Hybrid insecticide protein, nucleic acid molecule coding said protein, transgenic plants and seeds thereof containing said protein, method of producing protein and use thereof

FIELD: chemistry.

SUBSTANCE: present inventions relate to protein engineering, plant molecular biology and pest control, as well as a hybrid insecticide protein and use thereof. Described is a hybrid insecticide protein which includes from the N-end to the C-end an N-end portion of Cry3A protein which is fused with the C-end portion of Cry1Ab protein, wherein the position of the crossover of the Cry3A protein and the Cry1Ab protein is located in a conservative block 2, in a conservative block 3 or in a conservative block 4 and has anti-western corn rootworm activity. Also disclosed are nucleic acid molecules which code the novel proteins, methods of producing proteins, methods for use thereof, as well as transgenic plants and seeds thereof which contain such proteins.

EFFECT: inventions enable to obtain cheap means of controlling Diabrotica worms.

39 cl, 8 dwg, 9 tbl, 46 ex

 

The LEVEL of TECHNOLOGY

The present invention relates to industries protein engineering, molecular biology and plant pest control. More specifically, the invention relates to new constructed hybrid proteins having insecticidal activity, to nucleic acids, the expression of which are these insecticidal proteins, and to methods of obtaining and usage of these insecticidal proteins and related nucleic acids in insect control.

Insect pests are a major cause of crop losses. Only in the US, the annual loss caused by the invasion of various insect species, billions of dollars. Besides that insect pests cause losses to crops, they also cause trouble to the fruit and vegetables producers, annoy gardeners and homeowners.

The most malicious corn pests are different kinds of maize root of the bug. The most significant species in the U.S. are Diabrotica virgifera virgifera, Western corn root beetle, D.longicornis barberi, Northern corn root beetle and D.undecimpunctata howardi, southern corn root beetle. Major corn pest in the Corn belt of the USA are the only Western and Northern corn root beetles. An important corn Korneva what about the pest in the southern United States, is a Mexican corn root beetle, Diabrotica virgifera zeae. The most significant damage to the plant caused by the larvae of corn root beetle, because they only feed on the roots of corn. This harm is reflected in the increase in the lodging of the plants, the reduction of grain yield and vegetative mass, and change in the content of nutrients in the grain. The exercise of power by the larvae also has indirect effects on corn because it result in the roots of open passages for the invasion of bacteria and fungi that cause disease in terms of the rot root and stem. Adult corn root beetle exert their activity in corn fields in late summer, when they eat corn, "silk", and pollen, which prevents normal pollination.

The fight against the corn root beetles are mainly through the intensive use of chemical pesticides, which activity is manifested through impeding the growth of insects, preventing them from feeding or breeding or insects. In this way can be achieved good results in the fight against the corn root of the bug, but sometimes these chemicals can affect other beneficial organisms. Another problem, which is manifested by the widespread use of chemical pesticides is the emergence of insect species, the moustache is sustainable for them. Another problem caused by the fact that larvae of corn root beetle feed on the underground, which makes them difficult to make contact with insecticides. Therefore, in most cases, the application of insecticides is done prophylactically at the time of planting. The result of this practice is a great harm to the environment. Some of this damage has been reduced through the use of different methods of farming, however, the need for alternative mechanisms of pest control is constantly growing.

Biological agents to control pests, such as strains of Bacillus thuringiensis (Bt), expressing pesticidal toxins, such as δ-endotoxins (Delta-endotoxins, also called crystalline toxin or Cry proteins), were also used for cereals and showed satisfactory results mainly in the fight against lepidopteran insect pests. These δ-endotoxins are proteins that are enclosed in a crystalline matrix, known for its insecticidal activity, reflected if swallowed their specific insect species. Classification of different δ-endotoxin was performed on the basis of their spectrum of activity and homology sequences. Before 1990 the main classes was determined by their spectrum of activity, and Cry1 proteins possess the activity of the Yu against Lepidoptera (moths and butterflies), proteins SGU have activity against both lepidopteran and against Diptera (flies and mosquitoes), proteins SGU have activity against Coleoptera (beetles), and proteins SGU have activity against Diptera (Hofte and Whitely, 1989, Environ. Rev. 53:242-255). In 1998, has developed a new nomenclature, which gave a systematic classification of Cry-proteins based on homology amino acid sequence, and not on the activity in relation to certain insect species (Crickmore and others 1998, Environ. Molec. Biol. Rev. 62:807-813).

Spectrum insecticidal activity of individual δ-endotoxin of Bt is quite narrow, that is, given δ-endotoxin active against only a small number of species in this order of insects. For example, it is known that the toxin Shua has a very high toxicity against Colorado potato beetle, Leptinotarsa decemlineata, however, has a very low or zero toxicity in relation to related beetles of the genus Diabrotica (Johnson and others, 1993, J. Econ. Entomol. 86:330-333). According to Slaney and others (1992, Insect Biochem. Molec. Biol. 22:9-18) the toxicity of the toxin Shua at least 2000 times weaker against the larvae of the southern corn root beetle than against Colorado potato beetle. It is also known that the toxicity Shua against the Western corn root beetle or Northern corn root of the WMD beetle is very weak or even absent.

The specificity of δ-endotoxins is the result of the effectiveness of various steps taken in the manufacturing process of the active toxic protein, and its subsequent interaction with epithelial cells in the middle intestine of the insect. In order to possess insecticidal properties, most of the known δ-endotoxins should be included in the body of the insect by ingestion and proteoliticeski be activated to form the active toxin. Activation of insecticidal crystal (Cry) proteins is a multi-step process. After swallowing insects crystals must first dissolve in the digestive tract of the insect. After the dissolution of the δ-endotoxins are activated by specific proteolytic cleavage. Proteases in the digestive tract of the insect can play an important role in specificity by identifying places where it is converted δ-endotoxin. After δ-endotoxin dissolved and processed, it binds to specific receptors on the surface of the epithelium of the mid-gut of the insect, and subsequently integrated in the lipid bilayer membrane of the brush edges. Then form ion channels that violate the normal functioning of the mid-gut, ultimately leading to the destruction of the insect.

In Lepidoptera, the level H in the digestive channels which is alkaline, protease bowel processed δ-endotoxins, for example, Cry1Aa, Cry1Ab, Cry1Ac, Cry1B and Cry1F, from protoxins size 130-140 kDa toxic proteins in approximately 60-70 kDa. The processing of protoxin in the toxin, according to published data, is carried out by moving both N-terminal and C-terminal amino acids, and the exact location of the recycling process depends on the specific δ-endotoxin, and taking part in it specific fluid substances digestive tract of the insect (Ogiwara and others, 1992, J. Invert. Pathol. 60:121-126). Thus, the activation process requires removal of the entire C-terminal tail section of protoxin. This proteolytic activation of δ-endotoxin may play a significant role in determining its specificity.

Beetles have digestive channels with level of acidity from the more neutral to acid, and δ-endotoxin-specific beetles, similar in size activated toxins specific for Lepidoptera. So first it was thought that the processing of δ-endotoxins that are specific to the beetles, is not necessary for their toxicity. However, based on our data, we can assume that δ-endotoxins active against Coleoptera, dissolve and proteoliticeski processed in toxic polypeptides smaller is the size. Protein δ-endotoxin Shua size 73 kDa, produced by bacteria .thuringiensis var. tenebrionis, easy processing in bacteria at N-end, losing residues 49-57 in the process of crystal formation or after forming in the end a conventional isolated form size 67 kDa (Carroll and others, 1989, Biochem. J. 261:99-105). In our work, McPherson and others (1988, Biotechnology 6:61-66) also demonstrated that native coding sequence Shua contains two functional codon that initiates translation, in the same reading frame, one to encode a protein with a size of 73 kDa and the other to encode a protein with a size of 67 kDa, starting respectively with Met-1 and Met-48 decoded sequences of amino acids. Therefore, both proteins can be considered as a full-sized proteins Shua natural origin.

With the expansion of knowledge about how to operate δ-endotoxins, has increased the number of attempts to develop δ-endotoxins with new activities. Development of δ-endotoxins have been made more possible by determining the three-dimensional structure Shua in 1991 (Li and others, 1991, Nature 353:815-821). Li et al. determined that protein Chua has three structural domains: N-terminal domain I, residues from 58-290, consisting of seven α-helical domain II, residues 291-500 consisting of three β-sheets in the package of the so-called Greek key and the C-terminal domain, from the remnants of 501-644 representing the β-sandwich in the so-called packaging roll. Was also defined three-dimensional structure active against lepidopteran toxin Cry1Aa (Grochulski and others, 1995, J. Mol. Biol. 254:447-464). The Cry1Aa toxin has three domains: N-terminal domain I, residues from 33-253, domain II residues from 265-461, and domain III from the remnants of 463-609 additional external chain in one of the β-sheet residues from 254-264. If patterns Shua and Cry1Aa be transferred to other sequences Cry1, the domain I includes amino acid residues 28 to 260, domain II is from about 260 to 460, and domain III is from about 460 to 600. Cm. the work of Nakamura and others, Agric. Biol. Chem. 54(3): 715-724 (1990); Li and others, Nature 353: 815-821 (1991); Ge, etc., J. Biol. Chem. 266(27): 17954-17958 (1991); and Honee and others, Mol. Environ. 5(11):2799-2806 (1991); each of which is incorporated into the present application by reference. Thus, it is now known that on the basis of homology of the sequence of amino acids known Bt δ-endotoxins have similar three-dimensional structure consisting of three domains.

Toxin part of Bt Cry proteins are also characterized by the presence of five conservative blocks on their amino acid sequence, numbered from SW to SW located in this order for N-end-to-end (Hofte and Whiteley, see above). The conservative bloc 1 (SV) contains about 29 amino acids, conservative block 2 SW) contains approximately 67 amino acids, conservative block 3 (SV) contains approximately 48 amino acids, conservative block 4 (SW) contains approximately 10 amino acids, and conservative block 5 (SW) contains approximately 12 amino acids. Sequences located before these five conservative blocks and after them, are highly variable, and are therefore called "variable parts" V1-V6. In a typical scenario, the domain I of Bt δ-endotoxin consists of the variable segment 1, the conservative block 1, variable segment 2 and 52 N-terminal amino acids of the conservative block 2. Domain II in the typical case consists of C-terminal amino acids (approximately 15) of the conservative block 2, variable segment 3 and the N-terminal amino acids (approximately 10) conservative block 3. Domain III in the typical case consists of C-terminal amino acids (38) conservative block 3, variable phase 4, the conservative block 4, variable segment 5 and conservative block 5. Active against lepidopteran Cry1 toxins, as well as other Delta-toxins, have a variable section 6, consisting of approximately 1-3 amino acids, lying within domain III.

Many Bt strains and δ-endotoxins are active in relation to various kinds of insects and nematodes. However, the relatively small number of these strains and toxins have what aktivnosti against Coleoptera. In addition, most active against Coleoptera δ-endotoxins, are known at the present time, for example, Chua, Show, Sgus, Shua, Shua, Show and Sgus, lack of oral toxicity against corn root of the bug that does not allow for adequate measures against it in case feed them through bacteria and transgenic plants. There is therefore a need to develop other approaches for the production of new toxins active against corn root of the bug.

Were developed active against lepidopteran δ-endotoxins in attempts to improve the specific activity or expansion of the range of insecticidal activity. For example, the domain specificity for silkworm (Bombyx mori) of protein Cry1Aa moved in protein Shoes, thus giving a new insecticidal activity of the resulting hybrid Bt-protein (Ge and others, 1989, PNAS 86: 4037-4041). In addition, Bosch and others 1998 (U.S. Patent 5,736,131 included in the present application by reference) describes a hybrid toxins of Bacillus thuringiensis, which contains at its C-end of domain III of the first Cry protein, and at its N-end - domains I and II of the second Cry protein. Such hybrid toxins showed changed insecticidal specificity against lepidopteran insects. For example, the hybrid toxin N described in the source De Maagd, etc., Appl. Environ. Environ. 62(5): 1537-1543 (196), contains at its N-end domains I and II of Cry1Ab protein, and at its C-end of domain III Cry1C protein. According to the data obtained, N has a high toxicity against lepidopteran insect Spodoptera exigua (beet scoop) compared with the parent toxin Cry1Ab, and significantly greater toxicity than the parent toxin Cry1C. It was also shown that the replacement of domain III in the toxins that are not active against Cutworm beet, such as Cry1E and Cry1Ab on domain III of the Cry1C, which is active against Cutworm beet, you can get a hybrid toxins with activity against this insect. All the hybrids described in the work of Bosch and others, are the domains of Cry-proteins active against Lepidoptera, to produce new toxins with activity against Lepidoptera. The results suggest that domain III Cry1C protein is an important determinant of specificity regarding beet scoops. Cm. the sources of Bosch and others, FEMS Microbiology Letters 118: 129-134 (1994); Bosch and others, Bio/Technology 12: 915-918 (1994); De Maagd, etc., Appl. Environ. Environ. 62(8): 2753-2757 (1996); and De Maagd, etc., Mol. Environ. 31(2): 463-471 (1999); each of which is incorporated here by reference.

There is evidence of several attempts to develop a 5-endotoxin active against Coleoptera. Chen and Stacy (U.S. Patent 7,030,295 included here by reference) have successfully created the toxin, active against corn root beetle, by introducing not found in nature site of the protease recognition domain I, domain III, or as in domain I and domain III protein Shua. One of the resulting modified Chua-proteins identified Shua into a domain which I have entered the site of the protease recognition, proved to be active against different species of Diabrotica. Van Rie and others, 1997 (U.S. Patent No.. 5,659,123) was developed Shua by arbitrary substitution of amino acids that are considered important in the question of the availability of the solvent, in domain II of the amino acid alanine. Some of these arbitrary substitutions in domain II, according to the obtained data, appeared to increase the activity against the Western corn root of the bug. However, other researchers have shown that some substitutions by alanine in domain II of the protein Chua gave a result of the breakdown of the bond with the receptor or instability patterns (Wu and Dean, 1996, J. Mol. Biol. 255: 628-640). According to Deutsch and others, 1999 (publication Number of international patent applications WO 99/31248) the replacement of amino acids in ry3b increased toxicity against the southern and Western corn root of the bug. However, from the data obtained on 35 specimens ry3b shows that only three of them, with mutations mainly in the domain II and at the junction of the domain I - domain II, was active against Western corn root of the bug. The AOC is e, variations in the toxicity of the original ry3b against the Western corn root beetle in the same samples exceeded the differences between mutated toxins ry3b source and toxins ry3b. Shadenkov and others (1993, Mol. Biol. 27:586-591) received a hybrid protein with the merger of amino acids 48-565 protein Shua with amino acids 526-725 protein Cry1Aa. Thus, the crossing sequences Shua and Cry1Aa happened in the conservative block 4, located in domain III. Shua is very active against Colorado potato beetle (Leptinotarsa decemlineata). However, a hybrid protein described Shadenkov and others, was not active against Colorado potato beetle, despite the fact that a hybrid protein comprised of sequence Shua more than 75%. Thus, the addition of only 25% sequence Cry1Aa destroyed activity against Coleoptera, possessed by the parent protein Shua. This suggests that the hybrid proteins, obtained by merging part of the Cry protein, active against Coleoptera, for example protein Chua, active against lepidopteran Cry-protein, such as Cry1A, will not possess activity against Coleoptera insects, in particular against Coleoptera insect that does not have such a natural susceptibility to Shua as corn root of the bug.

Based on the above, it is obvious that the OST is raised the need to develop new effective means of pest control, which would be economically beneficial to farmers and sustainable for the environment. In particular, the right of such proteins with toxicity against Diabrotica species, the main pest of maize, the method steps which would differ from the existing means of pest control in mitigating the development of resistance. In addition, it is desirable that the supply of such funds struggle was carried out through the products that minimise harm to the environment, for example through transgenic plants.

The INVENTION

In light of these needs, the present invention is the provision of new engineered hybrid insecticidal protein (bend). These new folds is obtained by merging unique combinations of variable regions and conservative blocks, at least two different Cry proteins and, optionally, enable protoxin tail section of the Bt Cry protein in the end, or N-terminal peptidnogo fragment, or both. Non-limiting example is the combination of complete or incomplete variable regions and conservative blocks from the first Cry protein with activity against Coleoptera, with complete or incomplete variable plots and conservative blocs second Cry protein with activity against Lepidoptera and different from the PE the first Cry protein, and, optionally, the inclusion of protoxins the tail region of the active against lepidopteran Bt Cry protein or N-terminal peptidnogo fragment, or both, resulting in new engineered hybrid insecticidal proteins, which possess activity against a range of insects, different from the spectrum of the first or second parent Cry-proteins, or both. These bends may contain complete or incomplete variable plots, conservative blocks or domains of the modified protein Shua, and of the Cry protein that is different from the modified protein Shua. Pipidinny fragment can impart a bend insecticidal activity, or can do insecticidal activity fold higher than the bend without peptidnogo fragment, or may do fold more stable than bend without peptidnogo fragment. Toxicity bend according to the present invention against corn root beetle (Diabrotica sp.) unexpectedly turned out to be surprisingly high. The present invention also relates to nucleic acids coding for this bend or complementary to hybridities in harsh environments recombinant hybrid nucleic acids of the present invention.

