to insects paralytic neurotoxin under the regulatory control of the hybrid promoter, moreover, the specified AcMNPV is one of the vS-Tox4 and vCap/Polh-Toh.

Another objective of the present invention is to provide a method of biological control of insect-pest, which use toxic to insects composition containing toxic to insects, a number of agent for combating insects, genetically engineered for the expression of a gene specific to insects paralytic neurotoxin of parasitic d is rojiani or square, the plant or the environment that must be protected from insect-pest. The number of the specified agent against insects in a given composition and the level of expression of a specified gene neurotoxin using the agent for combating insects are such that the composition produces a toxic effect in relation to claims insects. Preferred agents for combating insects are insect viruses, including baculoviruses, especially disseminated viruses, such as NPVs and GVs, more specifically AcMNPV and its derivatives and close relatives. Occluded (disseminated) form of genetically modified nuclear polyhedrosis viruses will be most suitable in the present invention. The person skilled in the art understands that a genetically engineered virus expressing the toxin for insects, by itself, capable of occlusion, or that the occlusion can be achieved by other means, for example by co-infection with positive to occlusion virus.

In accordance with this present invention includes a method of combating insect pests, which cause toxic to insects amount equal to 16 and 42, habitat data insect in the Yat toxic to insects number, equal to 16 and 42, habitat data, insects, and habitat data insects is a plant.

The present invention includes a method of combating insect pests, which use bait containing toxic to insects amount equal to 16 and 42.

Similarly, the aim of the present invention to provide agents for combating insects, genetically modified day, gene expression, specific to insects paralytic neurotoxin, and these agents are effective against insect pests other than those that attack plants or are harmful to them. This agent can be introduced into toxic to insects, paralytic to insects or insecticidal compositions with acceptable in terms of environmental conditions media, and can be used in the method of dealing with in addition to insect pest susceptible to a particular type of agent used to combat insects.

In addition to the use in insecticidal compositions, which protects plants, agents for combating insects in accordance with the present invention can be used in the control of other insect pest is omogo paralytic neurotoxin, suitable for the searched pest. For example, there are the baculoviruses, which specifically infect and mosquitoes and fleas (see. Beard et al. (1989), J. Invertebrate Path. 54: 128-131 and Federici (1980), Virology 100:1-9). As with insect pests that attack plants, claim insect gives an opportunity to choose the agent for combating insects, used for the expression of paralytic toxin. The expert knows how to select the appropriate regulatory and/or promoter sequence for use with the agent against insects.

Another objective of the present invention is to provide a method of obtaining specific to insects paralytic neurotoxin in the cell in which it is not expressed in natural conditions. This method provides for the construction of recombinant DNA molecules, in which the coding sequence specific to insect neurotoxin is under the control of a regulatory sequence that provides the expression of the coding sequence in a selected host cell, the introduction of recombinant DNA molecules in a suitable cell host and culturing the obtained recombinant host cell anatoxin-encoding sequence incarcerous, for example, below a promoter expressed in the cell host or infected cell host. A DNA molecule containing expressed sequence neurotoxin, can be introductionat in a cage-the owner with the use of vector sequences that facilitate its introduction. Culturing the host cell may include the cultivation of single cells in liquid media, tissue culture cells or proliferation of cells using genetic engineering for the neurotoxin gene expression in multicellular organisms, including higher organisms, such as insects. In General, any method known in the art for the introduction of DNA into the cell-the owner, may be applied in accordance with the present invention. Know how to select cells-owners, sequencing, plasmid or viral vectors, promoters and genes of neurotoxins that are suitable for such receipt. Specific to insect neurotoxins obtained in such genetically modified cell cultures hosts when they are properly delivered to the desired insect, are toxic for this insect. Can be used neurotoxins obtained in these cultures.

With regard to the neurotoxin in the cell-master, where:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner, promoter, which functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, which is expressed in this cell the owner and specified promoter and specified the coding sequence are in the molecule within the specified vector so that a neurotoxin expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) culturing a genetically modified host to Express the specified encoding sequence and get specific to insects paralytic neurotoxin.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) constructing a recombinant DNA molecule that includes a vector part, able to intsoy sequence for specific insects paralytic neurotoxin, which is expressed in this cell the owner and specified promoter and specified the coding sequence are in the molecule within the specified vector so that a neurotoxin expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) culturing a genetically modified host to Express the specified encoding sequence and get specific to insects paralytic neurotoxin with subsequent recovery and purification of the specified neurotoxin after the implementation stage of cultivation.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner and the vector part is obtained from a virus insect, a promoter that functions in this cell the owner and the coding sequence for the special is asany promoter and specified the coding sequence are in the molecule within the specified vector parts, to neurotoxins expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) culturing a genetically modified host to Express the specified encoding sequence and get specific to insects paralytic neurotoxin.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner and the vector part is obtained from a virus insect, which is a baculovirus, a promoter that functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, which is expressed in this cell the owner and specified promoter and specified the coding sequence are in the molecule within the specified vector so that neurotoxin molecule of recombinant DNA in this cell the owner with the in order to produce a genetically modified cell is the master, and

(C) culturing a genetically modified host to Express the specified encoding sequence and get specific to insects paralytic neurotoxin.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner and the vector part is obtained from a virus insect, which is a baculovirus, and the baculovirus is a nuclear polyhedrosis virus, a promoter that functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, which is expressed in this cell the owner and specified promoter and specified the coding sequence are in the molecule within the specified vector parts, to neurotoxins expressively running the specified promoter in this cell the owner,

(b) being introduced ocasey cell-master, and

(C) culturing a genetically modified host to Express the specified encoding sequence and get specific to insects paralytic neurotoxin.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner and the vector part is obtained from a virus insect, which is a baculovirus, and the baculovirus is a nuclear polyhedrosis virus, which in turn is AcMNPV, a promoter that functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, which is expressed in this cell the owner and specified promoter and specified the coding sequence are in the molecule within the specified vector parts, to neurotoxins expressively running the specified promoter in this cell-host;

(b) being introduced the indicated recombinant molecule will tivirus genetically modified host so to Express the specified encoding sequence and get specific to insects paralytic neurotoxin.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner, promoter, which functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, and specified the coding sequence for specific insects paralytic neurotoxin obtained from predatory insect mite, which is expressed in this cell the owner, with the indicated promoter and specified the coding sequence are in the molecule within the specified vector parts, to neurotoxins expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) culturing the returns specific to insects paralytic neurotoxin.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) design a molecule rekombinantnoi DNA, which includes the vector part, capable of introduction and replication in this cell the owner, promoter, which functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, and specific to insects paralytic neurotoxin has at least about 70% amino acid identity to the amino acid sequences given in table. 2, from aspartate encoded in approximately 118 nucleotide to cysteine encoded in approximately 873 nucleotides, which is expressed in this cell the owner, with the indicated promoter and specified the coding sequence are in the molecule within the specified vector so that a neurotoxin expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) collect and get specific to insects paralytic neurotoxin.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner, promoter, which functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, and specific to insects paralytic neurotoxin has at least about 83% amino acid identity to the amino acid sequences given in table. 2, from aspartate encoded in approximately 118 nucleotide to cysteine encoded in approximately 873 nucleotides, which is expressed in this cell the owner, with the indicated promoter and specified the coding sequence are in the molecule within the specified vector so that a neurotoxin expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) cultist and get specific to insects paralytic neurotoxin.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner, promoter, which functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, and specific to insects paralytic neurotoxin has at least about 88% amino acid identity to the amino acid sequences given in table. 2, from aspartate encoded in approximately 118 nucleotide to cysteine encoded in approximately 873 nucleotides, which is expressed in this cell the owner, with the indicated promoter and specified the coding sequence are in the molecule within the specified vector so that a neurotoxin expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) cultist and get specific to insects paralytic neurotoxin.