In addition, the invention enabled the vectors containing such recombinant (or complementaries him) nucleic acid, a plant or microorganism, comprising such nucleic acids and enabling their expression, such as transgenic maize; the progeny of such plants, which contain nucleic acid is stably incorporated in it and is inherited according to Mendel, and/or seeds of such plants and such offspring.

The invention also includes compositions and formulations containing bends, with the ability to inhibit the insect's ability to survive, grow and reproduce, or to limit damage insect damage of crops, for example, by applying these bends or compositions or formulations in the areas of insect infestation or prophylactic treatment of susceptible to insects or plants to protect them from insect pests.

In addition, the invention relates to a method for producing bends and to methods of using the nucleic acids, for example, microorganisms, insects, or in transgenic plants to impart protection from insects.

Described here new folds possess high activity against insects. For example, the bends of the present invention can be used to fight serious pests-insects such as the Western corn root beetle (Diabrotica virgifera virgifera), Northern corn Korneva the beetle (D. longicornis barberi), and Mexican corn root beetle (D. virgifera zeae). Some bends can also be used to combat the European corn borer (Ostrinia nubilalis) and other lepidopteran insects. Bends can be applied separately or in combination with other strategies for combating insects to maximize the effectiveness of the pest with minimal harm to the environment.

Other aspects and advantages of the present invention will become clear to experts in the industry in the process of examining the following description of the invention, not limiting examples.

BRIEF DESCRIPTION of FIGURES

Below the pictures are part of the present description and are included to demonstrate certain aspects of the present invention. The understanding of the invention may be facilitated by reference to one or more of these figures in combination with the detailed description of specific versions of the invention presented here.

Figa-1E shows the comparison of the sequences in some implementations, a bend with a parent Cry-proteins or modified proteins Shua used to design these folds, including Chua, Cry1Ab and Shoa, and also indicates the percentage identity. Underlined N-terminal peptidyl fragments. Five conservative blocks marked Yar is Accame SW-SW. The location of the connections between domains I, II and III indicated by a vertical dashed line. The sequence AAPF protease recognition of cathepsin G shown in bold.

On Figa-2E shows the set of executions bend, active at least against the Western corn root beetle, and a specified percentage identity compared to bend 8AF. N-terminal peptidyl fragments are underlined with one line. C-terminal protoxin tail sections are underlined by a double line. Five conservative blocks marked with labels SW-SW. The location of the connections between domains I, II and III show the icon "↓" and have the appropriate labels. The location of the position of the crossover is indicated by "♦" Sequence AAPF protease recognition of cathepsin G shown in bold.

Figure 3 shows a map of recombinant vector 12207 used for transformation of maize and containing the expression cassette with a promoter, the maize ubiquitin, operable associated with the coding sequence FRCG, operable associated with the NOS-terminator.

Figure 4 shows a map of recombinant vector 12161 used for transformation of maize and includes an expression cassette with a promoter, the maize ubiquitin, operable associated with the coding sequence of FR8a, operable associated with the NOS-terminator the M.

Figure 5 shows the map of recombinant vector 12208 used for transformation of maize and includes an expression cassette with a promoter, which is a virus twisting yellow leaves cestrum (CMP), operable associated with the coding sequence FRCG, operable associated with the NOS-terminator.

Figure 6 shows the map of recombinant vector 12274 used for transformation of maize and includes an expression cassette with a promoter, which is a virus twisting yellow leaves cestrum (CMP), operable associated with the coding sequence of FR8a, operable associated with the NOS-terminator.

7 shows a map of recombinant vector 12473 used for transformation of maize and includes an expression cassette with a promoter, the maize ubiquitin (ubi), operable associated with the coding sequence FRD3, operable associated with the NOS-terminator.

On Fig shows the map of recombinant vector 12474 used for transformation of maize and includes an expression cassette with a promoter, which is a virus twisting yellow leaves cestrum (CMP), operable associated with the coding sequence FRD3, operable associated with the NOS-terminator.

A BRIEF description of the SEQUENCES CONTAINED the SEQUENCE LISTING

SEQ ID NO: 1 is the nucleotide sequence 2OL-8a.

SEQ ID NO: 2 sequence 2OL-8a, encoded SEQ ID NO: 1.

SEQ ID NO: 3 is the nucleotide sequence of FR8a.

SEQ ID NO: 4 sequence FR8a, encoded SEQ ID NO: 3.

SEQ ID NO: 5 is the nucleotide sequence FRCG.

SEQ ID NO: 6 sequence FRCG, encoded SEQ ID NO: 5.

SEQ ID NO: 7 is the nucleotide sequence of FR8a-9F.

SEQ ID NO: 8 sequence FR8a-9F encoded SEQ ID NO: 7.

SEQ ID NO: 9 is the nucleotide sequence of FR-9F-catg.

SEQ ID NO: 10 is the sequence FR-9F-catg encoded SEQ ID NO: 9.

SEQ ID NO: 11 is the nucleotide sequence of FR8a-12AA.

SEQ ID NO: 12 is the sequence of FR8a-12AA encoded SEQ ID NO: 11.

SEQ ID NO: 13 is the nucleotide sequence WR-9mut.

SEQ ID NO: 14 is the sequence WR-9mut encoded SEQ ID NO: 13.

SEQ ID NO: 15 is the nucleotide sequence of FRD3.

SEQ ID NO: 16 is a sequence FRD3 encoded SEQ ID NO: 15.

SEQ ID NO: 17 is the nucleotide sequence of FR-12-cg-dmS.

SEQ ID NO: 18 is the sequence FR-12-cg-dm3, encoded SEQ ID NO: 17.

SEQ ID NO: 19 is the nucleotide sequence 9F-cg-del6.

SEQ ID NO: 20 is a sequence 9F-cg-del6, encoded SEQ ID NO: 19.

SEQ ID NO: 21 is the nucleotide sequence of FR-cg-dmS.

SEQ ID NO: 22 is the sequence FR-cg-dm3, encoded SEQ ID NO: 21.

SEQ ID NO: 23 is the nucleotide sequence 9F-cg-dm3.

SEQ ID NO: 24 - posledovatelno the ü 9F-cg-dm3, encoded SEQ ID NO:23.

SEQ ID NO: 25 is the nucleotide sequence B8a.

SEQ ID NO: 26 is the sequence B8a, encoded SEQ ID NO: 25.

SEQ ID NO: 27 is the nucleotide sequence 5*B8a.

SEQ ID NO: 28 is the sequence 5*B8a, encoded SEQ ID NO: 27.

SEQ ID NO: 29 is the nucleotide sequence of V3A.

SEQ ID NO: 30 is the sequence V3A, encoded SEQ ID NO: 29.

SEQ ID NO: 31 is the nucleotide sequence V4F.

SEQ ID NO: 32 is a sequence V4F encoded SEQ ID NO: 31.

SEQ ID NO: 33 is the nucleotide sequence 5*V4F.

SEQ ID NO: 34 sequence 5*V4F, encoded SEQ ID NO: 33.

SEQ ID NO: 35 is the nucleotide sequence 2OL-7.

SEQ ID NO: 36 is a sequence 2OL-7 encoded SEQ ID NO: 35.

SEQ ID NO: 37 is the nucleotide sequence of T7-2OL-7.

SEQ ID NO: 38 is a sequence T7-2OL-7 encoded SEQ ID NO:37.

SEQ ID NO: 39 is the nucleotide sequence 5*2OL-7.

SEQ ID NO: 40 is the sequence 5*2OL-7 encoded SEQ ID NO: 39.

SEQ ID NO: 41 is the nucleotide sequence 2OL-10.

SEQ ID NO: 42 is a sequence 2OL-10, encoded SEQ ID NO: 41.

SEQ ID NO: 43 is the nucleotide sequence 5*2OL-10.

SEQ ID NO: 44 is the sequence 5*2OL-10 encoded SEQ ID NO: 43.

SEQ ID NO: 45 is the nucleotide sequence 2OL-12A.

SEQ ID NO: 46 is a sequence 2OL-12A, encoded SEQ ID NO: 45.

SEQ ID NO: 47 is the nucleotide sequence 2OL-13.

SEQ ID NO: 48 is the follower of the awn 201-13, encoded SEQ ID NO: 47.

SEQ ID NO: 49 is the nucleotide sequence V5u6.

SEQ ID NO: 50 sequence V56 encoded SEQ ID NO: 49.

SEQ ID NO: 51 is the nucleotide sequence 5*V5u6.

SEQ ID NO: 52 is the sequence 5*V56 encoded SEQ ID NO: 51.

SEQ ID NO: 53 is the nucleotide sequence 88A-dm3.

SEQ ID NO: 54 sequence 88A-dm3, encoded SEQ ID NO: 53.

SEQ ID NO: 55 is the nucleotide sequence FR(1Fa).

SEQ ID NO: 56 is the sequence FR(1Fa), encoded SEQ ID NO: 55.

SEQ ID NO: 57 is the nucleotide sequence FR(1Ac).

SEQ ID NO: 58 is the sequence FR(1Ac), encoded SEQ ID NO: 57.

SEQ ID NO: 59 is the nucleotide sequence FR(1Ia).

SEQ ID NO: 60 sequence FR(1Ia), encoded SEQ ID NO: 59.

SEQ ID NO: 61 is the nucleotide sequence DM23A.

SEQ ID NO: 62 sequence DM23A encoded SEQ ID NO: 61.

SEQ ID NO: 63 is the nucleotide sequence 8AF.

SEQ ID NO: 64 sequence 8AF, encoded SEQ ID NO: 63.

SEQ ID NO: 65 - nucleotide sequence 5*cry3A055.

SEQ ID NO: 66 sequence 5*Cry3 A055 encoded SEQ ID NO: 65.

SEQ ID NO: 67 is optimized for maize nucleotide sequence

Shua.

SEQ ID NO: 68 sequence Shua encoded SEQ ID NO: 67.

SEQ ID NO: 69 is the nucleotide sequence of cry3A055.

SEQ ID NO: 70 sequence Cry3A055 encoded SEQ ID NO: 69.

SEQ ID NO: 71 - optimizarea the Naya maize nucleotide sequence

cry1Ab.

SEQ ID NO: 72 sequence Cry1Ab, encoded SEQ ID NO: 71.

SEQ ID NO: 73 - optimized for maize nucleotide sequence

cry1Ba.

SEQ ID NO: 74 sequence Cry1Ba, encoded SEQ ID NO: 73.

SEQ ID NO: 75 - optimized for maize nucleotide sequence

cry1Fa.

SEQ ID NO: 76 sequence Cry1Fa, encoded SEQ ID NO: 75.

SEQ ID NO: 77 - nucleotide sequence cry8Aa.

SEQ ID NO: 78 sequence Cry8Aa encoded SEQ ID NO: 77.

SEQ ID NO: 79 - nucleotide sequence of the cry1Ac.

SEQ ID NO: 80 sequence Cry1 Ac encoded SEQ ID NO: 79.

SEQ ID NO: 81 - nucleotide sequence Shua.

SEQ ID NO: 82 sequence Cry1Ia, encoded SEQ ID NO: 81.

SEQ ID NO: 83 - 125-primernye sequence used in the invention.

SEQ ID NO: 126-134 - N-terminal peptidyl fragments.

SEQ ID NO: 135 full - length protein Shua.

SEQ ID NO: 136-143 - primernye sequence used in the invention.

SEQ ID NO: 144 is the coding sequence of the T7-8AF.

SEQ ID NO: 145 sequence T7-8AF, encoded ASEQ ID NO: 144.

SEQ ID NO: 146 - coding sequence-catG8AF.

SEQ ID NO: 147 sequence-CatG8AF encoded SEQ ID NO: 146.

SEQ ID NO: 148 - coding sequence 8AFdm3.

SEQ ID NO: 149 sequence 8AFdm3 encoded SEQ ID NO: 148.

SEQ ID NO: 150 is the coding posledovatelno the ü 8AFlongdm3.

SEQ ID NO: 151 sequence 8AFlongdm3 encoded SEQ ID NO: 150.

SEQ ID NO: 152 is the coding sequence of the cap8AFdm3.

SEQ ID NO: 153 sequence cap8AFdm3 encoded SEQ ID NO: 152.

SEQ ID NO: 154 - coding sequence; 8AFdm3.

SEQ ID NO: 155 sequence 8AFdm3T encoded SEQ ID NO: 154.

SEQ ID NO: 156 to the coding sequence of the 8AFlongdm3T.

SEQ ID NO: 157 sequence 8AFlongdm3T encoded SEQ ID NO: 156.

SEQ ID NO: 158 - coding sequence cap8AFdm3T.

SEQ ID NO: 159 sequence cap8AFdm3T encoded SEQ ID NO: 158.

SEQ ID NO: 160 - FR8a+34 bend.

DEFINITION

For clarity below are definitions of some terms used in this description:

"Activity" bend according to the present invention means that these bends are functioning as orally active means of combating insects, have toxic effects and can disrupt or impede the feeding insect that may or may not cause the death of the insect. If the fold of the present invention is delivered into the body of the insect, the result in the typical case is the death of the insect or the inability of the insect to feed on the source from which the bend goes into his body.

The term "associated with / operable linked" refers to two nucleic acids that are associated with each other physically isofunctional. For example, say that a promoter or regulatory DNA sequence "associated with" a DNA sequence that encodes a RNA, or protein, if these two sequences are operatively connected or located so that the regulatory DNA sequence will affect the expression level of the coding or structural DNA sequence.

In the context of the present invention "chimeric insecticidal protein" (HIB) is an insecticidal protein containing pipidinny fragment, which was introduced by merging the N-terminal part of the bend. This pipidinny fragment can give this fold insecticidal activity, can do the activity fold higher than that bend without peptidnogo fragment, or it can fold more stable than bend without peptidnogo fragment, in particular against at least, the Western corn root of the bug. Pipidinny fragment is a sequence of amino acids, which in a typical case, heterological Bt IG-protein (not derived from it), but can be obtained from the Bt Cry protein. Such peptidyl fragments are extended for N-terminal part of the insecticidal protein in nature do not occur in the N-terminal part of Bt Cry proteins. One of the examples of N-terminal peptidnogo fragment is a follower of the awn amino acids MTSNGRQCAGIRP (SEQ ID NO: 129), which is not derived from Bt Cry protein.

"Coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, mark, sense RNA or antisense RNA. Preferably, then this RNA was broadcast in the body for the production of protein.

In the context of the present invention "linking" nucleic acids means that the two or more nucleic acids are combined together using any means known in the industry. For example, non-limiting, for example, that the nucleic acid can be ligitamate together using, for example, DNA ligase or connect annealing using PCR. Nucleic acids also can be linked by chemical synthesis of nucleic acids using a sequence of two or more separate nucleic acids.

"Struggle" with insects means obstruction through toxic effects the ability of insect pests to survive, to grow, to eat and/or to reproduce or limit damage or loss of crops of cereals caused by insects. "Fight" with the insects might mean or not mean the destruction of them, although in the preferred embodiment, it means the destruction of insects.

In the context of the present invention, the term "corresponds" means that when the selected amino acids in certain proteins (e.g., Bt Cry proteins or modified Chua-proteins) to combine with each other, the amino acids, which coincide with certain numbered positions, for example, the toxin Shua (or SEQ ID NO: 68, or SEQ ID NO: 134); the toxin Cry3A055 (SEQ ID NO: 70) or Cry1Ab toxin (SEQ ID NO: 72), although they do not necessarily have to be exactly in these numbered positions in relation to the reference amino acid sequence, especially in relation to the identification of domains I, II and III, and/or conservative blocks and variable regions, the positions of these amino acids correspond" to each other. For example, in the scheme of domain I of the hybrid protein amino acids 11-244 Cry3A055 protein (SEQ ID NO: 70) correspond to amino acids 58-290 native protein Shua (SEQ ID NO: 135), or amino acids 11-243 native protein Shua (SEQ ID NO: 68), or amino acids 33-254 native Cry1Ab protein.

In the context of the present invention, the words "Cry-protein" may be used interchangeably with the words "Delta-endotoxin or Delta-endotoxin".

In the context of the present invention "engineered hybrid insecticidal protein" (bend) is an insecticidal protein, created by the merger of unique combinations of variable regions and conservative blocks from at least two different Cry proteins. Such newly created bend may contain complete or incomplete variabeln the e plots conservative blocks or domains of the modified protein Shua and of the Cry protein that is different from the modified protein Shua. The bends of the present invention may, optionally, include protoxin tail section of the Bt Cry protein or N-terminal pipidinny fragment, or both. In non-limiting example, the fold is created by combining (in the direction from N-Terminus to C-end) amino acids 1-468 Cry3A055 protein (SEQ ID NO: 70), which includes variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-648 Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acids of the conservative block 3, variable phase 4, the conservative block 4, variable area 5, the conservative block 5 and the variable area 6, and area of 38 amino acids of the tail section of protoxin Cry1Ab. Designed KILLED containing N-terminal pipidinny fragment, may also be called "chimeric insecticidal protein" (HIB).