The present invention also includes a method of obtaining specific to insects paralytic neurotoxin in the cell host, in which:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner, promoter, which functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, and specific to insects paralytic neurotoxin has at least about 70% amino acid identity to the amino acid sequences given in table. 4, which is expressed in this cell the owner, with the indicated promoter and specified the coding sequence are in the molecule within the specified vector so that a neurotoxin expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) culturing a genetically modified host to Express the specified encoding posledovatelnostei way of getting specific to insects paralytic neurotoxin in the cell-master, where:

(a) constructing a recombinant DNA molecule that includes a vector part, capable of introduction and replication in this cell the owner, promoter, which functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, and specific to insects paralytic neurotoxin has at least about 83% amino acid identity to the amino acid sequences given in table. 4, which is expressed in this cell the owner, with the indicated promoter and specified the coding sequence are in the molecule within the specified vector so that a neurotoxin expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) culturing a genetically modified host to Express the specified encoding sequence and get specific to insects paralytic neurotoxin.

The present invention also includes a method polucheniya recombinant DNA, which includes a vector part, capable of introduction and replication in this cell the owner, promoter, which functions in this cell the owner and the coding sequence for specific insects paralytic neurotoxin, and specific to insects paralytic neurotoxin has at least about 88% amino acid identity to the amino acid sequences given in table. 4, from aspartate encoded in approximately 118 nucleotide to cysteine encoded in approximately 873 nucleotides, which is expressed in this cell the owner, with the indicated promoter and specified the coding sequence are in the molecule within the specified vector so that a neurotoxin expressively running the specified promoter in this cell-host;

(b) being introduced specified molecule of recombinant DNA in this cell the owner in order to produce a genetically modified cell is the master, and

(C) culturing a genetically modified host to Express the specified encoding sequence and get specific to insects paralytic neurotoxin.

KRA is when constructing a recombinant AcMNPV vEV-Toh by homologous recombination between sequences of wild-type virus and plasmid polyhedrin. The EcoRI fragment size 933 base pair containing Toh, incarcerous in pEVmodXIV (described in application for U.S. patent N 353847, registered on may 17, 1989). The plasmid contains 2,6 kilobar basis vector sequences (open area), the region of 0.8 kilobar grounds of the AcMNPV genome, located immediately behind the polyhedrin gene, the region of 1.5 kilobar grounds of the AcMNPV genome from site kpni restriction sites within the polyhedrin gene and extending down from the gene (AcMNPV sequence shown in the form of oblique lines), and the modified polyhedrin promoter LSXIV shown as an arrowhead between the restriction sites EcoRV and Bgl II. Direction Toh-coding sequences (dotted line) from the promoter LSXIV shown by the arrow inside the circle.

Fig. 2 compares the effects on the increase of mass of insect larvae infected by injecting or AcMNPV wild-type (L-I), or a recombinant virus vEV-Toh depending on the time elapsed after infection. Also included control samples imaginary infection (TC-100). Larvae initially examined at 24 h after infection (shown as 0 h after infection (pi). Those who die as a result of physical impact injection, removed during this study period. Pageprivate in Fig. 2 in columns and remove from each column (left scale gives the average mass per larva in milligrams). The percentage of each group of larvae, paralyzed, depending on the time represented by the black Central column within a larger open column, which represents the average mass (right scale gives % paralyzed or dead).

Fig. 3 illustrates a roaming plasmid phc-dETL-RTK, which is used to construct a recombinant virus vETL-Toh, and this virus expresses TxP-I under the regulatory control of the early promoter ETL virus AcMNPV. Fragment Bgl II, Kpn I from pEV-Toh containing Toh-encoding sequence (oblique line), incarcerous in sites Bgl II and Kpn I plasmid phc-dET, which is obtained from phcwt (Rankin et al. (1988) supra), replacing the polyhedrin promoter between sites EcoRV and Bgl II promoter sequences ETL (Crawford and Miller (1988), J. Virology 62:2773-2781) extending from -300 to -6 (relative to the ATG codon of translation initiation, marked+1, +2, +3). The promoter ETL marked by the arrowhead. The genomic sequence of AcMNPV, which carry out homologous recombination and allelic substitution, shown in the form of oblique lines; these sequences flank purposes; it is located on the fragment Hind III-SalI at the junction of AcMNPV/vector, shown on the left side of the chart. Vector sequences are shown as open lines. Are relevant sites of the restriction enzyme.

Fig. 4 illustrates a roaming plasmid pSp-Toh, which is used to construct a recombinant virus vSp-Toh expressing Toh-coding sequence under the regulatory control of the synthetic hybrid very late promoter (labeled SPLSXIV and described in application for U.S. patent N 353847, registered on may 17, 1989). pSp-Toh contains full polyhedrin gene under its own promoter and additional AcMNPV sequences, flanking the polyhedrin gene. The sequence of AcMNPV shown in the form of oblique lines; vector sequence shown in an open field. The EcoRI fragment containing Toh-encoding sequence, incarcerous in the EcoRI site downstream from the promoter SPLSXIV and in the orientation opposite to the orientation of the polyhedrin gene. Gene expression directed hybrid promoter, the above expression under the control of the polyhedrin promoter. vSp-Toh has occluded the phenotype due to intact the 34-coding sequence under the regulatory control of the synthetic promoter Cap/Polh, described in the work Thiem et al. (1990), Gene 91:87-95. Fragment Bgl II, Kpn I from pEV-Toh containing sequence Toh replaces the CAT gene in pCap/Polh-CAT (Thiem et al. (1990) supra). Direction Toh-coding sequences (dot dashed line) from the promoter of cap/Polh (the area shaded diagonally) is shown by an arrowhead. The sequence of the wild-type virus and vector pUC8 specified areas with beveled lines and without any lines, respectively.

Fig. 6 compares the increment of the mass of larvae of T. ni late fourth and early fifth age stages after per os feeding culture medium (TC-100) and the phone off or AcMNPV wild-type or vSp-Tox34. The height of the histogram along the Y-axis denotes the average mass in milligrams of 30 larvae per test virus, unless specified otherwise. In the later stages of the study, the average weight is determined only in relation to living insects. Columns deviations give a plus or minus twice the standard deviation. Typically, the average standard deviation of plus or minus two times contains approximately 95% of cases of experimental measurements in the standard distribution. Larvae can withstand hungry for 24 h before feeding multiradius studies show it is the lethal dose LD100. After feeding for 24 h on the basis of the contaminated diet the larvae give a clean diet and weighed every 24 hours-Days after feeding deferred on axis X. day 0 indicates the day when the larvae were given clean food. Histograms are empty, beveled and obliquely shaded areas indicate uninfected larvae, larvae infected with a virus AcMNPV wild-type and larvae infected with a virus vSp-Tox34, respectively. The columns within each histogram column shows the percentage of larvae that were paralyzed (solid bars) or ukuleles (empty bars).

Fig. 7 compares the increment of the mass of larvae of the first stages of T. ni after the injection 4105plaque-forming units neutropenia AcMNPV (L-I - upward slant), vEV-Toh (empty), vCap/Polh-Toh (shaded), vETL-Toh (oblique hatching) or vSp-Tox34 (dotted). Uninfected larvae (downward slant) injected 2 μl of liquid tissue culture. Larvae weighed first, and then infect and placed on disease-free diet. Columns deviations represent 2x the standard deviation, which contains 95% of the experimental sabiki deviations do not overlap, can be considered as significantly different at a confidence level of 0.05. In those cases, when the bars are deviations overlap, use student test for pairwise comparison of the two middle values (DECalc-PLUS Routine Library, Digital Equipment Corp., Nerrimack, New Hampshire) or multiple criteria scope Duncan (SAS Base/Stat Release 6,04, SAS Institute, Cary, North Carolina). Mean values that are statistically different, are placed in ascending order and then they are given a letter designation (A, B, C . . ), to denote the highest and the lowest average mass in relation to treatments during the day outside of the column standard deviation.

Fig. 8 represents autoradiogram polyacrylamide gels with sodium dodecyl sulfate, which show proteins derived from wt-AcMNPV or vEV-Toh-infected cells S. frugiperda depending on the time after infection. After 1 h adsorption, the infected cells have been labelled by the type of pulse with (35S)-cysteine for 6, 12, 24, 36 and 48 h after infection. Proteins from the liquid tissue culture (intracellular, Fig. 8A) and from cell lysates (extracellular, Fig. 8B) are denatured and restore electrophoresis using 12% polyacrylamide GE the s protein is carried out in the far left lane of each gel, and celebrate molecular weight. Proteins from uninfected cells separated in the track labelled imaginary (imaginary infection). Sources of other proteins is indicated on each part of Fig. 8. Strands TxP-I TxP-II and protein polyhedrin indicated by arrows.

Detailed description of the invention.

Agent for biological control of insects is the agent, effective in the control of insect pests. Agents for combating insects can be modified with the purpose of expression specific to insects paralytic neurotoxin for use in the present invention, and these agents include viruses, insects, entomopathogenic bacteria and fungi, RestrictAnonymous bacteria, plants and plant viruses that are carried by insects, or could accidentally proglatavetsa them. The fight is mentioned in the sense of restrictions in eating behavior or destruction of the insect pest. Agent for biological control of insects in accordance with the present invention should have a toxic effect, which is an attribute of the at least partially the expression of the sequence coding specific to insect neurotoxin. Toxic to insects effect applies to any who CSOs insect or changing the normal behavior of the desired insect for example, eating behavior, the correct reaction or other stereotypical behaviours.

Parasitic to insects, mites is mites, which eat insects. Many of these ticks injected with poison owners insects on which they feed. This venom contains specific to insect neurotoxins designed to immobilized insect hosts. Ticks are most likely to Express genes specific to insects paralytic toxin include mites within the group ventricosus, including P. anobii, P. beckeri, P. emerqinatus, P. schwerdtfegeri, P. tuberculatus, P. tritici, P. and P. ventricosus zwoelferi.

Specific to insects paralytic neurotoxin is a polypeptide that causes paralysis sensitive insect. Larvae and/or adult insects can be affected agents for insect neurotoxin. The paralytic effect of the neurotoxin may initially be in the form of a destruction efficiency of the motor or other insect behavior, including feeding behavior. Specific to insect neurotoxins are those that have a detrimental effect on insects and have negligible effects on higher animals, especially mammals. Specific to gasoducto Toch-34 and Taha, protein TxP-I and TxP-II, produced by P. tritici. Deduced amino acid sequences of two specific paralytic to insects proteins are presented in table. 2 and 4. It should be understood that any protein with the amino acid sequence, to essentially identical (at least 70% identity, or at least 70% similarity) with the amino acid sequence from aspartate encoded in approximately 118 nucleotide to cysteine encoded in approximately 873 nucleotides or substantially identical to the sequence shown in the table. 4, which has a measurable toxic effect on insects, is the functional equivalent proteins Toh or Toga. Preferably, if the amino acid sequence specific to insects paralytic neurotoxin is at least about 83% identity (with differences in one sequence, considered as non-identical, compared with the amino acid sequence shown in table. 2 or 4), or at least about 83% similarity. The toxin is functionally equivalent to neurotoxins present invention, causes a similar sokrushitelny paralysis To some amino acid substitutions can be made in protein sequences without affecting the function of the protein. As a rule, conservative amino acid substitutions or substitutions of similar amino acids are maintained without influencing the function of proteins. Similar amino acids can be those that are similar in size and/or charge properties, such as aspartate and glutamate, on the one hand, and isoleucine and valine, on the other hand, are pairs of similar amino acids. The similarity between the amino acid pairs installed in science in a number of ways. For example, Dayhoff et al. (1978) in Atlas of the sequence and structure of proteins, volume 5, Supplement 3, Chapter 22, pages 345-352, entered into this description by reference, provides frequency table against amino acid substitutions that can be used as a measure of amino acid similarity. Frequency table Dayhoff et al. based on comparisons of amino acid sequences for proteins with the same function from a variety of evolutionary various sources.

Additional functional equivalents specific to insects paralytic neurotoxin include polypeptides with parts of the amino acid sequence characterized by significant identity with Toh or Toga, Sledovatelnot protein Toh or Toga, you can make insertions, and which retain biological activity specific to insects paralytic neurotoxin, affecting sokrushitelny paralysis of the muscles.

Genes specific to insects paralytic neurotoxin can be found in for predatory insects, mites, including but not limited to those presented in table. 1, especially those that belong to the group ventricosus, or other parasites or predators in relation to insects. Genes homologous to genes Toh and Taha of the present invention, can be identified in ticks or other sources using nukleinovokisly hybridization to sequences described in the present invention, or by using cross-reaction of toxin molecules with antibody specific for toxins of the present invention, or using other methods known in the art, including methods for PCR (polymerase-cableway reaction) is carried out using oligonucleotides corresponding to conservative or unequivocal regions of the gene(s) of the toxin contained in this description. In principle, it is possible to identify any gene specific to raceconscious protein can be easily identified, and performance of such genetically modified vector can be estimated using the doctrine of the present invention in combination with well known techniques.

The recombinant DNA molecule is one which can be obtained either by natural processes using known methods, managed to produce a desired result or artificial methods based parts, originating from heterologous sources, and these parts can be naturally occurring or chemically synthesized molecules, and these parts are connected by ligating or other well known means.

Genetic modification for the purpose of containing and gene expression specific to insect neurotoxin that causes paralysis of insects means that the nucleotide sequence encoding such protein and guides its synthesis, we are agent for biological control of insects or the host cell, which in its natural state does not contain this gene, with the purpose that the modified agent or the cell could produce this protein neurotoxin. For insertion expressed gene neurotoxin in the agent to combat tx2">

To control the transcription or translation of the nucleotide sequence that encodes a specific insects paralytic neurotoxin, it is possible to use any known regulatory sequences, promoter and/or promotora-associated sequences that direct gene expression in the sought infected and uninfected host or infected or uninfected cell host.