"To deliver" bend means that this bend comes into contact with insects, resulting in toxic effects and control of insects. Shipping fold can be done in different ways, for example by expression fold transgenic plant, using the left protein compositions (compositions), sprayable protein compositions (composition), using bait matrix, or by using any other known in the industry delivery systems of toxins.

"Amount effective for controlling insects" means such concentration of bending by toxic exposure interferes with the ability of insects to survive, to grow, to feed and/or reproduce, or limits caused by insect damage or loss of the harvest of cereals. "Amount effective for controlling insects" may mean or not mean the destruction of insects, although it is preferable that it meant the destruction of insects.

The term "expression cassette" is a nucleic acid sequences capable of directing expression of a particular nucleotide sequence in an appropriate cell host containing a promoter operable associated with significant nucleotide sequence that is operable linked to termination signals. In a typical case, it also contains sequences required for proper translation of the relevant nucleotide sequence. At least one of the components of the expression cassette, containing significant nucleotide sequence may be heterologous with respect to at least one of the other components is tov. This expression cassette may be such that occurs in nature, but was obtained in recombinant form, useful for heterologous expression. However, in a typical case, the expression cassette is heterologous in relation to the owner, i.e. a specific sequence of nucleic acids of this expression cassette does not occur in nature in the cell host, and must be entered in the cell is the owner or a predecessor of the host cell by transformation. The expression of the nucleotide sequence may be under the control konstitucijnogo promoter or inducible promoter that initiates transcription only when a host cell is opened for a specific external stimulus. In the case of a multicellular organism, such as plants, the promoter can also be specific to a particular tissue or organ or stage of development.

"Gene" is a defined region located within the genome, which, in addition to the above coding sequence of nucleic acids, also contains other, mainly regulatory, nucleic acids, responsible for the management of expression, that is, so to speak, for the transcription and translation of the coding part. The gene may also contain other 5' and 3' untranslated sequence and the termination settlement is egovernance. Others present elements can be, for example, the introns. The regulatory sequence of nucleic acids in a gene may not be normally operatively associated with an associated sequence of nucleic acids, as it occurs in nature, and in this case, the gene is a chimeric gene.

The term "significant gene" refers to any gene that when you move into the plant gives this plant the desired characteristics, such as resistance to antibiotics, resistance to viruses, resistance to insects, resistance to disease or resistance to other pests, herbicide tolerance, improved nutritional value, improved industrial significant qualities or altered reproductive capability. "Significant gene may be a gene that are transplanted in plants for the production of commercially valuable enzymes or metabolites in a plant.

"Heterologous" nucleic acid sequence is a nucleic acid sequence that is not naturally associated with the host-cell, in which it has been introduced, including artificial multiple copies of the natural nucleic acid sequence. The heterologous amino acid sequence is one that is not naturally associer the bathroom with a native amino acid sequence, for example, the amino acid sequence of SSU protein.

"Homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with the host-cell into which it has entered.

"Homologous recombination" is a mutual exchange of nucleic acid fragments between homologous molecules of nucleic acids.

The term "identity" or "percent identity" refers to the degree of similarity between two nucleic acid or protein sequences. For comparison of sequences in the typical case, one sequence serves as a reference (reference), which compares the test sequence. When using the comparison algorithm, test sequences and reference sequences are input into the computer, if necessary, the coordinates of the subsequence, and the parameters of the program algorithm. The algorithm then compare sequences calculates the percentage identity of the test sequence (sequences) with respect to a reference sequence based on the specified parameters.

Optimal alignment of sequences for comparison can be made, for example, using the algorithm of the local homology presented in the work of Smith Waterman, Adv. Appl. Math. 2: 482 (1981), algorithm combining homology presented in the work of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by way of finding details presented in the work of Pearson and Lipman, Proc. NAT'l. Acad. Sci. USA 85: 2444 (1988), computer implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the software package Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Ausubel and others, below).

One example of an algorithm suitable for determining percent sequence identity and similarity of sequences is the algorithm BLAST, described in Altschul and others, J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is available through the national center for biotechnology information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first finding the pairs with the highest degree of identity (RSI) by identifying short words of length W in the test sequence that either completely match or satisfy some positive threshold value T when combined with the word of the same length from the sequence obtained in the database. T is the threshold proximity of the word (Altschul and others, 1990). These initial finding proximity of words (matches) serve as a seed to initiate the search for more long of the MBC, containing these words Then these word match extended in both directions along each sequence for as far how can increase the total value of points for the match. Aggregate values are calculated using (for nucleotide sequences) of the parameters M (bonus points awarded for a pair of matching residues; he is always> 0) and N (penalty score for mismatching residues; he is always <0). To calculate aggregate values for the amino acid sequence applies a matrix scoring. The extension of matching words in each direction stops when the total value of points for the match falls from the maximum value achieved by the value of X when the cumulative value of the account falls to zero or below zero due to the accumulation of one or more negative results match, or when reaching the end of any of the sequences. The parameters W, T and X of the BLAST algorithm to determine the sensitivity and speed of convergence. The BLASTN program (for nucleotide sequences) default word length (W) equals 11, the expected value (E) is equal to 10, the fall (cutoff) is equal to 100, M=5, N=-4, and a comparison is performed on both chains. For amino acid sequences, the BLASTP program defaults a word length (W) is equal to 3, the expected value (E) is equal to 10, and uses the matrix BLOSUM62 scoring (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89 of 10,915 (1989)).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see,e.g., Karlin and Altschul, Proc. NAT'l. Acad. Sci. USA 90: 5873-5787 (1993)). One of the variables determining the similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), showing the probability of a match between two nucleotide or amino acid sequences may occur occasionally. For example, the test sequence of the nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequences with the reference sequence

nucleic acid is less than 0.1, more preferably less than 0.01, and most preferably less than about 0.001.

Another widely used and accepted computer program comparison of sequences is the CLUSTALW vl.6 (Thompson and others, Nuc. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids divided by the total number of bases or amino acids and multiplied by 100 to obtain the percentage identity. For example, if two sequences of 580 base pairs coincide 145 bases, these sequences will be identical by 25 percent. If two srawniwa the presented sequences have different lengths, the number of matches divided by the smaller of the two lengths. For example, if the comparison of proteins from 200 and 400 amino acids coincided 100 amino acids, the identity of their 50 percent relative to the shorter sequence. If a shorter sequence contains less than 150 bases or 50 amino acids, the number of matches divided by 150 (for nucleic acid bases) or 50 (for amino acids) and multiplied by 100 to obtain the percentage identity.

Another indication that two nucleic acids are substantially identical is that the two molecules hybridize to each other in tough conditions. The phrase "hybridized specific for" refers to the binding, dupliciranja or hybridizing of a molecule only to a particular nucleotide sequence in stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The phrase "associated in General" refers to complementary hybridization between the nucleic acid probe and nucleic acid-target and embraces minor mismatches that can be overcome by reducing the stiffness of the environment hybridization in order to achieve the desired detection of nucleic acid target sequence.

"Stringent hybridization conditions" and "ill which such conditions are hybridization washing" in the context of experiments on hybridization of nucleic acids, such as southern and Northern hybridization, are dependent on the sequence and are different under different environmental parameters. Longer sequences hybridize specifically to elevated temperatures. Extensive information on hybridization of nucleic acids is presented in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, new York. In General, the temperature for hybridization and washing under conditions of high stringency is chosen by approximately 5°C below the melting temperature (TPL) for the specific sequence at a defined ionic strength and pH. In the typical case, when "hard conditions" the probe will be hybridisierung to its target sequence, but to no other sequences.

Temperature TPLrepresents the temperature (at specific values of the ionic strength and pH)at which 50% of the target sequence's hybrid to perfectly matching probe. For very severe conditions, the temperature is chosen equal to TPLfor a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a southern or Northern blot is a: 50% forms the MFA with 1 mg of heparin at 42°C, hybridization is performed overnight. The example of washing under conditions of high stringency: 0.1 to 5M NaCl at 72°C for approximately 15 minutes. An example of stringent conditions washing: washing twice in standard SSC solution at 65°C for 15 min (for a description of SSC buffer, see, Sambrook, below). Often washing under conditions of high rigidity is preceded by washing under conditions of low stringency to remove the original probe signal. An example of a wash in terms of the average stiffness for the duplex containing, for example, more than 100 nucleotides: 1x SSC at 45°C for 15 minutes. The example of washing under conditions of low stringency for duplex containing, for example, more than 100 nucleotides: 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., from about 10 to 50 nucleotides) stringent conditions typically mean that the salt concentration is less than 1.0 M Na ions, typically from about 0.01 to 1.0 M Na ions (or other salts) at pH 7.0 to 8.3, and the typical value of the temperature of at least about 30°C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. In General, the signal-to-noise ratio greater than 2 (or more) times the value obtained for an indefinite probe in this particular sample for hybridization indicates detecting the position of a specific hybridization. Nucleic acid, not hybridizers to each other in tough conditions, however, are generally identical, if otherwise identical proteins encoded by them. This happens, for example, if a copy of a nucleic acid is created using the maximum degeneration codons, valid data of the genetic code.

The following are examples of sets of conditions of hybridization/wash that can be used to clone homologous nucleotide sequences that are generally identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence in the optimal variant's hybrid to a reference nucleotide sequence in 7% dodecyl sodium sulfate (LTOs), 0.5 M NaPO4, 1 mm EDTA at 50°C With washing: 2X SSC, 0.1% of LTOs at 50°C, more preferably in 7% dodecyl sodium sulfate (LTOs), 0.5 M NO4, 1 mm EDTA at 50°C With washing: IX SSC, 0.1% of LTOs at 50°C, more preferably in 7% dodecyl sodium sulfate (LTOs), 0.5 M NaPO4, 1 mm EDTA at 50°C With washing: 0,5X SSC, 0.1% of LTOs at 50°C, preferably in 7% dodecyl sodium sulfate (LTOs), 0.5 M NaPO4, 1 mm EDTA at 50°C With washing: 0,1X SSC, 0.1% of LTOs at 50°C, even more preferably B7% dodecyl sodium sulfate (LTOs), 0.5 M NaPO4, 1 mm EDTA at 50°C With washing: 0,1X SSC, 0.1% of LTOs at 65°C.

Gave the further evidence, the two nucleic acids or two proteins are substantially identical is that the protein encoded first nucleic acid is immunologically cross-reactive with protein, encoded second nucleic acid, or specifically binds with him. Thus, in a typical case, one protein is substantially identical to another protein, if these two proteins differ only by conservative substitutions.

The term "insecticide" means toxic biological activity capable of combating insects, preferably destroying them.

The sequence of nucleic acids is isakovavenue with a reference sequence if the sequence of the nucleic acid encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference sequence of nucleic acids.

"Isolated" nucleic acid molecule or an isolated toxin represent the nucleic acid molecule or toxin that due to man's actions are separate from their natural environment and therefore are not a product of nature. An isolated nucleic acid molecule or an isolated toxin may exist in a purified form or may exist in pastest the military environment, for example (which means no limitations) such as recombinant microbial cell, a plant cell, plant tissue or plant.

"The modified toxin Shua" or "Shua" the present invention relates to a toxin derived from Chua and having at least one secondary site recognition protease that is recognized by a protease of the intestine of the insect target and does not occur in nature in the toxin Shua, as described in U.S. patent 7,030,295 included here by reference.

"Modified the coding sequence Shua" according to the present invention can be obtained from the native coding sequence Shua or synthetic sequence Shua and contains the coding sequence for at least one additional site recognition protease that does not occur naturally in an unmodified gene Shue.

"Nucleic acid molecule" or "nucleic acid sequence" is a segment of a single - or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention the nucleic acid molecule in a typical case is a segment of DNA.

The term "plant" refers to any plant at any stage of development, in particular to seed plants.

"Rastitel the I cell is a structural and physiological unit of a plant, consisting of protoplast and the cell wall. The plant cell may be in the form of an isolated single cell or cultivated cells or be part of a more complex unit such as plant tissue, organ, plant or the whole plant.

"The plant cell culture" means a culture of the structural units of the plant, such as, for example, protoplasts, cells, cell cultures, cells in plant tissues, pollen, pollen tubes, ovules, embryo bags, eggs and embryos at different stages of development.

"Plant material" refers to the leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, slices, cell cultures or tissue cultures, or any part or product of a plant.

"Plant organ" is distinguishable and visually structured and differentiated part of a plant such as root, stem, leaf, flower Bud, or embryo.

The term "plant tissue" refers to a group of plant cells organized into structural and functional unit. This includes any plant tissue in planta or in culture. This term includes (not limited to) whole plants, plant organs, plant seeds, tissue culture, and any group of plant cells organized in a structure the structural and/or functional units. The use of this term in conjunction with (or without) any particular type of plant tissue listed above or otherwise falling under this definition, does not have as its purpose the exclusion of any other type of plant tissue.

"Promoter" - netransliruemye DNA sequence located to the coding region and containing space for binding RNA polymerase and initiating transcription of DNA. The promoter region may also include other elements, working with regulators of gene expression.

The term "regulatory elements" refers to sequences that are included in the control expression of the nucleotide sequence. Regulatory elements include a promoter operable associated with significant nucleotide sequence, and termination signals. In the typical case, they also encompass sequences required for proper translation of the nucleotide sequence.

"Transformation" is a process for introducing heterologous nucleic acid into the cell is the owner or in the body. In particular, the "transformation" means stable integration of the DNA molecule into the genome of the organism of interest.

"Transformed / transgenic / recombinant" refers to the body of the host, such as a bacterium or plant, the who introduced a heterologous nucleic acid molecule. This nucleic acid molecule can be stably integrated into the host genome, or the nucleic acid molecule can also be present as an extrachromosomal molecule. This extrachromosomal molecule may be reproduce or infect other programs. It should be understood that the transformed cells, tissues or plants include not only the final products of the transformation process, but also their transgenic offspring. The terms "untransformed", "transgenic" or "recombinant" refer to a natural organism, the host, such as bacteria or plant that does not contain the heterologous nucleic acid molecule.

Nucleotides are indicated by their bases by the following standard shorthand: adenine (a), cytosine (C), thymine (T) and guanine (G). Similarly, the amino acids are indicated by the following standard shorthand: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (No; 1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), Proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tight; Y) and valine (Val; V).

Description of the INVENTION

The present invention relates to new constructed hybrid insect cidnum proteins (bend), designed to have activity at least against the Western corn root of the beetle, but can also be active against Northern corn root beetle, Mexican corn root beetle and/or the Colorado potato beetle. Some bends have activity against lepidopteran pest, the European corn borer. These new folds are produced by merging unique combinations of complete or incomplete variable regions and conservative blocks, at least two different Cry proteins and, optionally, include protoxin tail region of the Bt Cry protein at the C-terminal part, or N-terminal pipidinny fragment, or both. Non-limiting example is the combination of complete or incomplete variable regions and conservative blocks from the first SGU-protein with activity against Coleoptera, with complete or incomplete variable plots and conservative blocks from the second SGU-protein with activity against Lepidoptera and different from the first Bt SGU-protein, as well as the inclusion of choice protoxin tail section of the Bt SGU-protein at the C-terminal part, or N-terminal peptidnogo fragment, or both, resulting in a new engineered hybrid insecticidal protein, the activity against because a whole range of insects and different from the first or second parent Cry-proteins or both. These bends can also contain a complete or incomplete variable plots, conservative blocks or domains of the modified Chua-protein and SSU protein that is different from the modified Chua protein. N-terminal pipidinny fragment or protoxin tail section can give bend insecticidal activity, can do insecticidal activity fold higher than that of the bend, not containing peptidnogo fragment or protoxin tail section, and/or may make the fold more stable than bend, not containing peptidnogo fragment or protoxin tail section, in particular against at least, the Western corn root of the bug. The amino acid sequence of peptidnogo fragment in a typical case is heterologous to the Bt Cry protein (i.e., not derived from it). However, on the basis of the material presented here by a qualified specialist will see that the N-terminal pipidinny fragment can be created using the amino acid sequence derived from a Bt Cry protein. It turned out that bends according to the present invention have unexpectedly remarkable toxicity against corn root beetle, especially the Western, Northern and Mexican corn beetles. The present invention also relates to nucleic acids, the result is the expression of which are the bends, and to the production and application of bends to combat insect pests. The result of the expression of these nucleic acids are bends that can be applied to combat beetles insects such as Western, Northern and Mexican corn root beetles, or to apply for the fight against lepidopteran insects such as European corn borer, especially when they are expressed in transgenic plants, such as transgenic corn.