It is clear that the specialist can easily determine the choice of the necessary regulatory sequences or promoters. For example, the virus insect, such as a baculovirus, suitable promoters, especially late and very late promoters, synthetic promoters or hybrid promoters, if you require high levels of expression. However, if the goal is getting paralytic neurotoxin to limit in the diet of insect larvae to the shortest possible periods or to extend the effective range of the hosts of the virus insect, then it is desirable to place the gene paralytic neurotoxin under the regulatory control of a baculovirus or Nabuchodonosor (e.g., insect) promoter, expressed earlier in the process sargerus insects, shown as an example in the present description. Terms AcMNPV and AcNPV used in relation to the same virus. I believe that the term AcMNPV currently more common in the field of Virology. It is noted that the infectious ability of the majority of NPVs is restricted to members of the genus or family of the original owner (see Groner (1986) supra). It is also noted that baculoviruses AcMNPV replicated in several families of Lepidoptera, but their infectious ability is limited to this order. Other entomopathogens viruses that are suitable in the present invention include, but are not limited to, naturally, other baculoviruses, iridoviruses, parvovirus, nodemobile, CPVs, entomopoxvirus, ascovirus and retroviruses. Experts know how to incarcerate expressed gene in the viral genome in a site that does not impede the exercise of the viral replicative functions. Similarly, the technician can select a promoter with the desired efficiency and expression in order to control the expression of specific to insects gene paralytic neurotoxin in desirable viral vector. Search the insect will dictate the selection of the virus and the specific type of virus, p is the description of the agent against insects is a composition or an active component of the composition, which has a detrimental effect on insect pests. In response to the action agent for combating insects in the expression paralytic neurotoxin reduced feeding insects or other aspects of behavior and death. Agent for combating insects of the present invention preferably is a virus insects, genetically modified to Express a heterologous gene encoding specific to insects paralytic neurotoxin, but it can be entomopathogenic fungus or a bacterium that is genetically made for the purpose of expression of a heterologous gene encoding specific to insects paralytic neurotoxin. Preferably the toxin secretorum in the hemolymph of the insect-infected entomopathogens constructed by methods of recombinant DNA.

Insecticidal compositions suitable for application to plants to control insect pests, contain suitable agricultural carrier and an agent for combating insects. The use of insecticidal compositions of the present invention can protect plants from insect pests by reducing the power and destruction of susceptible insects.

< a struggle with a certain type of pest. The specialist also knows how to direct expression of specific insects paralytic neurotoxin in a specific agent for combating insects or in the cell-master.

It should be understood that insect pests can be affected by the agent against insects in accordance with the present invention with the use of traditional methods, including digestion, inhalation or direct contact agent for combating insects.

Parasites on insects, including bacteria, viruses, fungi, mites, nematodes, protozoa and insects, can also be genetically modified to Express the gene, specific to insects paralytic neurotoxin. Parasitism or contamination of the respective insects such parasites on insects leads to paralysis of these insects, in addition to symptomatology typically associated with the unmodified parasite. Paralysis infected insects exacerbates disease state insect. Feeding and infection of insect-pest reduces the level of power and accelerates death. Specific examples of these toxin proteins are (not limited to) Toh and Toga.

DNA sequences encoding specific to NASELENIJA, can be used for genetic modification of the organism for the production of an agent for combating insects. Claim organisms for such genetic modifications include parasites on insects, plants and neutopenia restrictiontype bacteria.

The main use of genetically derived agents for combating insects, preferably baculoviruses, in accordance with the present invention is their use as components of agricultural compositions for application to plants, plant environment or distributed in baits for the implementation of biological control of insect pests. You can also use agents for combating insects in accordance with the present invention when carrying out control of other insect pests with the appropriate choice of a specific organism, genetically modified for expression specific to insects paralytic neurotoxin. For example, there are the baculoviruses, which specifically infect and mosquitoes, and fleas. The desired insect directs specialist in the selection agent for combating insects, expressing paralytic toxin, and predisposto many options for obtaining such agricultural suitable and/or acceptable in relation to the surrounding conditions of songs to fight insects.

The concentration of the agent against insects necessary for obtaining insecticide effective compositions for the implementation of the fight against insect-pest depends on the type of organism and neurotoxin, as well as the formulation of the composition. Insecticide effective concentration of the agent against insects within the composition can easily be determined. For example, effective insecticidal terms of the concentration of the virus can be easily identified using well-known techniques in the field of Virology.

Agricultural compositions for the control of insect pests of plants, must be suitable for agricultural use and dispersion in liquid media. Similarly compositions for control of other insect pests should be acceptable from the point of view of environmental conditions. Typically, the components of the composition must be nepatologickymi and not detrimental to the integrity of disseminated virus. Foliar making must not damage or destroy the leaves. In addition to suitable solid or, more preferably, liquid carriers agricultural compositions may include adhesives, emulsifiers and wetting, but not those components, is desirable to add components, which protect the agent against insects from inactivation by ultraviolet light, or components, which serve as stimulants to increase potency and/or virulence of entomopathogen. Agricultural compositions for combating insect pests may also include agents that stimulate feeding insects.

There are reviews related to the usage of agents for biological control of insects, as well as to methods and compositions for agricultural use (see. for example, Couch and Ignoffo (1981) Microbial. Control of Pests and Plant Disease 1970-1980, Burges (ed.) chapter 34, p. 621-634; Corke and Rishbeth, ibid, chapter 39, p. 717-732; Brockwell (1980) in Methods for Evaluating Nitrogen Fixation, Bergersen (ed.) p. 417-488; Burt on (1982) in Biological Nitrogen Fixation Technology for Tropical Agriculture, Graham and Harris (eds.) p. century. pp. 105 -- 114; and Roughley (1982) ibid., p. 115-127; The Biology of Baciloviruses, v. 11, supra, and references cited above).

The invention is illustrated by the following examples, which in no case should not be construed as limiting its scope. You should understand that you can access other options, modifications, alternatives and equivalents of the materials and methods described in this invention that after reading the descriptions may seem pickup the alleged claims.

Example 1. Cloning of the gene TxP-l P. tritici.

All polyadenylation DNA extracted from combinations looking for the owner and pregnant female ticks, using the techniques described in the work of Davis et al. (1986), basic Methods in Molecular Biology, Elsevier Science Publishing Co. , New York; Jacobson (1987), Meth. Enzymol. 152:254. cDNA produced using the specified polyadenylation DNA as template. EcoRI linkers are added to the recipe, and the cDNA clone in the lambda ZAP II (Stratagene, La Jolla, California) to produce cDNA library.

Six million plaques are subjected to screening using polyclonal antibodies raised against the above TxP-I in accordance with the manufacturer's recommendations (Stratagene, La Jolla, California). Identify that numerous clones exhibit significant cross-react with this antibody.

Starting with the most pronounced in terms of immunologically cDNA clones eight different antimalarials cDNA inserts subcloning of lambda phage in the bacterial plasmid is sequenced. Predicted open reading frames are used to search GENBANK (emphasis 60,0) on homology to protein sequences. Four of these cDNA inserts, including the most immunologically cDNA coding is lipase C and jitsukawa protein 70. One clone contains many fragments of EcoRI and believed, and therefore contains many of the cDNA inserts. Attention is drawn to one of the fragments of this recombinant plasmid, because he was weak hybridization to partially degenerate probe 62 grounds. Hybridizers EcoRI fragment of this plasmid is located below the other three EcoRI fragments and therefore is in a relatively remote location in the original lambda clone from the phage promoter, which is when the construction of this vector system was supposed to direct gene expression to the detection by antibodies. It is therefore surprising that the original lambda clone produces sufficient amounts of protein for weak cross-reaction with anti-TxP-I antibody, which is observed in this system, because you would expect that from a remote insert expression directed by the phage, would be negligible or absent. Then a is sequenced hybridizers EcoRI fragment; it contains an open reading frame, which, as predicted, encodes a protein with amino acid sequence identical to the sequence of the 21 N-terminal amino acids, empirically determined for the Mature toxin TxP-I.