In one implementation the present invention encompasses engineered hybrid insecticidal protein containing the amino acid sequence from the first Bacillus thuringiensis (Bt) Cry proteins, including full or part-variable plots and conservative blocks of the first Cry protein, fused with the amino acid sequence from the second Bt Cry protein, different from the first Bt Cry protein, including full or part-variable plots and conservative blocs second Cry protein, and, optionally, containing: (a) protoxin tail plot of Bt Cry protein, located at the s-end; or (b) N-terminal pipidinny fragment, or both (a)and (b), and this fold has activity against at least the Western corn root of the bug.

In another execution of the present invention comprises a fold containing the N-terminal part of the first Bt SGU-the tree, fused with the C-terminal region of the second Bt SGU-protein, different from the first Bt SGU-protein, and at least one position of the crossover between the first and second Bt Cry protein is located in the conservative block 2, conservative block 3, variable area 4 or conservative block 4, and, optionally, containing (a) protoxin tail section Bt SGU-protein, located at the s-end; or (b) N-terminal pipidinny fragment, or both (a)and (b)and this fold has activity against at least the Western corn root of the bug.

In another implementation bend according to the invention contains (in the direction from N-Terminus to C-end) variable plot 1 or C-terminal part of the variable segment 1, the conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal part of the conservative block 3 of the first Bt SGU-protein, broshennye with the C-terminal part of the conservative block 3, variable plot 4, conservative block 4, variable plot 5, conservative block 5 and variable section 6 of the second Bt SSU protein.

In another implementation bend according to the invention contains at least two positions of the crossover between the amino acid sequence of the first Bt SGU-protein and the amino acid sequence of the second Bt SSU protein. In one implementation, the first position of the crossover is located in the conservative block 2, and the second position of the crossover is located in the conservative block 3. In another execution of the first connection crossover is located in the conservative block 3, and the second position of the crossover is located in the conservative block 4.

In another implementation bend according to the invention contains C-terminal part of the tail section Bt SSU protein. Protoxin tail section can impart a bend insecticidal activity (this means that this fold without protoxin tail section would not be active), you may do the activity fold higher than the bend without protoxin tail section, or may make the fold more stable than bend without protoxin tail section. In one version of the invention protoxin tail section is taken from Bt SGU-protein with activity against Lepidoptera. In another implementation protoxin tail section is taken from Cry1A protein. In another implementation protoxin tail section is taken from the protein Cry1Aa or Cry1Ab. Protoxin tail section according to the present invention may include protoxin tail Bt Cry protein or any fragment. In one aspect of this performance protoxin tail bend area contains at least 38 amino acids from the N-Terminus protecting tail Cry1Ab protein. In another aspect of this performance protoxin the tail section contains an amino acid sequence that corresponding to amino acids 611-648 sequence SEQ ID NO: 72. In another aspect of this performance protoxin tail section contains amino acids 611-648 sequence SEQ ID NO: 72.

In another implementation fold contains N-terminal pipidinny fragment. This N-terminal pipidinny fragment can give this fold insecticidal activity (that is, without the N-terminal peptidnogo fragment of this protein will not possess insecticidal activity), or N-terminal pipidinny fragment can do insecticidal activity fold higher than the bend without N-terminal peptidnogo fragment, or N-terminal pipidinny fragment can do fold more stable than bend without N-terminal peptidnogo fragment. In one aspect of this performance pipidinny fragment contains the amino acid sequence that is heterologous Bt Cry protein (i.e., not derived from it). In another aspect of this performance N-terminal pipidinny fragment contains at least 9 amino acids. In another aspect of this performance pipidinny fragment contains at least, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90 or 100 amino acids. In another aspect of this performance pipidinny fragment contains more than 100 amino the slot. In another aspect of this performance N-terminal pipidinny fragment contains the amino acid sequence of YDGRQQHRG (SEQ ID NO: 133) or TSNGRQCAGIRP (SEQ ID NO: 134). In another aspect of this performance N-terminal pipidinny fragment contains an amino acid sequence selected from the group consisting of sequences SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132.

Another performance variable plots and conservative blocks of the first Cry protein, active against Coleoptera, for obtaining bend according to the invention are used in combination with variable sections and conservative blocs second Cry protein, active against lepidopteran insect. The list of Cry-proteins active against Coleoptera, includes (not limited to) proteins Shu, Shu, Shu and Shu/Shu. The list of SSU proteins which are active against lepidopteran insects, includes (not limited to) proteins Cry1 and SGU. In one aspect of the performance of the first SGU-protein is a protein Shua, and the second Cry protein - protein Shua. In another aspect, the protein Shua may be replaced by a modified Chua, such as protein Chua described in U.S. patent 5,659,123 included here by reference. In another aspect of this performance protein Shua is a protein Shua and protein Cry1A protein Cry1Aa or Cry1Ab. In another aspect of this performance b the Lok Shua is selected from the following group and has the specified access number in GenBank: Cry3Aa1 (M), Cry3Aa2 (J02978), Shua (Y00420), Cry3Aa4 (M), Cry3Aa5 (M), Shua (U10985), Cry3Aa7 (AJ237900), Cry3Aa8 (AAS79487), Cry3Aa9 (AAW05659), Shua (AAU29411) and Shua (AY882576). In another aspect of this performance protein Cry1Aa is selected from the following group and has the specified access number in GenBank: Cry1Aa1 (M11250), Cry1Aa2 (M10917), Cry1Aa3 (D00348), Cry1Aa4 (X13535), Cry1Aa5 (D17518), Cry1Aa6 (U43605), Cry1Aa7 (AF081790), Cry1Aa8 (126149), Cry1Aa9 (AB026261), Cry1Aa10 (AF154676), Cry1Aa11 (Y09663), Cry1Aa12 (AF384211), Cry1Aa13 (AF510713), Cry1Aa14(AY197341) and Cry1Aa15 (DQ062690). In another aspect of this performance Cry1Ab protein is selected from the following group and has the specified access number in GenBank: Cry1Ab1 (M), Cry1Ab2 (M), Cry1Ab3 (Ml5271), Cry1Ab4 (D00117), Cry1Ab5 (X04698), Cry1Ab6 (M37263), Cry1Ab7 (X13233), Cry1Ab8 (M16463), Cry1Ab9 (X54939), Cry1Ab10 (A29125), Cry1Ab11 (112419), Cry1Ab12 (AF059670), Cry1Ab13 (AF254640), Cry1Ab14 (U94191), Cry1Ab15 (AF358861), Cry1Ab16 (AF37560), Cry1Ab17 (AAT46415), Cry1Ab18 (AAQ88259), Cry1Ab19 (AY847289), Cry1Ab20 (DQ241675), Cry1Ab21 (EF683163) and Cry1Ab22 (ABW87320). In another aspect of this performance of the first Cry protein contains an amino acid sequence selected from the group consisting of: SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 135, and the second Cry protein contains an amino acid sequence that is recorded in SEQ ID NO: 72.

In one implementation the present invention comprises a fold in which there is at least one position of the crossover between the N-terminal site of the first SGU-protein and C-terminal part of the second SGU-protein, located in the conservative block 3, variable section 4 or in the conservative block 4. In one aspects is E. this performance this position crossover in the conservative block 3 is located immediately behind the amino acid the corresponding Ser451, Phe454, or Leu468 sequence SEQ ID NO: 70. In another aspect of the execution of this position crossover is located in the conservative block 3 immediately after Ser451, Phe454 or Leu468 sequence SEQ ID: 70, or after Ser450, Phe453 or Leu467 sequence SEQ ID NO: 68; or Ser497, Phe100, Leu114 sequence SEQ ID NO: 135. The position of the crossover in some implementations, bend with Shua/Cry1Ab or modified Chua/Cry1Ab execution of the bends of the present invention shown in figure 2, indicating the percentage identity.

In another implementation bend according to the present invention contains at least two positions of the crossover between the amino acid sequence from the first Bt Cry protein and the amino acid sequence from the second Bt Cry protein. In one aspect of this performance one position crossover between Shua or modified Chua and Cry1Ab or Cry1Aa is conservative block 2 immediately after the amino acids corresponding to Asp232 sequence SEQ ID NO: 70, and the second position of the crossover between Cry1Ab and Shua or modified Chua is conservative block 3 immediately after the amino acids corresponding to Leu476 sequence SEQ ID NO: 72. In another aspect of the execution position of crossing over between Shua or modified Chua and Cry1Ab or Cry1Aa is conservative block 2 immediately pic the e amino acids, the corresponding Asp232 sequence SEQ ID NO: 70, or Asp231 sequence SEQ ID NO: 68, or Asp278 sequence SEQ ID NO: 135 and the second position of the crossover between Cry1Ab and Shua or modified Chua is conservative block 3 immediately after the amino acids corresponding to Leu476 sequence SEQ ID NO: 72.

In another aspect of this performance the first position of the crossover between proteins Shua or modified Chua and Cry1Ab is conservative block 3 immediately after the amino acid corresponding to Leu468 sequence SEQ ID NO: 70, and the second position of the crossover between Cry1Ab and Shua or modified Chua is conservative unit 4 immediately after the amino acids corresponding to Ile527 sequence SEQ ID NO: 72. In another aspect of this performance the first position of the crossover between proteins Shua or modified Chua and Cry1Ab is conservative block 3 immediately after the amino acid corresponding to Leu468 sequence SEQ ID NO: 70 or Leu467 sequence SEQ ID NO: 68, or Leu114 sequence SEQ ID NO: 135 and the second position of the crossover between Cry1Ab and Shua or modified Chua is conservative unit 4 immediately after the amino acids corresponding to Il527 sequence SEQ ID NO: 72. In another aspect of this execution fold contains the amino acid sequence of SEQ I NO: 28 or SEQ ID NO: 34.

In one implementation the present invention comprises a bend, in which the first Cry protein is represented by a protein Shua or modified Chua, and the second Cry protein is represented by a protein Cry1Aa or Cry1Ab, and this fold contains an amino acid sequence at least 80% identical to SEQ ID NO: 64. In another implementation fold contains the amino acid sequence, at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 64. The combination of various versions of the present invention with the sequence SEQ ID NO: 64 is presented in figure 2, which shows the percentage of identity.

In another execution of the present invention comprises a bend, in which the first Cry protein is represented by a protein Shua or modified Chua, and the second Cry protein is represented by a protein Cry1Aa or Cry1Ab, and this fold contains an amino acid sequence at least 75% identical to SEQ ID NO: 70. In another implementation fold contains the amino acid sequence, at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 70. The combination of various versions of the present invention with the sequence SEQ ID NO: 70 is presented in figure 1, which shows the percentage of identity.

In another execution of the present invention comprises a bend, which has a first position of the crossover is between Shua or modified Chua and Cry1Aa or Cry1Ab in the conservative block 2 and the second position of the crossover between Cry1Aa or Cry1Ab and Shua or modified Chua in the conservative block 3 and which contains the sequence amino acids, at least 56% identical to SEQ ID NO: 64. In one aspect of the execution of the bend is at least 60, 70 or 80% identity with the sequence SEQ ID NO: 64. In another aspect of the execution of the bend is at least, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 64.

In another execution of the present invention comprises a fold containing an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 62; SEQ ID NO: 64, SEQ ID NO: 147, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 160.

In one implementation bend according to the present invention has activity against other insect pests, including but not limited to) Northern corn root beetle, Mexican corn root beetle, Colorado potato beetle and/or the European corn borer.

In another implementation, the invention encompasses a nucleic acid molecule containing a nucleotide sequence that encodes a bend according to the present invention. In one aspect of the execution of this molecule nucleic acid contains a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID O: 29, SEQ ID NO: 33, SEQ ID NO: 61; SEQ ID NO: 63, SEQ ID NO: 146, SEQ ID NO: 152, SEQ ID NO: 154 and SEQ ID NO: 158. Explanation of specific examples of ways to create a nucleic acid molecule which encodes a bend, can be found in the Examples 1-41. Professionals in this industry will see what modifications you can make in these examples, the methods for creating bends according to the present invention.

The present invention also encompasses the expression cassette containing the nucleic acid molecule and recombinant vectors and transgenic cells are the masters, not from the human body, such as bacterial cells or plant cells, in which are placed the expression cassettes according to the invention.

The present invention also encompasses recombinant vectors containing the nucleic acids of the present invention. In such vectors, the nucleic acid is optimally contained in the expression cassette comprising the regulatory elements for expression of the nucleotide sequences in the cell-master, suitable for the expression of such nucleotide sequences. Such regulatory elements typically include a promoter and termination signals, and ideally also contain elements that allow you to perform effective translation of polypeptides encoded by nucleic acids of the present and the gain. Vectors containing these nucleic acids may be capable of replication in a specific cell-hosts, preferably as an extrachromosomal molecule, and thus are used to increase nucleic acids of the present invention in the cells of the host. In one performance cells-owners for such vectors are microorganisms, such as bacteria, particularly Bacillus thuringiensis or E. coli. Different cells-hosts for such recombinant vectors are the endophyte or epiphytes. In another execution of such vectors are viral vectors and used for replication of nucleotide sequences in a particular cell-hosts such as insect cells or plant cells. Recombinant vectors are also used for transformation of the nucleotide sequences of the present invention in cells of the host, whereby these nucleotide sequence is stably integrated into the DNA transgenic host. In one version of such transgenic host is a plant, such as corn.

Test bends according to the present invention in the bioassay showed that they have activity against insects. In one version of the bends according to the invention is active against Coleoptera, or against lepidopteran insects, or against those and others who. In one aspect of this execution bends according to the invention is active against Western corn root beetle, Northern corn root beetle, Mexican corn root beetle and/or the Colorado potato beetle. In another aspect of this performance bends according to the invention is active against European corn borer. Properties of bends on insect is illustrated in the Examples below, 43, 45 and 46.

The present invention also encompasses compositions containing a bend in a quantity effective against insects.

In another implementation, the invention encompasses a method for the production of bend, active against insects, comprising that: (a) receive a cell host containing a gene which itself contains heterologous sequence is a promoter operatively associated with a nucleic acid molecule according to the invention; and (b) growing the transgenic cell is a host in such a way as to Express the bend, active against insects.

In another implementation of the invention encompasses a method for the production of transgenic plants resistant to insects, consisting in the introduction of the nucleic acid molecules according to the invention in a transgenic plant, whereby the nucleic acid molecule induces the expression fold in the transgenic plant in an amount effective to combat what becomemy. In one aspect of the execution of such insects are beetles or lepidopteran insects. In another aspect of the execution of such beetles insects are Western corn root beetle, Northern corn root beetle, Mexican corn root beetle and/or the Colorado potato beetle. In another aspect of the execution of this lepidopteran insect is the European corn borer.

In another implementation of the invention encompasses a method of combating insects comprising feeding insects an effective amount of bend according to the invention. In one aspect of the execution of such insects are beetles or lepidopteran insects. In another aspect of the execution of such beetles insects are Western corn root beetle, Northern corn root beetle, Mexican corn root beetle and/or the Colorado potato beetle. In another aspect of the execution of this lepidopteran insect is the European corn borer. In the typical case filing bend insects is carried out orally. In one aspect, the bend is served by the oral via transgenic plant containing the amino acid sequence expressing a bend according to the present invention.

The present invention also covers a method of combating n is Contracting out, in which transgenic plant also contains a second nucleic acid molecule or group of molecules of the nucleic acid encoding the second pesticide element. Examples of this second nucleic acid can be a nucleic acid encoding a Bt Cry protein, and encoding vegetative insecticidal protein described in U.S. patent 5,849,870 and 5,877,012 included here by reference, or those that encode the channel for the production of non-protein insecticidal elements.

The present invention also encompasses a method of obtaining engineered hybrid insecticidal protein (fold), namely that: (a) receive the first Bt Cry protein or modified Bt Cry protein; (b) receive a second Bt Cry protein that is different from the first Bt Cry protein or modified Bt Cry protein; (C) combine full or part-variable plots and conservative blocks of the first Bt Cry protein or modified Bt Cry protein with complete or incomplete variable plots and conservative blocs second Bt SGU-protein for the formation of a bend having activity against at least the Western corn root beetle; and, optionally, (d) impose pipidinny fragment in the N-terminal part of the bend, or protoxin tail section Bt SGU-protein C-terminal part of the fold, or both, and these N-terminal pipidinny fragment, and the C-terminal protoxin plot, or both give the bend insecticidal activity, or increase the insecticidal activity of bend, or do fold more stable than bend without peptidnogo fragment, or protoxin tail section, or both.