Toxia and techniques provided by the manufacturer (SequenaseTM, US Biochemical, Cleveland, Ohio). The nucleotide sequence of the first identified EcoRI fragment containing the TxP-I-homologous sequence (denoted Toh), are presented in table. 2 together with the derived amino acid sequence. Amino acid sequence, linking the N-terminal amino acid sequence underlined. The open reading frame extends upward from the underlined sequence, suggesting that the toxin is synthesized as pramoxine or reprotoxin. Synthesis TxP-I in the form of pramoxine or reprotoxin may facilitate secretion, folding of the toxin and/or activation of the toxin outside the cage. An empirical comparison of amino acid composition and derived amino acid composition is presented in table. 3. Deduced amino acid sequence TxP-I does not show significant homology with any other protein in GENBANK (emphasis 60,0).

Believe that the EcoRI fragment, probably encodes TxP-I on the following criteria: being a part of the lambda clone exhibiting cross-reactivity with TxP-I-specific anticorodal containing a DNA sequence that includes otkryto is tivane, the coding sequence that is identical to the 21 N-terminal amino acids TxP-I and amino acid composition comparable with the composition of the TxP-I. This fragment is designated as Toch-34. Deduced amino acid sequence allows to ascribe the sixteenth amino acid of the Mature toxin properties of cysteine.

Example 2. Oligonucleotide probes and hybridization experiments.

Two oligonucleotide probe, the sequence of which is based on N-terminal sequence TxP-I, as published Tomalski et al., (1989), supra, synthesize, as shown in table. 7. Probe Pt-N1 is a mixture of 16 semnadtsatiletnem using partially degenerate nucleotide sequence, derived from N-terminal six amino acids, and contains three inosine residue in the provisions of degenerate codons. Calculated that the probe has Td(the temperature at which dissociated half duplex), equal to from 38 to 43oC, where Td= 2oC (number of residues of the A+T) + 4oC (number of residues of the G+C) for duplexes length 11-23 base pairs in 1 M Na+. Plasmid DNA withdraw from immunologically plaques using the methods described in recommendations Stratagen, La Jolla, California, cut with EcoRI (which otsaketiedoston membrane Zeta-ProbeTM(Bio-Rad Laboratories, Richmond, Califonia) alkaline capillary transfer using 0.5 M NaOH/1.5 M NaCl. The filter is washed in 2 x SSC/0.1% sodium dodecyl sulfate at room temperature (RT), pre-hybridizing in hybridization buffer (6x SSC/5 x solution Denhardt/0.5% sodium dodecyl sulfate/20 μg/ml DNA calf thymus/ 5% doctranslate) without probe for 1 h, hybridizing in fresh hybridization buffer containing approximately 1106cycles of 1 min radioactiveman of the oligonucleotide in 1 ml for 14 hours at a temperature of 28-30oC (about 10oC lower than that calculated for Tdin relation to Pt-N1). Then the blot was washed twice in 2 x SSC/0.1% sodium dodecyl sulfate for 15 min each time at a temperature of hybridization, and then subjected to autoradiography at a temperature of -80oC in the presence of intensifying screens. After the exposure of the blot again washed in the same buffer at an elevated temperature in 5oC lower than expected for Tdand again subjected to autoradiography. Washing also repeated using a temperature Tdon the minimum expected level (38oC) and the maximum expected level (43oC).

Fragments of the inserts showing hybridization in n bridezilla insert also is sequenced, since partial cDNA TxP-I can lose sequence homologous to the probe Pt-N1. Receive cDNA, which show cross-reactivity to anticigarette TxP-and I can hybreed to probe Pt-N1, but do not encode TxP-I.

Design a longer probe, as longer and minimally degenerate probes have a higher specificity than a short probes, and therefore more suited to sensing more complex sequences. Probe Pt-N2 synthesized in a mixture of 32 shestidesyatidvuhletiya; it is a complete N-terminal sequence of 21 amino acids (see table. 7). When building Pt-N2 degeneration being introduced to the fluctuating situation in some cases, when there is a choice of two codons. I believe that this limited degeneration would ensure the creation of long regions of homology that would increase the specificity of the probe. The assumption is made in accordance with the preferred use of codons in Drosophila in cases where there are three or four elections in swing positions. Conditions of hybridization for sensing either a lambda cDNA library, or fragments subcloned cDNA in plasmid DNA using Pt longer probes: 40-42oC for hybridization with a low degree of severity, and initial flushing with subsequent leaching at higher temperatures (52 and 68oC) to increase the degree of severity of hybridization.

The use of any probe for screening lifts lambda phage was not successful; hybridization in less harsh conditions resulted in the receipt of plaques with high backgrounds, whereas the severe conditions led to obtaining a false positive plaques. No probe was unable to successfully identify Toh-containing lambda phage or its purified form, or from a cDNA library. Probe Pt-N1 it hybridises to the sequence of the vector BluescriptTMseveral clones with less strict conditions and hsp70-homologous sequences, and sequences Toh. Pt-N2 it hybridises to the sequence Toh at relatively low terms of severity, as well as to the sequence of the vector BluescriptTMToh is sequenced despite the lack of convincing hybridization result. As stated above, Toh encodes a protein thr-I.

Example 3. Cloning of other genes associated with TxP-I cDNA libraries, analyze order to determine whether it contains other sequences, ioactive been labelled to high specific activity (Feinberg et al. (1983). Anal. Biochem. 132:6-13; Addendum (1984) Anal. Biochem. 137; 266-267) and used as a hybridization probe for additional TxP-I-homologous sequences from a cDNA library (lambda ZAP-II. The hybridization conditions are identical to those used above, except that the hybridization is carried out at a temperature of 65-68oC.

Allocate approximately 40 additional cDNA clones that exhibit significant homology to the probe TxP-I. These cDNA inserts analyzed by digestion with restriction endonucleases using the fragment EcoRI and hybridization on Southern. Most of the cDNA inserts similar in size to the fragment EcoRI Toch - 34 used as a probe. None of them has the same "top" fragments of 1.5 kilobar grounds that found in the initial lambda isolate containing a sequence Toh, which confirms the conclusion that these "top" EcoRI fragments exist due cloning artifact. It is noted, however, that several of the newly selected EcoRI fragments containing cDNA inserts that hybridizing to EcoRI fragment containing the cDNA Toh, vary in size from EcoRI fragment Toh. Smaller cDNA can theoretically be objurgative cDNA insert, which is larger than the initial TxP-I-isolate, 5'- and 3'-end of the cDNA is sequenced as described above using the sequence to the oligonucleotide near endings cDNA inserts Toh as primers. 5'-ends of the four larger cDNA inserts is sequenced using primers located within the 5'-end of the open reading frame Toh. 5'-end of one cDNA clone essentially identical in size and nucleotide sequence of cDNA clone Toh (missing one nucleotide at the site of cloning that is a trivial difference), 5'-ends of the other three cDNA clones differ in length and sequence from a cDNA clone Toh. Of these three clones contain two identical N-terminal 13 amino acids that Toh, but differ in length and sequence, above netransliruemoi leader region. The third of these clones (clone Toha) differs in the N-terminal sequence (discussed below), and above 5'-netransliruemoi leader region.

the 3'end of the seven toxin-related cDNA is sequenced using primers located within the 3'-end of the open reading frame Toh, the 3'ends of all the clones differ NII open reading frame TxP-I sequence inserts deviate in significant ways. This is not due to polyadenylation, or differences in the vector sequences. On the contrary, it seems that TxP-I-related genes differ due to genetic heterogeneity in ticks from which RNA is extracted for construction of cDNA libraries, or by the existence of mnogogrannogo family of toxins within the genome of ticks. Table. 5 gives a comparison of the 3'- endings of several toxin-related cDNA clones that are identified by probing cDNA libraries ticks using fragment Toh. Sequence align sequence Toh. Approximately 50 bases downstream from the end of the region that encodes a toxin (+873 Toh), sequences diverge.