In another implementation, the invention encompasses a method of manufacturing the engineered hybrid insecticidal protein (fold), namely that: (a) receive a first nucleic acid encoding a first Bt Cry protein or modified Bt Cry protein and a second nucleic acid encoding a second Cry protein that is different from the first SGU-protein or modified Bt SGU-protein; (b) separated from the first and second nucleic acid nucleotide sequence encoding a full or part-variable plots and conservative blocks of the first Bt SGU-protein or modified Bt SGU-protein and the second Bt SGU-protein; (C) connecting obtained in step (b) nucleic acid so as to obtain a new hybrid nucleic acid encoding a protein, and, optionally, perform the hybridization of the nucleic acid that encodes pipidinny fragment from the 5' end of the aforementioned hybrid nucleic acid, resulting in the lengthening of the 5' end, or perform the hybridization of the nucleic acid that encodes protoxin tail section Bt SGU-protein with 3' end above the aforementioned hybrid nucleic acid, resulting in elongation of the 3' end, or both; (g) in the expression cassette introduced a hybrid nucleic acid, with or without one or both of the lengthening of the ends of the 5' or 3'; (d) transform this expression cassette into a cell of the host, resulting in above-mentioned a host cell produces a bend; and (e) perform bioimpedance bend against at least, the Western corn root beetle, resulting insecticidal activity against the Western corn root of the bug.

In the following embodiments of methods according to the invention the first Bt Cry protein or modified Bt Cry protein is represented by a protein Shua or modified Chua, and the second Bt Cry protein is represented by A protein Cry1Aa or Cry1Ab.

In another implementation of the methods according to the invention is N-terminal pipidinny fragment contains 9 amino acids. In one aspect of this performance N-terminal pipidinny fragment contains the amino acid sequence of YDGRQQHRG (SEQ ID NO: 132) or the amino acid sequence of TSNGRQCAGIRP (SEQ ID NO: 133). In another aspect of this performance N-terminal pipidinny fragment is selected from the group of sequences consisting of: SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132.

In another execution of the methods of the invention protoxin tail section is taken from Cry1Aa or Cry1Ab. In one aspect of this is the execution of this protoxins plot contains, at least 38 amino acids. In another aspect of this performance protoxin tail section contains a sequence of amino acids corresponding to amino acids 611-648 sequence SEQ ID NO: 72. In another aspect of this performance protoxin tail section contains amino acids 611-648 sequence SEQ ID NO: 72.

Explanation of specific examples of ways to create a hybrid nucleic acid and bends can be found in the Examples 1-41.

In subsequent versions of the nucleotide sequence according to the invention, in particular a sequence encoding pipidinny fragment, protoxin tail and/or conservative blocks 2, 3 and 4, may be modified by the inclusion of random mutations on the technology known as in vitro recombination or DNA shuffling. This technology is described in Stemmer and other, Nature 370:389-391 (1994) and in U.S. patent 5,605,793, included here by reference. Millions of mutant copies of the nucleotide sequence are based on the original nucleotide sequence of the present invention, and selected those options, which have improved properties such as increased insecticidal activity, increased stability or different specificity or range of target pests. The method includes forming mathenesserlaan voicemachine of polynucleotide of the double-stranded template of polynucleotide, containing the nucleotide sequence of the present invention, and double-stranded template polynucleotide was split at random double-stranded fragments of desired length, furthermore, the method includes the steps of: adding to the resulting population of double-stranded random fragments one or more single - or double-stranded oligonucleotides, and the above-mentioned oligonucleotides contain the area of identity and an area of heterology to master the double-stranded polynucleotide; denaturation mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resulting population of single-stranded fragments with a polymerase under conditions which result in annealing of the above-mentioned single-stranded fragments at the areas mentioned above identity for the formation of pairs of annealed fragments, and these the field of identity is sufficient for one member of the pair to perform accurate replication of the second, thus forming mutagenically the double-stranded polynucleotide; and at least two cycles of repetition of the second and third stages, and the mixture in the second stage of the next cycle includes mutagenically the double-stranded polynucleotide of the third stage of the previous cycle, and following the cycle creates the following mutagenically the double-stranded polynucleotide. In a preferred embodiment of the invention, the concentration of single-stranded species of double-stranded random fragments in a population of double-stranded random fragments is less than 1% by weight of the entire DNA. In an even more preferred embodiment, shablony the double-stranded polynucleotide contains at least about 100 species of polynucleotides. In another preferred version the size of double-stranded random fragments is from about 5 BP to 5 KB. In another predpochtitelno performance certworthy step of the method includes repeating the second and third steps for at least 10 cycles.

As a biological insecticide controlling means bends are produced by expression of nucleic acids in heterologous cells hosts suitable for the expression of these nucleic acids. In one version derived cells Century. thuringiensis containing modifications of the nucleic acids of the present invention. Such modifications include mutations or the elimination of existing regulatory elements, leading to altered expression of this nucleic acid, or the inclusion of new regulatory elements controlling the expression of this nucleic acid. The other version to the cells of Bacillus thuringiensis are added up one or more nucleic KIS is from by or insertions in the chromosome, or the introduction of containing nucleic acid molecules that replicate extrachromosomal.

In another implementation, at least one of the nucleic acids according to the invention is introduced into an acceptable expression cassette containing the promoter and signal termination. The expression of the nucleic acid is constituitive, or used inducible promoter that responds to different types of stimuli for initiation of transcription. In another execution of the cage in which is expressed the fold, is a microorganism such as a virus, bacterium or fungus. In another execution of the virus such as baculovirus contains a nucleic acid according to the invention in its genome and expresses large quantities appropriate insecticidal protein after infection with the respective eukaryotic cells, suitable for virus replication and expression of nucleic acids. Thus obtained protein is used as an insecticidal agent. Alternatively, engineered baculoviruses containing the nucleic acid used to infect insects in vivo and kill them or by the expression of insecticidal toxin, or a combination of viral infection and expression of insecticidal toxin.

Cells bacteria cells are also hosts for the expression of kleinova acids according to the invention. In one version used non-pathogenic, symbiotic bacteria that can live and multiply inside the plant tissue, the so-called endophyte, or non-pathogenic, symbiotic bacteria that can colonize phyllosphere or the rhizosphere, the so-called epiphytes. Such bacteria include bacteria of the species Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia, Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas. Symbiotic fungi, such as Trichoderma Gliocladium, can also be hosts for expression invented nucleic acids with the same purpose.

Technology these genetic manipulations are specific for different current owners and is well known in the industry. For example, expressing vectors RK-3 and RK-2 can be used for the expression of heterologous genes in E. coli either transcriptional or translational fusion for the tac-promoter or trc-promoter. For the expression of operons that encode multiple proteins ORF, the simplest procedure is the introduction of the operon in the vector, such as RCC-3, translational fusion that allows you to use the binding site of cognate ribosome heterologous genes. Technology overexpression in gram-positive species, such as Bacillus, also known in the industry and can be used in the context of the present invention (Quax and Dr. who. In: Industrial Microorganisms: Basic and Applied Molecular Genetics, the authors Baltz and others, American Society for Microbiology, Washington (1993)). Alternative systems for overexpression based, for example, in yeast vectors include the use of Pichia, Saccharomyces and Kluyveromyces (Sreekrishna, In: Industrial microorganisms: basic and applied molecular genetics, Baltz, Hegeman, and Skatrud authors, American Society for Microbiology, Washington (1993); Dequin and Barre, Biotechnology L2:173 - 177 (1994); van den Berg and others, Biotechnology 8:135-139 (1990)).

In one implementation, at least one bend according to the invention is expressed in a higher organism such as a plant. In this case, transgenic plants expressing an effective amount of bend, protect themselves from insect pests. When the insect begins to eat such a transgenic plant, it also absorbs fold downregulation. This will keep the insect from further gryzenia in this plant, and can even lead to damage or death of the insect. Nucleic acid of the present invention is inserted in the expression cassette, which can then be stably integrated into the genome of plants. In another implementation, such a nucleic acid is included in non-pathogenic samoreplitsirujushchihsja virus. Plants transformed according to the present invention may be monocots or dvudolnymi and include, but are not limited to, maize, wheat, barley, rye, sweet potato, bean, pea, chicory,lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, zucchini, pumpkin, hemp, zucchini, Apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, black currant, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, alfalfa, rice, potato, eggplant, cucumber, resasco tal, as well as coniferous and deciduous trees.

After moving the desired nucleic acid in a particular kind of plant she can use traditional technologies of cultivation spread in this form or go into other varieties of the same species, particularly including commercially important cultivars.

Preferably, expression of a nucleic acid of the present invention was carried out in transgenic plants, thus causing the biosynthesis of the corresponding bend in these transgenic plants. Thus obtained transgenic plants resistant to insects, in particular corn root beetle. For expression in transgenic plants of nucleic acids according to the invention may require other modifications and optimization. Although in many cases, genes from microbial organisms can be expressed in plants at high levels, the s and without modification, but in transgenic plants may occur and weak expression of microbial nucleic acids with codons that are not preferred in plants. In this industry it is known that all organisms have specific preferences on the use of codons and the codons of the nucleic acids described in the present invention, can be modified to fit the preferences of the plants, at the same time supporting the coded amino acids. Moreover, high expression in plants is achieved from the coding sequence, GC content is at least 35%, more preferably 45%, even more preferably more than 50%, and most preferably more than 60%. Microbial nucleic acids with low GC-content can weakly expressed in plants due to the existence of motives ATTA, which can destabilize messages and motives of AATAAA, which can cause improper, polyadenylation. Although the preferred gene sequences can be adequately expressed in monocotyledonous and dicotyledonous plant species, sequences can be modified to reflect the specific preferences for codons and GC-content in monocots and dicots, as these preferences differ (Murray and others, Nucl. Acids Res. 17:477-498 (1989)). In addition, the implementation of which is screening nucleic acids for the presence of non-standard places splicing, which can cause truncation of messages. All the changes that you want to perform inside of nucleic acids, such as those described above, are performed using well-known technology site-directed mutagenesis, PCR and construction of synthetic genes using the methods described in the published patent applications EP 0385962, EP 0359472 and WO 93/07278.

In one version of the invention the coding sequence of the bend and/or the coding sequence of the parent Bt Cry protein is obtained/received in accordance with the procedure described in U.S. patent 5,625,136 included here by reference. This procedure uses codons that are preferred for corn, that is, a single codon, the most frequently encodes the amino acid in corn. Preferred maize codon for a particular amino acid can be obtained, for example, from known gene sequences for maize. The use of codon corn for 28 genes from maize plants found in the work of Murray and others, Nucleic Acids Research 17:477-498 (1989), the description of which is included here by reference.

In this way, the nucleotide sequence may be optimized for expression in any plant. It is recognized that the entire gene sequence, or any part thereof may be optimized or synthetic. That is, who may also be used synthetic or partially optimized sequence.

For efficient translation initiation sequences adjacent to the initiating methionine, modification may be required. For example, they can be modified by inclusion of sequences that are known for their efficiency in plants. Joshi suggested appropriate typical sequence for plants (NAR 15:6643-6653 (1987)), a Clonetech offers further common initiator broadcast (1993/1994 catalog, page 210). These consensuses are suitable for use with nucleic acids of the present invention. These sequences included in constructs containing nucleic acid to ATG inclusive (leaving unmodified the second amino acid) or alternatively, up to and including GTC, following the ATG (with the possibility of modification of the second amino acid of this transgene).

Expression of nucleic acids in transgenic plants are promoters that function in plants. The choice of promoter will vary depending on the spatial-temporal requirements expression, and depending on the kinds of targets. Therefore, the preferred is the expression of the nucleic acids of the present invention in the leaves, stems or trunks, on the cob, in the inflorescences (e.g., spikes, panicles, bones and the like), in the roots and/or seedlings. However, in many cases seek the protection from more than one type of pest insects, and so desirable expression in many tissues. Although many promoters of dicotyledonous has demonstrated its effectiveness in monocots and Vice versa, in the ideal case for expression in dicotyledonous selected promoters dicots and monocot promoters for expression in monocots. However, there are no restrictions regarding the origin of the selected promoters; enough for them to be effective expression of nucleic acids in the desired cell.

In one version used promoters with constitutive expression, including actin or ubiquitin, or promoters CMP, or the promoters of CaMV 35S and 19S. Nucleic acids of the present invention can also be expressed under the control of promoters with chemical regulation. This allows the bends be synthesized only when the seed plants are processed inducing chemicals. Optimal technology for chemical induction of gene expression is described in published application EP 0332104 (Ciba-Geigy) and in U.S. patent 5,614,395. The optimal promoter for chemical induction is a tobacco PR-1A.

The other version can be used category promoters induced damage. Described many promoters expressed at the site of an injury or infection by pathogenic fungi. In the ideal case is such a promoter should be activated locally in areas of infection, and in this way bend accumulate only in those cells that need to synthesize fold to kill the invading pest. To the best promoters of this type are described in the works of Stanford and others Mol. Gen. Genet. 215:200-208 (1989), Xu and other Plant Molec. Biol. 22:573-588 (1993), Logemann and other Plant Cell 1:151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22:783-792 (1993), Firek and other Plant Molec. Biol. 22:129-142 (1993), and Warner and other Plant J. 3:191-201 (1993).

Preferred tissue or your preferred fabric promoters applicable for the expression of genes encoding bends in plants, particularly maize, are the promoters of performing direct expression in the root, the core, the sheet or the pollen, especially in the root. Such promoters are, for example, selected from RERS or trpA described in U.S. patent No..5,625,136, or from MTL described in U.S. patent No. 5,466,785. Both of these patents are included here fully by reference.

The following performances are transgenic plants expressing the nucleic acid by the method of induction of damage or pathogenic infection.

The development process of hybrid nucleic acids containing genes bend according to the present invention in addition to the promoters used a number of transcription terminators available. The transcription terminators are responsible for the termination of transcription beyond the transgene and for the correct polied melirovanie. Acceptable transcription terminators terminators are known for their function in plants, including the CaMV 35S terminator, the terminator nopalin synthase (NOS), pea rbcS terminator I and others known in the industry. They can be used both in monocots and dicots. In the context of the present invention can be applied to any existing terminator, known for its function in plants.

In the expression cassette described in this invention may be incorporated numerous other sequences. These include sequences that demonstrated the ability to increase the expression, such as intron sequences (for example, from the Adhl and bronzel) and leading viral sequence (e.g., from TMV, MCMV and AMV).

It would be desirable to direct the expression of the nucleic acids of the present invention to different cellular localizations in the plant. In some cases it may be desirable to localize in the cytosol, and in other cases it may be preferred localization in some the subcellular organelle. Subcellular localization of transgene-encoded enzymes is well known in the technology industry. In a typical case, DNA encoding a peptide target of known gene product, aimed at the organelle, directed and killed desireda before nucleic acid. There are many such sequences targeted to the chloroplast, and their function in heterologous structures was shown. The expression of the nucleic acids according to the invention is also directed to the endoplasmic reticulum or in vacuoles of cells of the hosts. Technologies to achieve this are also well known in the industry.

Vectors suitable for transformation of plants, described in different places of this description. To migrate through agrobacteria suitable binary vectors, or vectors carrying at least one border sequence of T-DNA, while for direct gene transfer any suitable vector, and may be preferred linear DNA, including relevant design. In the case of direct gene transfer can be used transformation with a single species of DNA or co-transformation (Schocher and other Biotechnology 4:1093-1096 (1986)). As for direct gene transfer and Agrobacterium-mediated transfer transformation usually (but not necessarily) is selectable marker, which can provide resistance to antibiotics (kanamycin, hygromycin or methotrexate) or a herbicide (basta). Vectors transformation of plants containing genes bend according to the present invention may also contain genes (for example, the isomerase phosphomannose; PMI), which provides a positive CE is the under of transgenic plants, as described in the U.S. patents 5,767,378 and 5,994,629 included here by reference. However, the choice of such a marker is not critical to the present invention.

In another execution of the nucleic acid of the present invention is directly transformed into the plastid genome. The big advantage of plastid transformation is that plastids in General capable of the expression of bacterial genes without significant optimization of codons, and plastids are capable of expression of multiple open reading frames under the control of a single promoter. Technology plastid transformation is widely described in U.S. patent No. 5,451,513, 5,545,817 and 5,545,818, in PCT application no WO 95/16783 and in the work of McBride and others (1994) the OEWG. Nati. Acad. Sci. USA 91,7301-7305. The underlying technology of chloroplast transformation involves the injection sites cloned plastid DNA, bounding a selectable marker together with significant gene in the appropriate tissue target site, for example, using biolistic or protoplast transformation (for example, transformation through calcium chloride or PEG). Bounding areas ranging in size from 1 to 1.5 KB, sequences target, carry out homologous recombination with the genome of plastids and thus allow the replacement or modification of specific sections of the plastome. Initially, point mutations in hannahholocaust 16S rRNA and rps12, giving resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) Proc. Nati. Acad. Sci. USA 87, 8526-8530; Staub, J.M., and Maliga, P. (1992) Plant Cell 4, 39-45). The result is sustainable homoplasmy transformants at a rate of approximately one per 100 bombing leaves target. The presence of cloning sites between these markers has enabled the creation of a plastid-directed vector for introducing foreign genes (Staub, J.M., and Maliga, P. (1993) EMBO J. 12, 601-606). A significant increase in the frequency of transformation was obtained by replacement of the recessive genes for resistance to antibiotics rRNA or R-protein in a dominant selectable marker, the bacterial aadA gene encoding the enzyme aminoglycoside - 3'-adinistrator, neutralizing spectinomycin (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Earlier this token was successfully used for high-frequency transformation of the plastid genome of the green Alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Other selectable markers useful for plastid transformation, also known in the industry and are covered by the scope of the present invention. In a typical case, to achieve homoplastic state takes approximately 15-20 cycles of cell division after transformation. Plastid expression, in which the gene is introduced by homologous recombination in all several thousand copies of the circular plastid genome, present in every cell, takes advantage of the huge number of copies on nuclear gene expression, allowing you to gain levels of expression, easily exceeding 10% of the total soluble plant protein. In a preferred execution of the nucleic acid of the present invention is introduced into the plastid-directed vector and transformed into the plastid genome of the desired plant host. Get homoplasies plant plastid genomes containing nucleic acid of the present invention, preferably capable of high level expression of this nucleic acid.