The second cDNA insert (Tox21a) is sequenced in its entirety with the aim of exploring the nature of variations in the nature of the toxin gene. Oligonucleotide primers homologous Toh are synthesized with the aim of facilitating the process of sequencing. Because the nucleotide divergence between the two genes primers specific to certain internal areas of the insert Taha, synthesize, in order to complete the construction of the sequence. Table. 4 represents the data in this sequence. E. is it Toh, that indicates the existence of a diversity of toxin genes in the cDNA library, each of which encodes TxP-I-related toxin, but differs in amino acid sequence. Table. 6 represents a comparison of the deduced amino acid sequence for Toh and Taha. Sequence by 88.9% and similar to 82,8% identical. Two sequences of five amino acids "incarcerous" in the sequence Toha compared with the sequence Toh. In the predicted N-terminal sequence of the Mature gene product Toha would not be the same as N-terminal sequence determined empirically for TxP-I. in Addition, the first in frame ATG sequence Toja is located in line with the second frame ATG sequence Toh. Thus, effective could be the deletion of 13 amino acids at the N-end putatively reprotoxin-form Tox21a. A deletion can be predicted without any impact on the amino acid sequence of the Mature toxin, whereas the first insertion could be at the N-end of the toxin. All cysteine residues are retained between the "Mature" protein, consistent with those amino acids, which perform the role of mediator is yuusei sequence.

To demonstrate that the cloned gene Toh actually encodes the toxin insects, the sequence Toh incarcerous in baculovirus genome (AcMNPV) running very strong late promoter LSXIV, as described in the international patent application PCT/90/02814, registered on may 17, 1990; Ooi et al. (1989), J. Molec. Biol. 210: 721-736; Rankin et al. (1988), Gene 70:39-49, all of which are introduced in this description as reference materials.

All viruses originally come from AcMNPV L-I, (Lee and Miller (1978), J. Virol. 27: 754), purified from platelets and multiply in the cells of the IPLB-SF-21 Spodoptera frugiperda (Sf cells) (Vaughn et al. (1977) in vitro 13:213-217) in the environment of the TC-100 (GIBCO, Grand Island, New York), as described previously (Lee and Miller (1978) supra; Miller et al. (1986) work in Genetic Engineering, Principles and Methods, vol. 8 (eds. J. Setlow and A. Hollaender), Plenum Press, New York, p. 277-298).

The first stage consists of constructing a floating plasmid pEV-rtog (shown in Fig. 1). This roaming plasmid allows allelic replacement of the polyhedrin gene of AcMNPV genome Toh under regulatory control of a strong promoter LSXVI.

pEV-Toh design by insertion of the EcoRI fragment containing Toh-coding sequence, into EcoRI-cut pEVmodXIV, which delivers a powerful promoter LSX cells insects, as described by Miller et al. (1986) supra, and the recombinant virus is isolated and represent vEV-Toh after selection on the basis of his vkraplennoe-negative phenotype and screening for appropriate events allelic replacement by analysis of restriction endonucleases and hybridization on Southern.

Gene expression Toh in vEV-Toh-infected insect cells are examined as follows. Cells separately Sf infect toxins AcMNPV and vEV-Toh, as described in Lee et al. (1978) supra in the work of Miller et al. (1986) supra, and the liquid cell cultures from control (uninfected), AcMNPV and vEV-Toh - infected cells harvested at 48 h after infection. Larvae of the moth is a great wax Galleria mellonella injected 5 μl of the aliquot of culture fluid. The larvae, which enter the culture fluid from the vEV-Toh-infected cells, become paralyzed for 2 min, whereas insect larvae, which enter cells infected with AcMNPV wild-type, do not show any paralytic reactions over a long period of time (several days). Paralyzed larvae during visual examination of fixed, they have no reaction straightening (the ability to rise in the rack after they turned on the back) and they can't weave silk DL is given in the movement, the reaction straightening and spinning of silk. These results show that neuroparalytic toxin is produced in vEV-Toh-infected cells but not in cells infected with AcMNPV wild-type, by the expression of a sequence that encodes a cDNA Toh, and that this toxin secretiruetsa in the extracellular environment. The type of paralysis shown under the action of the gene product Toh reminds paralysis observed with the introduction of the larvae TxP-I TxP-II and/or TxP-III.

To study the ability of baculovirus carrying the gene Toh, to control the feeding behaviour of the larvae of insects during infection, infect insects by using vEV-Toh by injection of purified virus in the hemolymph of the tested larvae. Larvae Trichoplusiani early fourth age is injected environment TC-100 (Mnemosyne) or medium containing particles padding-virus from cell cultures infected with either AcMNPV wild-type or vEV-Toh (4105plaque-forming units of virus per larva). Control larvae include those larvae that are infected cultural environment, or those infected with AcMNPV wild-type. Infected insects (vEV-Toh) become paralyzed (immobilization and lack of reaction straightening) at 36 h after infection.

Prematurity vEV-Toh (Sf cells), explore autoradiography after pulse-labeling and electrophoresis in polyacrylamide gels with sodium dodecyl sulfate. After 1 h after infection or period of the imaginary infected cells have been labelled with (35S)-cysteine for 6, 12, 24, 36 and 48 h after infection. Then the solutions for labeling removed and cells cover popolnenie environments TC-100 and incubated for 2 h Cells containing intracellular proteins) and cell-free medium (containing sekretiruemyi proteins) are collected separately. Cells are lysed. Intracellular and sekretiruemyi proteins are denatured and restore in buffer containing sodium dodecyl sulphate and dithiothreitol, and then separated by electrophoresis in polyacrylamide gels with sodium dodecyl sulfate, using 12% polyacrylamide gels. Gels impregnated with fluorine, dried and subjected to autoradiography. The results are presented in Fig. 8. Protein standards are shown in the far left lanes, and their molecular masses are given in kilodaltons. Proteins from uninfected cells collect in the tracks marked with "imaginary". In addition, the same change in mobility detected by electrophoresis in polyacrylamide gels with sodium dodecyl sulfate, observed in related the shows what TxP-I (approximately 27 kilodaltons) and TxP-II (about 28 kilodaltons and about 29 kilodaltons) are related as molecules of the Mature toxin protoxin and reprotoxin. TxP-I TxP-II and stripes polyhedrin protein is shown in Fig. 8 with the arrow keys. TxP-III contains three bands within TxP-I and TxP-II. Fig. 8 shows that the three protein produced vEV-Tox34-infected cells, correspond in size to the three toxin-related proteins described Tomalski et al. (1988) supra; Tomalski et al. (1989) supra. Thus, the expression of sequences encoding Toh results TxP-I and TxP-II, which together comprise TxP-III. It is also possible, as Toh has two methionine codon near the 5'-end of the coding sequence, one of which is not found in the sequence, the coding Toha that two protein bands TxP-II reflect alternating sites translational start. These possibilities can be distinguished N-terminal sequencing of each protein component TxP-II or site-directed mutagenesis of any one or both of the respective codons ATG.

Example 6. Additional derivatives of AcMNPV, genetically obtained for gene expression of a mite neurotoxin.

To assess improves if the expression Thesurgery insects (TC-100) and insects, infected with AcMNPV wild-type (L-I) or vEV-Toh. The comparison results are presented in Fig. 2. At 24 h after infection (the time taken for 0 h after infection (pi) in this study) insect larvae from each group of control or subjects larvae weigh, on average, 60-80 mg At 24 h after infection of insect larvae infected with AcMNPV wild-type, show a much larger increase of mass than Mnemosyne or vEV-Tox34-infected larvae. Earlier it was observed that infection with wild-type virus really enhances larval feeding within the first days of infection (application for U.S. patent N 373952, registered on June 29, 1989). At 36 h after infection, all the insects in the group infected with a virus vEV-Toh, become paralyzed (immobility, lack of response and rectification), but none of the numbers mnemotrauma or infected with a virus AcMNPV wild-type larvae are not paralyzed. Weight vEV-Toh-infected larvae significantly lower than other groups; mass vEV-Toh-infected larvae actually reduced in the period from 24 to 36 h after infection. This may be the result of dehydration due to loss of feeding behavior. This trend continues for 60 h after infection. At 96 h after infection all mnim is described. All virus-infected insects are typical symptoms of viral infection by this time. Thus, the gene expression of tick toxin improves the properties of the baculovirus AcMNPV as a pesticide due to inhibition of food during infection. The expression of the toxin does not block viral replication, since all members of the group of larvae infected vEV-Toh die from a typical viral infection.

In the above-described viral building Toh-coding sequence is expressed under the regulatory control of the very late baculovirus promoter, which is expressed not earlier than about 18 h after infection in cells infected at high multiplicity of infection (moi, that is, 10 viruses per cell) or at least approximately 24-30 h after infection in cells infected at moi of 1. So don't expect paralytic effects of baculovirus-mediated expression Toh will be observed up to 36 h after infection.

In order to accelerate the time period, which could be observed paralytic effects of gene product Toh, AcMNPV virus genetically construct for the expression of Toh-encoding sequence under uragoda sequence, expressed under the regulatory control of the ETL AcMNPV promoter (described in Crawford et al. (1988), J. Virol 62-2773-2778, which is introduced in this description by reference). Toh-containing EcoRI fragment incarcerous in the EcoRI site of plasmid phc-dET, which is obtained from the phcwt (Rankin et al. (1988) supra), replacing the polyhedrin promoter between the EcoRI site and the site Bgl II promoter sequences ETL extending from -6 (relative to ATS translation initiation ETL near +1, +2, +3) to approximately 300 base pairs from above ETL coding sequences. Portable plasmid and AcMNPV wild-type together transferout with subsequent isolation and characterization. Fig. 7 results the data on the infected insect virus vETL-Toh.

The promoter Cap/Polh described Thiem and Miller (1990) Gene 91:87-95; this is the same promoter, as indicated vp39/LSXIV in Fig. 19 application for U.S. patent N 07/353847 registered 17 may 1989. The DNA sequence of the promoter Cap/Polh are given in table. 8. Fragment Bgl II, Kpn I plasmid pEV-Toh containing Toh-encoding sequence, incarcerous in place of the CAT gene in pCap/Polh-CAT (Thiem and Miller (1990) supra), the relevant pEV vp39/LSXIV CAT. pCap/Polh-Toh together transferout using vDA26Z (O'reilly et al. (1990) supra ) in Sf cells, and the virus obtained in resalat, confirming the correctness of its genetic structure analysis by restriction endonucleases.

Effects vCap/Polh-Toh on the increase of mass is determined by the injection 4105plaque-forming units of the larvae of T. ni fifth stages of the first day or 2 μl of liquid tissue culture without virus. Larvae weighed, injected and put on a virus-free diet. Larvae infected vCap/Polh-Toh, gain significantly less weight and show paralysis earlier than larvae infected with other recombinant viruses, in the comparative analysis. The maximum mass that you gain larvae infected with AcMNPV wild-type, almost three times higher than the maximum mass increase vCap/Polh-Toh-infected larvae on the first day after infection. Over 50% of the tested population infected vCap/Polh-Toh, paralyzed on the first day, whereas 10 or less other tested populations paralyzed or dead by this time after infection. vCap/Polh-Toh-infected larvae lose a small amount of mass between the first and second day after infection, when the paralysis of the tested population, committed, possibly due to dehydration paralyzed larvae (cm. Fig. 7).

Fig. 7 is a CPA shall outermost, the increase of mass of larvae infected insects and on mortality or paralysis of the infected larvae. On the first day after infection, the increase of mass AcMNPV - and vETL-Toh-infected larvae are approximately equivalent, whereas vEV-Toh, vSp-Toh and vCap/Polh-Toh-infected larvae have a lower increment of the mass. Two days after infection of mass increment infected insects in decreasing order are AcMNPV, clean vETL-Toh and pEV-Toh and vSp-Toh (both are equivalent) and vCap/Polh-Toh. By this time all larvae infected vCap/Polh-Toh, vEV-Toh and vSp-Tox34, paralyzed or dead, whereas only about 10-15% of the larvae infected with AcMNPV or vETL-Toh, paralyzed or dead.

On the third day after infection uninfected larvae pupate, a vETL-Toh-infected larvae become significantly less weight compared to AcMNPV-infected larvae. On the fourth day after infection, the remaining infected larvae either die or become paralyzed.

Fig. 7 shows that vCap/Polh-Toh is the most effective means for combating insects of those who tested against limits weight gain of larvae in relation to an earlier death or paralysis that ETL-Toh comes earlier than other toxin-producing recombinant viruses, it turns out that the power of the promoter may be less than adequate for delivery of paralytic doses of the toxin to the infected insect early period of infection. Late/very late hybrid promoter Cap/Polh, as it turns out, is much more powerful, and therefore it is a preferred promoter of this group to determine the expression of specific insects paralytic neurotoxin in a baculovirus vector.

Example 7. Expression Toh directed by the promoter SPLSXIV positive for Taurus enable the baculovirus.

Recombinant baculoviruses expressing Toh in example 4, are not containing bullock include viruses and therefore maloinvazivnye for insects in oral method of infection. Interspersed with baculovirus expressing Toh-coding sequence under the regulatory control of the very late promoter and polyhedrin protein under the control of its own promoter construct using the transferred plasmids S-Toh shown in Fig. 4. Promotor sequence, denoted here by the symbol Sp, yedavalli promoter Sp are presented in table. 9. S-Toh together transferout in Sf cells with DNA derived from AcMNPV using gene beta-galactosidase, inserzioni in the polyhedrin gene (vSynVI-gal). This virus has occlusion-negative phenotype and forms a blue plaque, when it is sown on a chromogenic substrate for beta-galactosidase, 5-bromo-4-chloro-3-indolyl-beta-D galactopyranoside (X-gal). Other AcMNPV derivative with a deletion of the polyhedrin gene, may also be suitable for construction of recombinant virus, as the polyhedrin substituted sequences S-Toh during allelic substitution at designing. Recombinant virus vSp-Tox34 separated from the joint offspring of transfection in the form of a virus with a positive in calves include beta-galactosidase-negative phenotype, and he expresses TxP-I. 19 of 20 larvae of T. ni, ate vSp-Toh on the fourth and fifth age stages are paralysis and stop to eat at 52 h after their initial contact with contaminated food, whereas larvae who eat infected with AcMNPV wild-type, continue to eat within 5 days (120 hours) after ingestion of contaminated food. Similar results showing the increase of mass in relation to the tested population 30 larvae, preparazione and decreases in weight as compared with larvae, fed wild-type virus. Thus, the viruses carrying and expressing the gene specific to insects paralytic neurotoxin, for example Toh-encoding sequence, have significantly improved pesticide properties.