Bends according to the invention can be used in combination with other pesticide elements (for example, with Bt Cry proteins) with the aim of expanding the range of pests on which they are directed. Moreover, the use of bends according to the invention in combination with modified Chua-toxins, Bt Cry proteins or other elements, active against CCI (corn root beetle), such as Rnci with another type of activity or aimed at other receptors in the digestive tract of an insect, especially useful to prevent and/or regulation of resistance in corn root of the bug. List of other chemical elements includes, but is not limited to, lectins, α-amylase, p is oxidase and cholesterol oxidase. Also useful in combination with bends according to the present invention are Vip genes described in U.S. patent No. 5,889,174 included here by reference.

This coexpressed more than one insecticidal element in one transgenic plant can be achieved by making one recombinant vector containing the coding sequence of more than one chemical element in the so-called molecular stack and the genetic construction of the plant, which would be contained and expressed all of these insecticidal elements. Such molecular stacks can also be obtained by the use of mini-chromosomes, as described, for example, in U.S. patent 7,235,716. In an alternative embodiment, a transgenic plant containing a single nucleic acid encoding a first insecticidal element may be retransformation another nucleic acid that encodes a different insecticidal element, etc. In an alternative embodiment, the plant, Parent 1, can be genetically engineered for expression of genes of the present invention. The second plant, Parent 2, can be genetically engineered for expression of an additional element in controlling insects. The crossing of Parent 1 with Parent 2 gives the progeny plant expressing all genes introduced into Parents 1 and 2.

Transgenic seeds on astasia the invention may also be treated with insecticidal coating, as described in U.S. patent No. 5,849,320 and 5,876,739 included here by reference. If insecticidal coating seed and transgenic seed according to the invention are active against the same insect, such a combination is useful (i) in the method of increasing the activity of the bend according to the invention against such insect and (ii) in the method of preventing the development of resistance to bend according to the invention due to the presence of a second mechanism of action against insect target. Thus, the invention provides a method of increasing the activity against insect-target or prevent the development of resistance to this insect, such as corn root beetle, which consists in applying insecticidal coating on transgenic seeds containing one or more bends according to the invention.

Even if insecticidal coating is actively against another insect, this insecticidal coating is useful to extend the range of insects against which the struggle, for example, by applying insecticidal coating, active against lepidopteran insects on transgenic seeds according to the invention is active against Coleoptera, resulting transgenic seeds with coating will be active against lepidopteran and against Coleoptera insects.

EXAMPLES

Then op is a description of an invention submitted to the following detailed examples. These examples are only illustrative and not intended to be any limitation if it is not specified. Used here is standard recombinant DNA technology or cloning molecules are well known in the industry and are described in J. Sambrook, and other, Molecular Cloning: A Laboratory Manual, third edition, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001); T.J. Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, new York (1984) and Ausubel, F.M. and other, Current Protocols in Molecular Biology, new York, John Wiley and Sons Inc., (1988), Reiter, and other, Methods in Arabidopsis Research, World Scientific Press (1992), and Schultz and others, Plant Molecular Biology Manual, Kluwer Academic Publishers (1998).

Example 1. Parent coding sequences 1

Optimized for maize coding sequence Shua, cry1Ab, cry1Ba, and cry1Fa, denoted here as mashua, mocry1Ab, mocry1Ba and mocry1Fa, were obtained according to the procedure described in U.S. patent 5,625,136, fully included here by reference.

The sequence cry3A055 (SEQ ID NO: 67), encoding a Cry3A055 protein (SEQ ID NO: 68)was obtained by modification of the coding sequence mashua the introduction of a nucleotide sequence that encodes a site of the protease recognition of cathepsin G in domain I, according to the U.S. patent 7,030,295, fully included here by reference.

Sequence mocry AA (SEQ ID NO: 67), encoding a protein represented in SEQ ID NO: 68 cry3A055 (SEQ ID NO: 69), encoding a protein represented in SEQ ID NO: 70, mocry1Ab (SEQ ID NO: 71), encoding a protein represented in SEQ ID NO: 72, mocry1Ba (SEQ ID NO: 73), encoding a protein represented in SEQ ID NO: 74, mocry1Fa (SEQ ID NO: 75), encoding a protein represented in SEQ ID NO: 76, cry8Aa (SEQ ID NO: 77), encoding a protein represented in SEQ ID NO: 78, cry1Ac (SEQ ID NO: 79), encoding a protein represented in SEQ ID NO: 80, and cry1Ia (SEQ ID NO: 81), encoding a protein represented in SEQ ID NO: 82, were used in the construction of coded hybrid nucleic acids and proteins, as described in the following examples.

Example 2. The use of PCR primers for constructing hybrid nucleic acids.

Polymerase chain reaction (PCR) is a recurring, enzyme-initiated primer synthesis of nucleic acid sequences. This procedure is well known and is widely used by specialists in this field (See. Mullis, U.S. patent No. 4,683,195, 4,683,202 and 4,800,159; Saiki, Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim [1985] "Enzymatic Amplification of.beta.-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia," Science 230:1350-1354). PCR-based enzyme significant increase of DNA fragment, the edges of which are two oligonucleotide primers, hybridizers to the opposite chains to a target sequence. These primers are oriented so that their 3' ends facing each other. P is Vtorushina cycles of denaturation by heating, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase result in the increase of the segment defined by the 5' ends of the PCR primers. Because the product of the elongation of each primer can serve as a template for the other primer, each cycle almost doubles the number of DNA fragments obtained in the previous cycle. The result is an exponential accumulation of a specific fragment of the target in several million times in a few hours. Using thermostable DNA polymerase, for example Taq polymerase isolated from thermophilic bacteria Thermus aquaticus, increase process can be done fully automatic.

Chimeric coding sequences described in the following examples were developed using various combinations of examples of primers shown in Table 1. Mixture for PCR reactions and protocols PCR temperature cycles used in the experiments are presented in Tables 2 and 3, respectively. In each of the following examples, the PCR primers are indicated by name and SEQ ID NO:, and the mixture for PCR reactions and protocols thermal cycles of PCR are marked by their respective rooms. A qualified specialist will see that to construct a chimeric coding sequences for real what the invention can be applied to other PCR primers, and other PCR conditions, and that the list of examples of primers and PCR conditions used in the present invention, in any case is not restrictive.

Table 4 shows the mutual relationship between the three domains Cry3A055 protein, Cry1Ab and Shua with their respective variable plots and conservative blocs. For each protein shows which amino acids are contained in the domain, conservative blocks and variable parts.

Example 3. Development 2OL-8a.

The first fragment of the nucleic acid encoding the N-terminal part of a Cry3A055 protein (SEQ ID NO: 70), increased by way of PCR from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers 5 A-1-bm (SEQ ID NO: 83) and C3-3A-6 (SEQ ID NO: 84), and a Mixture of 1 and a temperature cycle Profile 1. This reaction PCR introduced a point mutation by deletion of nucleotide 28 of the sequence SEQ ID NO: 69 (sgua), which has led to a shift of the reading frame Shua, and removed the BamHI site and a Kozak sequence (Kozak, M., 1986. Cell 44:283-92) on the 5' end of the resulting amplicon.

The second fragment of the nucleic acid encoding C-terminal part of the Cry1Ab protein (SEQ ID NO: 72), increased by way of PCR from a plasmid containing mocrylAb (SEQ ID NO: 71), using primeros-1b-3 (SEQ ID NO: 85) and 1Ab-6-Sac (SEQ ID NO: 86), and also a Mixture of 1 and a temperature cycle Profile 1.

The above-described first and second nucleic acids were coupled by using them as templates in the extended (overlap) PCR reaction (Horton and others, 1989. Gene 77: 61-68) with primers 5'3A-1-bam (SEQ ID NO: 83) and 1Ab-6-Sac (SEQ ID NO: 86), and a Mixture of 2 and a temperature cycle Profile 1, with the difference that as the annealing temperature used gradient 45-65°C.

The resulting amplicon was provided as a fragment with a blunt end into the vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA), cut with SmaI, to form plasmid p2OL8a/CR2.1. Then the fragment BamHI-SacI from p2OL8a/CR2.1 provided to RETA (EMD Biosciences, Inc., San Diego, CA), which was cut with BamHI-SacI and transformed into E. coli. Fragment BamHI-SacI from p2OL8a/CR2.1 contained 40 nucleotides derived from the vector pCR2.1-TOPO, adjacent to varnocna the amplicon from the first PCR reaction. Ligation of this fragment BamHI-SacI to rate created an open reading frame start codon (ATG) T7 tag and ending with the SacI site introduced DNA. This open reading frame is indicated 2OL-8a (SEQ ID NO: 1) and encodes a chimeric insecticidal protein 2OL-8a (SEQ ID NO: 2). Thus, chimeric insecticidal protein 2OL-8a from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MASMTGGQQMGRGSTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 126), amino acids 10-468 b the LCA Cry3A055 (SEQ ID NO: 70), components of variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-648 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids of the conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6; and 38 amino acids protoxin tail section Cry1Ab protein.

The nucleotides encoding amino acids 1-14 peptidnogo fragment derived from the T7 tag and cleavage site BamHI vector RET. The nucleotides encoding amino acids 15-26 peptidnogo fragment derived from the vector pCR2.1-TOPO. And the nucleotides encoding amino acids 27-35 peptidnogo fragment obtained from a slice Shua beyond the rest of the coding sequence Shua.

Example 4. Development Of FR8a.

The coding sequence of FR8a was developed by placing the Kozak sequence (ACC) and the start codon (ATG) immediately after the N-terminal BamHI site in 2OL-8a (See. Example 3). In addition, the EcoRI site in 2OL-8a was destroyed to help future vectorization FR8a. All these changes were made during one PCR using 2OL-8a as a template and as primers: 8a-atg-delRI (SEQ ID NO: 87) and C2-3A-4 (SEQ ID NO: 88), Mixture 2 and the temperature is tion cycle Profile 2.

The resulting amplicon ligated to the vector pCR2.1-TOPO (Invitrogen). Fragment BamHI-PpuMI of the cloned PCR product is then ligated to the fragment PpuMI-Ncol from 2OL8a/pCR2.1 (Cm. Example 3) and the fragment NcoI-BamHI from 2OL8a/pCR2.1 to create FR8a (SEQ ID NO: 3)which encodes a chimeric insecticidal protein FR8a (SEQ ID NO: 4). Thus, chimeric insecticidal protein FR8a from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 Cry3A055 protein (SEQ ID NO: 70), the components of the variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-648 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids of the conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6; and 38 amino acids protoxin tail section Cry1Ab protein.

This fold FR8a has a very high activity against the Western corn root beetle, as shown in Table 5. Therefore, removal of the amino acid sequence T7 of the N-terminal peptidnogo fragment bend 2OL-8a does not have an adverse effect on insecticidal activity.

Adding additional 34 amino acids to the N-end the FR8a created bend, the designation of FR8a+34 (SEQ ID NO: 160), with N-terminal pipidinny a fragment of 56 amino acids (SEQ ID NO: 131). This N-terminal pipidinny a fragment of 56 amino acids did not have any negative impact on the activity of FR8a against the Western corn root beetle (Cm. Table 5).

Example 5. Development FRCG.

In order to determine whether the site of the protease recognition of cathepsin G required for insecticidal activity of the hybrid protein containing the N-terminal fragment from Shua, model was made available for site of cathepsin G from hybrid protein FR8a (Example 4). The first fragment of the nucleic acid MluI-PpuMI from a plasmid containing the FR8a (SEQ ID NO: 3), and the second fragment of the nucleic acid PpuMI/MluI from plasmids containing mashua (SEQ ID NO: 67), ligated using standard molecular biology techniques to create FRCG (also called FR8a-catg) (SEQ ID NO: 5), which encodes a hybrid protein FRCG (SEQ ID NO: 6). Thus, chimeric insecticidal protein FRCG from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-467 protein Shua (SEQ ID NO: 68), the components of the variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477648 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids of the conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6; and 38 amino acids protoxin tail section Cry1Ab protein.

Protein FRCG showed the same activity against the Western corn root beetle as protein FR8a (see Table 5), suggesting that the site of the protease cathepsin G is not necessary for insecticidal activity of the bend.

Example 6. Development of FR8a-9F.

The first fragment of a nucleic acid approximately 323 BP increased by way of PCR from a plasmid containing the FR8a (SEQ ID NO: 3), using reverse primers (SEQ ID NO: 89) and FR8a-OL-l (SEQ ID NO: 90), a Mixture of 2 and a temperature cycle Profile 2. The second fragment of the nucleic acid of approximately 470 BP increased by way of PCR from a plasmid containing the FR8a, using primers FR8a-OL-2 (SEQ ID NO: 91) and C1-3A-2 (SEQ ID NO: 92), and a Mixture of 2 and a temperature cycle Profile 2. The resulting two amplicon connected by using them as templates in the extended PCR reactions using primers 5'FR8a (SEQ ID NO: 93) and C1-3A-2 (SEQ ID NO: 92), using PCR Mixture 2 and the temperature cycle Profile 2, to increase the 5' end of the sequence FR8a-9F. Product extended PCR cloned in the vector pCR2.1-TOPO (Invitrogen)labeled 5'FR-F/pCR2.1. Fragment BamHI/PpuMI sequence 5'FR-9F/pCR2.1 then ligated to the fragment PpuMI/BamHI sequence FR8a to create a sequence FR8a-9F (SEQ ID NO: 7)that encodes a chimeric protein FR8a-9F (SEQ ID NO: 8). Thus, chimeric insecticidal protein FR8a-9F from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRP (SEQ ID NO: 129), amino acids 1-468 Cry3A055 protein (SEQ ID NO: 70), which includes variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-648 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6; and 38 amino acids protoxin tail section Cry1Ab protein.

Activity bend FR8a-9F against the Western corn root beetle was slightly lower than the bend FR8a (Cm. Table 5), suggesting that the C-terminal 9 amino acids peptidnogo fragment of SEQ ID NO: 127 play a role in the empowerment of protein FR8a full insecticidal activity.

Example 7. Development of FR-9F-catg.

The coding sequence of the FR-9F-catg was created in order to return from Shua warnockii nucleotides FR8a back to the frame and to fix the site of the protease recognition of cathepsin G. Fragment BamHI/PpuMI out of 5'FR-9F/pCR2.1 (Cm. Example 6) ligated with a fragment of PpuMI/BamHI sequence FRCG (Cm. Example 5) to generate the coding sequence FR-9F-catg (SEQ ID NO: 9), which encodes a chimeric protein FR-9F-catg (SEQ ID NO: 10). Thus, in the chimeric protein FR-9F-catg from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRP (SEQ ID NO: 129), amino acids 1-467 of protein Shua (SEQ ID NO: 68), which includes variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-648 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6; and 38 amino acids protoxin tail section Cry1Ab protein.

The level of activity of the bend FR8a-9F-catg against the Western corn root beetle was the same as the FR8a (Cm. Table 5), this shows that the bend can be obtained from sequence or modified Chua or native Shu.

Example 8. Development of FR8a-12aa.

The nucleotides encoding amino acids 2-13 peptidnogo fragment contained in FR8a (SEQ ID NO: 4), extracted using PCR. Fragment increased by the PCR method from p is asmide, containing FR8a (SEQ ID NO: 3), using the primers 5'FR8a-12aa (SEQ ID NO: 94) and C1-3A-2 (SEQ ID NO: 90), a Mixture of 1 and a temperature cycle Profile 1. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen). Fragment BamHI-PpuMI from clone pCR2.1-TOPO and then ligated with a fragment of PpuMI-BamHI from a plasmid containing the FR8a to create a sequence FR8a-12aa (SEQ ID NO: 11), which encodes the chimeric insecticidal protein FR8a-12aa (SEQ ID NO: 12). Thus, chimeric insecticidal protein FR8a-12aa from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MYDGRQQHRG (SEQ ID NO: 128), amino acids 10-468 Cry3A055 protein (SEQ ID NO: 70), which includes variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-648 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6; and 38 amino acids protoxin tail section Cry1Ab protein.

The level of activity of the bend FR8a-12aa against the Western corn root beetle was the same as the FR8a (Cm. Table 5), based on which we can assume that for full insecticidal activity FR8a not require the N-terminal 12 amino acid n is privlege fragment of the sequence SEQ ID NO: 127.

Example 9. Development Wr-9mut.

The fragment of the nucleic acid is increased by the PCR method of FR8a/pCR2.1 (Example 2) using the primers 5 TR8a-12aa (SEQ ID NO: 94) and C1-3A-2 (SEQ ID NO: 92), and a Mixture of 1 and a temperature Profile 2. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen). Then the fragment BamHI/PpuMI ligated to the fragment PpuMI/BamHI sequence FR8a (SEQ ID NO: 3) to create a sequence Wr-9mut (SEQ ID NO: 13), which encodes a protein WR-9mut (SEQ ID NO: 14), in which N-end-to-end are pipidinny fragment containing the amino acid sequence of MYDGRQQHRG (SEQ ID NO: 128) and amino acids 10-598 Cry3A055 protein (SEQ ID NO: 70). Thus, protein WR-9mut is Shua with N-terminal peptidyl fragment according to the invention.

Protein WR-9mut does not have activity against the Western corn root of the bug. Therefore, the addition of N-terminal peptidnogo fragment to non-hybrid modified protein Shua destroyed insecticidal activity. From this we can conclude that there might be some correlation between the C-terminal part of the hybrid protein Cry1Ab FR8a and N-terminal peptidyl fragment, giving the insecticidal activity of the protein FR8a.

Example 10. Development of FRD3.

3' end of the coding sequence was created by increasing method PCR fragment from a plasmid containing the FR8a (SEQ ID NO: 3), with the use of primer is C2-3A-3 (SEQ ID NO: 95) and 3'1Ab-dm3 (SEQ ID NO: 96), as well as a Mixture of 2 and a temperature cycle Profile 2. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen). Fragment ApaI/SacI cloned amplicon size 364 BP, marked 3'FRD3/pCR2.1, ligated with a fragment SacI/ApaI sequence FR8a to create a FRD3 (SEQ ID NO: 15), which encodes a chimeric protein FRD3 (SEQ ID NO: 16). In this chimeric protein FRD3 from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 Cry3A055 protein (SEQ ID NO: 70), which includes variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-610 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids of the conservative block 3, variable area 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6. Thus, the chimeric insecticidal protein FRD3 is a variant chimeric insecticidal protein FR8a, which removed the plot of 38 amino acids, representing protoxin tail Cry1Ab.

FOLD FRD3 showed the same level of activity against the Western corn root beetle as the FR8a (Cm. Table 5), this suggests that the tail protoxins plot of 38 amino acids FR8a is not necessary for full insecticidal activity.

Example 11. Development of FR-12-cg-dm3

The SacI fragment/PpuMI size 3082 BP from the plasmid containing FR8a-12 (See. Example 8), PpuMI fragment/MluI size 721 BP from the sequence FRCG (Cm. Example 5) and MluI fragment/SacI size 923 BP from the sequence FRD3 (Cm. Example 10) were combined to create the coding sequence FR-12-cg-dm3 (SEQ ID NO: 17), which encodes a chimeric protein FR-12-cg-dm3 (SEQ ID NO: 18). In this chimeric protein FR-12-cg-dm3 from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MYDGRQQHRG (SEQ ID NO: 129), amino acids 10-467 protein Shua (SEQ ID NO: 70), which includes the full variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-610 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6. Thus, the chimeric protein FR-12-cg-dm3 is a variant of FR8a, which removed the 12 N-terminal amino acids peptidnogo fragment, the site of the protease recognition of cathepsin G, and 38 amino acids protoxin tail section Cry1Ab.

This fold FR-12-cg-dm3 is not as active against Western corn root beetle, as FR8a (Cm. Table 5), and is what we can conclude, for full insecticidal activity requires some interaction between the C-terminal part of FR8a and N-terminal peptidyl fragment.

Example 12. Development 9F-cg-del6

5 Konec this coding sequence was obtained by increasing method PCR fragment from a plasmid containing FR-9F-catg (Cm. Example 7), using primers 5'FR-del6 (SEQ ID NO: 97) and C1-3A-2 (SEQ ID NO: 92), a Mixture of 3 and a temperature cycle Profile 3. The resulting amplicon was cloned into pCR2.1-TOPO. Then the fragment BamHI/PpuMI size 215 BP ligated with a fragment of PpuMI/BamHI size 4668 BP from the sequence FR-9F-catg to create FR-9F-cg-del6 (SEQ ID NO: 19), which encodes a chimeric protein FR-9F-cg-del6 (SEQ ID NO: 20). In this chimeric protein FR-9F-cg-del6 from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MCAGIRP (SEQ ID NO: 130), amino acids 1-467 of protein Shua (SEQ ID NO: 68), which includes variable sites 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-648 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6, and 38 amino acids protoxin tail section Cry1Ab. That is they way chimeric protein FR-9F-cg-del6 is a variant of FR8a-9F-catg, which deleted amino acids 2-7 peptidnogo fragment.

This FR-9F-cg-del6 does not have activity against the Western corn root beetle, from which we can conclude that for activity against the Western corn root beetle N-terminal pateisinamu the desired fragment of at least 7 amino acids of the 9 C-terminal amino acid sequence of SEQ ID NO: 127.

Example 13. Development of FR-cg-dm3

The MluI fragment/SacI size 3839 BP from FRCG (Example 5) and MluI fragment/SacI size 923 BP from FRD3 (Example 10) ligated to create the FR-cg-dm3 (SEQ ID NO: 21), which encodes a protein FR-cg-dm3 (SEQ ID NO: 22). In this chimeric insecticidal protein FR-cg-dm3 from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-467 protein Shua (SEQ ID NO: 68), which includes variable sites 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-610 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6.

The same level of activity bend FRD3 and FR8a against Western ku is ulusnogo root beetle (Cm. Table 5) confirms that the site of cathepsin G and protoxin tail protein site FR8a not required for full insecticidal activity against the Western corn root of the bug.

Example 14. Development 9F-cg-dm3.

The MluI fragment/SacI plasmid containing FR-9F-cg (Cm. Example 7), ligated with the MluI fragment/SacI size 923 BP of the plasmid containing FRD3 (Cm. Example 10) to create 9F-cg-dm3 (SEQ ID NO: 23), which encodes a chimeric protein 9F-cg-dm3 (SEQ ID NO: 24). In this chimeric protein 9F-cg-dm3 from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRP (SEQ ID NO: 129), amino acids 1-467 of protein Shua (SEQ ID NO: 68), which includes variable sites 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-610 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 24 amino acids of the conservative block 3, the flexible section 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6.

The level of activity of the bend 9F-cg-dm3 against the Western corn root beetle was the same (see table 5), it confirms that the C-terminal 9 amino acids peptidnogo fragment can give the activity in the case, if the domain bend consists of either a variable regions and conserved the main blocks of the modified Chua (Chua), any of the variable regions and conservative blocs Shua.

Example 15. Development VA.

The fragment of the nucleic acid encoding the N-terminal part of a Cry3A055 protein (SEQ ID NO: 70), increased by way of PCR from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers 5 A-1-bam (SEQ ID NO: 83) and C3-3A-8 (SEQ ID NO: 99), and a Mixture of 1 and a temperature cycle Profile 1. The fragment of the nucleic acid encoding C-terminal part of the Cry1Ab protein (SEQ ID NO: 72), containing variable sections 4-6, increased from plasmids containing mocrylAb (SEQ ID NO: 71), using primers C3-3A-7 (SEQ ID NO: 100) and 1Ab-6-sac (SEQ ID NO: 86), and a Mixture of 1 and a temperature cycle Profile 1. The resulting amplicon was identified 2OL-8b.

The fragment of the nucleic acid encoding the N-terminal part of a Cry3A055 protein (SEQ ID NO: 70), increased by way of PCR from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers 5 A-1-bam (SEQ ID NO: 83) and C2-3A-4 (SEQ ID NO: 88), and a Mixture of 1 and a temperature cycle Profile 1.

The fragment of the nucleic acid encoding C-terminal portion of the protein Cry1Ba (SEQ ID NO: 74), increased by way of PCR from a plasmid containing mocrylBa (SEQ ID NO: 73), using primers 1-5 (SEQ ID NO: 101) and 1B-10 (SEQ ID NO: 102), and a Mixture of 1 and a temperature cycle Profile 1, with the difference that the applied temperature annealing 60°C.

Then two of the above PCR product was used as template in the extended PCR reaction with the PRA which measures 5 A-1-bam (SEQ ID NO: 83) and 1-In-10 (SEQ ID NO: 102) and using a Mixture of 1 and a temperature cycle Profile 2. The resulting amplicon was outlined in [10].

Next, the fragment of the nucleic acid Shua (SEQ ID NO: 69) increased by the PCR method using 2OL-8b (see above) as template, with primers 5'3A-1-bam (SEQ ID NO: 83) and 3A-22 (SEQ ID NO: 103) under the following PCR conditions: 1 Mixture, the temperature cycle profile: 94°C for 45 seconds, gradient 50°C-70°C - 45 seconds, 72°C for 90 seconds, 30 cycles. A fragment of another nucleic acid is increased by the method of PCR, using as template V10 (see above), primers 1-7 (SEQ ID NO: 104) and 1 In-10 (SEQ ID NO: 102), using a Mixture of 1 PCR reaction and the temperature cycle Profile 2, with the difference that applied the annealing temperature 60°C. the resulting two PCR product is then used as templates in the extended PCR reactions with primers 5'3A-1-bam (SEQ ID NO: 83) and 1-10 (SEQ ID NO: 102), using the following PCR conditions: a Mixture of 2, the profile of the temperature cycle: 94°C - 30 seconds, gradient 40°C-60°C 30 seconds, 72°C - 60 seconds, 30 cycles.

The resulting PCR product is ligated to the vector pCR2.1-TOPO (Invitrogen) and designated B10/pCR2.1. Fragment BamHi-SacI from B8a/pCR2.1 then ligated to RETA (Novagen), which was cut with BamHi/SacI to create the coding sequence VA (SEQ ID NO: 25), which encodes a hybrid protein VI (SEQ ID NO: 26). This hybrid protein VI contains from N-Terminus to C-Terminus of amino acids 1-468 Cry3A055 protein (SEQ ID NO: 70), and amino acids 505-656 b the LCA Cry1Ba (SEQ ID NO: 74).

Example 16. Development 5*VA.

Fragment BamHI-XbaI from plasmids containing 2OL-8a (See. Example 3), and the fragment XbaI-SacI from plasmids containing VA (Cm. Example 15), ligated to create 5*VA (SEQ ID NO: 27)that encodes a chimeric protein 5*VA (SEQ ID NO: 28). This protein 5*WA from N-Terminus to C-end are: pipidinny fragment containing the sequence of amino acids

MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-467 Cry3A055 protein (SEQ ID NO: 70) and amino acids 505-656 protein Cry1Ba (SEQ ID NO: 74). Thus, the chimeric protein 5*WA is a hybrid protein WA, to which was added N-terminal pipidinny fragment.

Example 17. Development V3.

This gene is increased by the PCR method using 3 fragments together as patterns: the first pattern is increased from a plasmid containing Shua (SEQ ID NO: 69) using primers 5'3A-1-bam (SEQ ID NO: 83) and C2-3A-4 (SEQ ID NO: 88), and a Mixture of 1 and a temperature cycle Profile : 1; the second segment has increased from plasmids containing mocry1Ab (SEQ ID NO: 71), using primers C2-3A-3 (SEQ ID NO: 95) and C3-1Ab-2 (SEQ ID NO: 105), and a Mixture of 1 and a temperature cycle Profile : 1; and the third piece is increased from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers C3-3A-5 (SEQ ID NO: 106) and 3A-12-sac (SEQ ID NO: 107), and a Mixture of 1 and a temperature cycle Profile 1. These three PCR product is then used as templates in the extended PCR reaction with primers 5'3A-1-bam (SEQ ID NO: 8) and 3A-12-sac (SEQ ID NO: 107), and also using a Mixture of 1 Profile and 1 for the coding sequence v3A (SEQ ID NO: 29), which encodes a hybrid protein V3A (SEQ ID NO: 30). In a hybrid protein V3A from N-Terminus to C-end are: amino acids 1-226 Cry3A055 protein (SEQ ID NO: 70), which includes variable plot 1, conservative block 1, variable 2 and the N-terminal 34 amino acids of the conservative block 2, amino acids 237-474 Cry1Ab protein (SEQ ID NO: 72), which includes the C-terminal 33 amino acids of the conservative block 2, variable 3 phase and N-terminal 20 amino acids of the conservative block 3, and amino acids 467-598 Cry3A055 protein (SEQ ID NO: 70), where includes the C-terminal 28 amino acids of the conservative block 3, variable phase 4, the conservative block 4, variable area 5 and the conservative bloc 5.

In the crook of V3A are two positions crossover. The first cross between Chua and Cry1Ab is located in the conservative block 2, and the second crossing between Cry1Ab and Shua is conservative in block 3. Thus, V3A is a variant Shua in which all variable plot 3 was replaced by variable 3 phase protein Cry1Ab. This fold V3A was not as active against Western corn root beetle, as FR8a, on what basis we can assume that the sequence Cry1Ab in the conservative block 3, variable plot 4, conservative block 4, var is abelina section 5, conservative block 5 and/or variable section 6 is important for full insecticidal activity of the protein FR8a.

The coding sequence v3A ligated to the vector pCR2.1-TOPO and then was subcloned into RETA using fragment BamHI/SacI. Protein V3A, expressed by the vector RETA has a T7 tag at N-end. This protein was identified T7-V3A.

Example 18. Development V4F.

The first fragment of a nucleic acid encoding sections 1-3 protein Chua, increased by way of PCR from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers 5'3A-1-bam (SEQ ID NO: 83) and C3-3A-6 (SEQ ID NO: 84), and a Mixture of 1 and a temperature cycle Profile 1.

The second fragment of the nucleic acid encoding the variable phase 4 Cry1Ab protein, PCR increased from plasmids containing mocrylAb (SEQ ID NO: 71), using primers C3-1b-3 (SEQ ID NO: 85) and C4-3A-10 (SEQ ID NO: 108), and a Mixture of 1 and a temperature cycle Profile 1.

The third fragment of the nucleic acid encoding the variable sections 5-6 protein Chua, increased by way of PCR from a plasmid containing Shua (SEQ ID NO: 69), using primers C4-3A-9 (SEQ ID NO: 109) and 3A-12-sac (SEQ ID NO: 107), and a Mixture of 1 and a temperature cycle Profile 1.

All three PCR amplicon combined and used as template in the extended PCR with primers 5 A-1-bam (SEQ ID NO: 83) and 3A-12-sac (SEQ ID NO: 107) using the following PCR conditions: a Mixture of 1 and profile temperatures cycle:94°C - 30 seconds, gradient 50°C-70°C - 30 seconds, 72°C - 30 seconds for 20 cycles. The resulting amplicon v4F sequence (SEQ ID NO: 31)encoding a hybrid toxin V4F (SEQ ID NO: 32), cloned in the vector pCPv2.1-TOPO and outlined v4F/pCR2.1. A hybrid protein V4F contains from N-Terminus to C-Terminus of amino acids 1-468 Cry3A055 protein (SEQ ID NO: 70), amino acids 477-520 constituting the variable phase 4 Cry1Ab protein (SEQ ID NO: 72), and amino acids 512-598 Cry3A055 protein (SEQ ID NO: 70).

Protein V4F has two positions crossover. The first position of the crossover between Chua and Cry1Ab is conservative block 3, and the second crossing between Cry1Ab and Shua - conservative block 4. Therefore, protein V4F is a variant Shua, in which the flexible section 4 is replaced by the variable phase 4 Cry1Ab protein. A hybrid protein V4F not active against Western corn root beetle, this suggests that the sequence of the Cry1Ab in the C-terminal part of FR8a contribute to the insecticidal activity of FR8a.

Fragment BamHI-SacI from v4F/was subcloned into the pCR2.1 RET. Protein downregulation resulting plasmid was designated T7-V4F.

Example 19. Development 5*V4F.