Example 8. Expression Toh-coding sequence that is sent by the promoter Cap/Polh positive for Taurus enable the baculovirus.

To obtain the occlusion-positive baculovirus that expresses Toh-coding sequence under the control of the promoter Cap/Polh, first construct a plasmid containing between sites EcoRI and Kpn I mnogosloinaya site Bluescript plusTM(BSKS+, Stragene, La Jolla, California) the left end of the 4.6 kilobar reason EcoRI fragment AcMNPV wild-type from the EcoRI site at 0,0 units of genetic maps to AcMNPV site Kpn I within the polyhedrin gene. This plasmid (pERI-K) then digested with EcoRV to linearize the plasmid, slicing it from the EcoRV site above polyhedrin gene. Fusion promoter Cap/Polh-Toh and gene removed from pCap/Polh-Tox34 (see Fig. 5), cutting off the bottom Toh using the site Kpn I (then get the blunt end), then cut off the upper portion of the promoter Cap/Polh using EcoRV site. Fragment nesushiesya orientation of the gene for the toxin is directed counterclockwise relative to the sequences of AcMNPV. The obtained plasmid denote pCap/Polh-Tox34VI+.

Cap/Polh-Tox34VI+ used as a portable plasmids for allelic replacement occlusion-negative derivative with AcMNPV deletion or replacement mutation in the polyhedrin gene. Suitable deletion mutant is vSynVI-gal as described in example 7, or deletion mutant obtained by allelic replacement using plasmids pEVmedXIV.

Recombinant viruses are selected on the basis of their occlusion-positive Petipa and sceneroot restriction fragments length polymorphism analysis due to allelic substitution.

As modified baculoviruses examples 5 and 8 contain bullock inclusion, they can be incorporated into insecticide effective agricultural acceptable compositions which can be used for the infected cultures. Ingestion of such occluded viral particles leads to the propagation of these viruses in the field and the dissemination agent for combating insects. It should be understood that the recombinant virus itself unable to direct the synthesis of Polikanov may be occluded by other methods, e.g. co-infection with helper virus, which expressi, which could stabilize neutropenia viruses and thereby increase the efficiency or productivity of their use.

Example 9. Expression Tohu-coding sequences derived AcMNPV.

Recombinant AcMNPV able to Express Toha-coderush sequence under the control of a strong promoter LSXIV, design using the following steps.

First EcoRI cDNA fragment containing Taha, cut off from the corresponding lambda ZAPII phage and clone into plasmid Bluescript described above, to obtain the pBSK-Tox21a.

Then it is desirable to mutagenicity behind the frame ATG codon (which controls the beginning of the broadcast using methionine) at position -49, -48, -47, as shown in the table. 4. This site-directed mutagenesis carried out using the technique of polymerase-cableway reaction (Innis et al. (eds. ) (1990) PCR Protocols, Academic Press, San Diego, California; H. Erlich (ed. ) (1989) PCR Tecnology, Stockton Press, New York) and the following primers:

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PCR is also used for amplification of the fragment containing the desired mutation AT CC. Mutagenesis creates a BamHI site in the upper pane of the sequence Toga. The resulting product is digested with BamHI and AccI for visualaid the LASS="ptx2">

pBSK-Toha cut with BamHI, which is wedged in the vector, and partially cut with AccI, which cuts off an area of about 300 nucleotide, as in table. 4, and in the sequence Toga. This leads to the removal of the N-terminal part of the sequence Toha from pBSK-Toha. Search vector fragment, which extends for a distance of about 300-400 base pairs below linear pBSk-Toha, purified using gel filtration.

The mutated PCR product then clone in the purified vector fragment of pBSK-Toha using standard molecular biology techniques, getting pBSK-PCR21a. A modified sequence Toha then cut from pBSK-PCR-21a by digestion with BamHI and EcoRI, make blunt, clear gel-filtration and clone in portable plasmid pSPXIVVI+X3which is cut with EcoRI and make obtuse. A modified sequence Toha incarcerous in pSRXIVVI+X3in proper orientation relative to the promoter LSXIV. The obtained plasmid pSPXlVPCR-Toha essentially identical pSPTCx32 in Fig. 4, except that the toxin gene is Toha instead Toh. Plasmid pSPXIVPCRTox21a then used for allelic replacement in AcMNPV, as described in privatelist to obtain specific paralytic to insects protein Toga.

The toxicity of the expression product Toha have mainly according to the description in example 6, except that vSPXIVPCRTox21a used to infect insects instead vSPTox34. Get results similar to those shown in. Fig. 7. Therefore, Taha encodes specific to insects paralytic neurotoxin.

The specialist will understand the modifications of the techniques that are required to build a similar disseminated virus or viruses that Express Toha running other baculovirus promoters for use in agents for biological control of insects.

1. The recombinant DNA fragment corresponding to the gene Toh and encoding active against insects neuroparalytic toxin with the following sequence: (PL.2) or with sequences taking into account the degeneracy of the code.

2. The recombinant DNA fragment corresponding to the gene Toha encoding active against insects neuroparalytic toxin sequence, are shown in table.4 in the description, or sequences, taking into account the degeneracy of the code.

3. Recombinant baculovirus vector vCaP/Po1h - Toh size 7,66 KB for the expression of naranasan insects neuroparalytic toxin Pyemotes tritici under item 1; the promoter Cap/Po1h virus ACMNPV between restriction sites EcoRV and Bg1n, HVA, EcoR1; multiple sites EcoR1 restriction, HVA, Cla1, KPn1.

4. Recombinant baculovirus vector phc-dETL Toh for expression neuroparalytic toxin (Toh) containing: DNA fragment corresponding to the gene Toh encoding active against insect toxin Pyemotes tritici, p. 1; early promoter ETL ACMNPV between sites EcoRV and Bg1n; restricciones sites EcoR1, KPn1 at the end of the gene Toh.

5. Recombinant baculovirus vector pEV-Toh size of 5.9 KB for expression neuroparalytic toxin (Toh) containing: DNA fragment corresponding to the gene Toh encoding active against insect toxin Pyemotes tritici, p. 1; the modified polyhedrin promoter LSXIV between restriction sites EcoRV and Bg1n; restriction sites EcoR1, KPn1 at the end of the gene Toh.

6. Recombinant baculovirus vector pSp-Toh for expression neuroparalytic toxin (Toh), comprising: a DNA fragment corresponding to the gene Toh encoding active against insect toxin Pyemotes tritici, synthetic hybrid late promoter SPLSHIV between sites EcoR1 and BamH1; EcoR1 sites, Pst1, Sst1 at the end of the gene Toch.

 

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