Fragment BamHI-XbaI from a plasmid containing the FR8a (Cm. Example 4), and the fragment XbaI-SacI H3V4F/pCR2.1 (Cm. Example 18) ligated to RET, cut with BamHI-SacI, he got 5*V4F/pET21. The coding sequence of the 5*V4F (SEQ ID N: 33) encodes a chimeric protein 5*V4F (SEQ ID NO: 34). This chimeric insecticidal protein 5*V4F contains from N-Terminus to C-end pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-491 Cry3A055 protein (SEQ ID NO: 70), amino acids 501-520, the components of the variable phase 4 Cry1Ab protein (SEQ ID NO: 72), and amino acids 512-598 Cry3A055 protein (SEQ ID NO: 70).

FOLD 5*V4F is a hybrid protein V4F added N-terminal peptidyl fragment (SEQ ID NO: 127). Activity against Western corn rootworm beetle bend 5*V4F has, though not at the same level that FR8a. Consequently, the N-terminal part gave insecticidal activity of the protein V4F, confirming that there may be contributing to the interaction between the C-terminal part and the N-terminal peptidyl fragment FR8a.

The protein expressed by the plasmid 5*V4F/pET21, denoted T7-5*V4F and has a T7 tag N-terminal to peptidnogo fragment 5*V4F.

Example 20. Development 2OL-7.

The fragment of the nucleic acid encoding the variable region 1 protein Chua, increased by way of PCR from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers 5'3A-1-bam (SEQ ID NO: 83) and C1-3A-2 (SEQ ID NO: 92), and a Mixture of 1 and a temperature cycle Profile 1.

The fragment of the nucleic acid encoding the variable sections 2-6 Cry1Ab protein, increased by way of PCR from a plasmid containing mocry1Ab (SEQ ID NO: 71), using primers C1-1Ab-1 (SEQ ID NO: 11) and 1Ab-6-sac (SEQ ID NO: 86), and also a Mixture of 1 and a temperature cycle Profile 1.

The resulting two amplicon was used as template for the extended PCR reaction with primers 5'3A-1-bam (SEQ ID NO: 83) and 1b-6-sac (SEQ ID NO: 86) with application of a Mixture of 2 and a temperature cycle Profile 1 to create the coding sequence 2OL-7 (SEQ ID NO: 35), which encodes a hybrid protein 2OL-7 (SEQ ID NO: 36). In this hybrid protein 2OL-7 from N-Terminus to C-end are amino acids 1-156 Cry3A055 protein (SEQ ID NO: 70)comprising the variable region 1 and the N-terminal 14 amino acids of the conservative block 1, and amino acids 167-648 Cry1Ab protein (SEQ ID NO: 72), the components of the C-terminal 15 amino acids of the conservative block 1, variable 2, conservative block 2, variable 3 phase, conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and variable plot 6, and 38 amino acids protoxin tail section Cry1Ab. Thus, 2OL-7 is a variant of the Cry1Ab protein in which the variable plot 1 has been replaced by variable plot 1 protein Shua.

The coding sequence 2OL-7 cloned in pCR2.1-TOPO (Invitrogen), and then moved in rat using BamHI/SacI and outlined 2OL-7/pET21a. The coding sequence in 2OL-7/pET21 was labeled T7-2OL-7 (SEQ ID NO: 37). The protein expressed by the vector 2OL-7/pET21a was labeled T7-2OL-7 (SEQ ID NO: 38)

Example 21. Development 5*2OL-7.

A fragment of the BamHI/XbaI sequence FR8a (Cm. Example 4), PpuMI fragment/SacI sequence 2OL-7 (Cm. Example 20) and a fragment of the BamHI/SacI from RETA ligated to obtain 5*2OL-7/pET21a. The coding sequence of the 5*2OL-7 (SEQ ID NO: 39) encodes a chimeric protein 5*2OL-7 (SEQ ID NO: 40). Protein 5*2OL-7 from N-Terminus to C-end contains pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-156 Cry3A055 protein (SEQ ID NO: 70), and amino acids 167-643 Cry1Ab protein (SEQ ID NO: 72). Thus, a hybrid protein 5*2OL-7 is a hybrid protein 2OL-7, to which are added an N-terminal pipidinny fragment.

Example 22. Development 2OL-10.

The fragment of the nucleic acid encoding the N-terminal part of the protein Chua, increased by way of PCR from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers 5 A-1-bam (SEQ ID NO: 83) and C2-3A-4 (SEQ ID NO: 88), and a Mixture of 1 and a temperature cycle Profile 1. The fragment of the nucleic acid encoding C-terminal portion of the protein Cry1Ab, increased by way of PCR from a plasmid containing mocrylAb (SEQ ID NO: 71), using primers C2-3A-3 (SEQ ID NO: 95) and 1Ab-6-sac (SEQ ID NO: 86), a Mixture of 1 and a temperature cycle Profile 1. These two PCR product is then used as templates in the extended PCR reaction with primers 5 A-1-bam (SEQ ID NO: 83) and 1Ab-6-sac (SEQ ID NO: 86) under the following PCR conditions: a Mixture of 2, the profile thermocycle is: 94°C - 30 seconds, the gradient of 45°C-65°C 30 seconds, 72°C - 30 seconds, 20 cycles, resulting in a coding sequence 2OL-10 (SEQ ID NO: 41), which encodes a hybrid protein 2OL-10 (SEQ ID NO: 42). A hybrid protein 2OL-10 contains from N-Terminus to C-Terminus of amino acids 1-232 Cry3A055 protein (SEQ ID NO: 70) and amino acids 243-648 Cry1Ab protein (SEQ ID NO: 72). Thus, a hybrid protein 2OL-10 represents essentially the domain I protein Shua and domains II and III of the Cry1Ab protein.

The coding sequence 2OL-10 was cloned in the vector pCR2.1-TORO (Invitrogen), which is then moved in RETA using BamHI/SacI. Protein expressed 2OL-10/pET21a, received the designation T7-2OL-10.

Example 23. Development 5*2OL-10.

Fragment BamHI-XbaI from a plasmid containing the FR8a (Cm. Example 4), and the fragment XbaI-SacI from 2OL-10/pCR2.1 (Cm. Example 22) ligated to RET, cut with BamHI-SacI to obtain 5*2OL-10/pET21. The coding sequence of the 5*2OL-10 (SEQ ID NO: 43) encodes a chimeric protein 5*2OL-10 (SEQ ID NO: 44). Protein 5*2OL-10 contains from N-Terminus to C-end pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-232 Cry3A055 protein (SEQ ID NO: 70) and amino acids 243-648 Cry1Ab protein (SEQ ID NO: 72). Thus, the chimeric protein 5*2OL-10 is a hybrid protein 2OL-10 added to N-terminal peptidyl fragment.

Example 24. Development 2OL-12A.

The first fragment of a nucleic acid encoding-terminal part of the protein Cry1Ab, increased by way of PCR from a plasmid containing mocry1Ab (SEQ ID NO: 71), using primers 5'1Ab-bam (SEQ ID NO: 98) and C3-1Ab-2 (SEQ ID NO: 105), and a Mixture of 1 and a temperature cycle Profile 1.

The second fragment of the nucleic acid encoding C-terminal portion of the protein Chua, increased by way of PCR from a plasmid containing Shua (SEQ ID NO: 69), using primers C3-3A-5 (SEQ ID NO: 106) and 3A-12-sac (SEQ ID NO: 107), and a Mixture of 1 and a temperature cycle Profile 1.

The above-described first and second nucleic acid fragments connected by using them as templates in the extended PCR reaction with primers 5'1Ab-bam (SEQ ID NO: 98) and 3A-12-sac (SEQ ID NO: 107), using a Mixture of 1 and a temperature cycle Profile 1, to generate the coding sequence 2OL-12A (SEQ ID NO: 45), which encodes bend 2OL-12A (SEQ ID NO: 46). Protein 2OL-12A comprises from N-Terminus to C-Terminus of amino acids 1-476 Cry1Ab protein (SEQ ID NO: 72) and amino acids 469-598 Cry3A055 protein (SEQ ID NO: 70).

The bend 2OL-12A no activity against the Western corn root beetle, but it is active against European corn borer (see Table 6). This demonstrates that the bend can be developed from proteins that are active against lepidopteran and proteins active against Coleoptera, without losing activity against lepidopteran insects.

The coding sequence 2OL-12A cloned in pCR2.1-TOPO (Invitrogen), and then moved in RET is using BamHI/SacI. The protein expressed by the vector 2OL-12A/pET21a, marked T7-2OL-12A.

Example 25. Development 2OL-13.

Four nucleic acid fragment obtained as follows: fragment 1 UPS way PCR from a plasmid containing Shua (SEQ ID NO: 69), using primers 5 A-1-bm (SEQ ID NO: 83) and C1-3A-2 (SEQ ID NO: 92), and a Mixture of 1 and a temperature cycle Profile : 1; fragment 2 has increased by way of PCR from a plasmid containing mocry1Ab (SEQ ID NO: 71), using primers C2-3A-3 (SEQ ID NO: 95) and C3-1Ab-2 (SEQ ID NO: 105), a Mixture of 1 and a temperature cycle Profile : 1; fragment 3 increased by way of PCR from a plasmid containing mocry1Ab (SEQ ID NO: 71) C3-1b-3, using primers (SEQ ID NO: 85) and C4-3A-10 (SEQ ID NO: 108), and a Mixture of 1 and a temperature cycle Profile : 1; fragment 4 increased by way of PCR from a plasmid containing Shua (SEQ ID NO: 69), using primers C4-3A-9 (SEQ ID NO: 109) and 3A-12-sac (SEQ ID NO: 107), and a Mixture of 1 and a temperature cycle Profile 1.

Then all four fragments were used as templates in the extended PCR reactions using primers 5'3A-bam (SEQ ID NO: 83) and 3A-12-sac (SEQ ID NO: 107), and a Mixture of 1 and a temperature cycle Profile 1, to generate the coding sequence 2OL-13 (SEQ ID NO: 47), which encodes a hybrid toxin 2OL-13 (SEQ ID NO: 48). Protein 2OL-13 contains from N-Terminus to C-Terminus of amino acids 1-159 Cry3A055 protein (SEQ ID NO: 70), amino acids 170-522 Cry1Ab protein (SEQ ID NO: 72), and amino acids 515-598 b the LCA Cry3A055 (SEQ ID NO: 70). Thus, the hybrid toxin 2OL-13 contains VI and N-terminal part of SW of protein: Shua; C-terminal portion of the block SW, V2, SW, V3, SW and V4 of the Cry1Ab protein; and SW, V5 and SW of bulkachuwa.

The coding sequence 2OL-13 cloned in pCR2.1-TOPO (Invitrogen), and then moved in rat using BamHI/SacI. The protein expressed by the vector 2OL-13/pET21a was labeled T7-2OL-13.

Example 26. Development 2OL-20.

Fragment BamHI/NspI from plasmids containing togusa (SEQ ID NO: 67), NspI fragment/HindIII from plasmids containing 2OL-8A (SEQ ID NO: 1), and the fragment HindIII/BamHI from RETA ligated to obtain 2OL-20/pET21a.

Example 27. Development of V5 and 6.

The fragment of the nucleic acid encoding the N-terminal part of the protein Chua, increased by way of PCR from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers 5 A-1-bam (SEQ ID NO: 83) and C4-3A-10 (SEQ ID NO: 108), and a Mixture of 1 and a temperature cycle Profile 1.

The fragment of the nucleic acid encoding C-terminal portion of the protein Cry1Ab, increased by way of PCR from a plasmid containing mocry1Ab (SEQ ID NO: 71), using primers C4-3A-9 (SEQ ID NO: 109) and 1Ab-6-sac (SEQ ID NO: 86), and a Mixture of 1 and a temperature cycle Profile 1.

These two PCR product is then used as templates in the extended PCR reactions using primers 5 A-1-bm (SEQ ID NO: 83) and 1Ab-6-sac (SEQ ID NO: 86), and a Mixture of 1 and a temperature cycle Profile 2 to create the coding sequence V5 and 6 (SEQ ID NO 49), which encodes a hybrid toxin V5 and 6 (SEQ ID NO: 50). Protein V5 and 6 contains from N-Terminus to C-Terminus of amino acids 1-524 Cry3A055 protein (SEQ ID NO: 70), the components V1, CW, V2, SW, V3, SW, V4 and SW, and amino acids 533-648 Cry1Ab protein (SEQ ID NO: 72), comprising V5, CB5 and V6, and 38 amino acids protoxin tail section Cry1Ab.

The coding sequence of the V5 and 6 were cloned in pCR2.1-TOPO, and then moved in rat using BamHI/SacI. Protein expressed V5 and 6/pET21a, marked T7-V5 and 6.

Example 28. Development 5*V5 and 6.

A fragment of the BamHI/XbaI sequence FR8a (Cm. Example 4), a fragment XbaI/SacI sequence V5 and 6 (See. Example 27) and a fragment of the BamHI/SacI of RETA ligated to obtain 5*V5 and 6/RET. The coding sequence of the 5*V5 and 6 (SEQ ID NO: 51) encodes a chimeric protein 5*V5 and 6 (SEQ ID NO: 52). In chimeric insecticidal protein 5*V5 and 6 from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-524 Cry3A055 protein (SEQ ID NO: 70), the components V1, CW, V2, SW, V3, SW, V4 and SW, amino acids 533-648 Cry1Ab protein (SEQ ID NO: 72), comprising V5, CB5 and V6, and 38 amino acids protoxin tail section Cry1Ab. Thus, the chimeric insecticidal protein 5*V5 and 6 represents a hybrid protein V5 and 6, to which are added an N-terminal pipidinny fragment.

Example 29. Development 88A-dm3

Fragment of nucleic acid, coderush the th C-terminal part of the protein Cry8Aa (SEQ ID NO: 78), increased by way of PCR from a plasmid containing cry8Aa (SEQ ID NO: 77), using primers 5'8Aa-dm3 (SEQ ID NO: 111) and 3'8Aa-dm3 (SEQ ID NO: 112), and a Mixture of 2 and a temperature cycle Profile 2. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen) and designated 88A-dm3/pCR2.1.

The MluI fragment/SacI of 88A-dm3/pCR2.1 and SacI fragment/MluI from a plasmid containing the FR8a (Cm. Example 4), ligated to generate the coding sequence 88A-dm3 (SEQ ID NO: 53), which encodes a hybrid protein 88A-dm3 (SEQ ID NO: 54). In this protein 88A-dm3 from N-Terminus to C-end includes: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 Cry3A055 protein (SEQ ID NO: 70) and amino acids 532-664 protein Cry8Aa (SEQ ID NO: 78).

The coding sequence 88A-dm3 also transformed in rate by using the enzyme BamHI/SacI and ligation. Protein, expressed 88A-dm3/pET21 marked T7-88A-dm3.

Example 30. Development of FR(1Fa)

The fragment of the nucleic acid encoding the N-terminal part of FR8a (Cm. Example 3), increased by way of PCR from a plasmid containing the FR8a (SEQ ID NO: 1), using primers C2-3A-3 (SEQ ID NO: 95) and tant-OL-2 (SEQ ID NO: 113), and a Mixture of 3 and a temperature cycle Profile 3.

The fragment of the nucleic acid encoding the C-terminal part of Cry1Fa protein (SEQ ID NO: 76), increased by way of PCR from a plasmid containing mocry1Fa (SEQ ID NO: 75), using primers tant-OL-1 (SEQ ID NO: 114) and tant-3'sac (SEQ ID NO: 115), and With whom thou 3 and the Temperature cycle Profile 3.

These two PCR product is then used as templates in the extended PCR reaction using primers C2-3A-3 (SEQ ID NO: 95) and tant-3'sac (SEQ ID NO: 115), and a Mixture of 3 and a temperature profile of the Profile 3. The resulting PCR product was cloned in pCR2.1-TOPO (Invitrogen). Fragment BamHI/MluI from a plasmid containing the FR8a, MluI fragment/SacI of product extended PCR pCR2.1, as well as a fragment of the BamHI/SacI of RETA then ligated to create FR(1Fa)/pET21a. The coding sequence of the FR(1Fa) (SEQ ID NO: 55) encodes a chimeric protein FR(1Fa) (SEQ ID NO: 56). In the protein FR(1Fa) from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 Cry3A055 protein (SEQ ID NO: 70) and amino acids 470-649 protein ry1F (SEQ ID NO: 76).

Example 31. Development of FR(1 ° C)

Domains I and II protein FR8a increased by way of PCR from a plasmid containing the FR8a (SEQ ID NO: 1), using primers C2-3A-3 (SEQ ID NO: 95) and 1Ac-OL-2 (SEQ ID NO: 116), and a Mixture of 3 and a temperature cycle Profile 3. Domain III of Cry1Ac protein (SEQ ID NO: 80) increased by way of PCR from a plasmid containing cry1Ac (SEQ ID NO: 79), using primers 1Ac-OL-1 (SEQ ID NO: 117) and 1 ° C-3'sac (SEQ ID NO: 118), and a Mixture of 3 and a temperature cycle Profile 3.

These two PCR product was used as template in the extended PCR using primers C2-3A-3 (SEQ ID NO: 95) and 1 ° C-3'sac (SEQ ID NO: 118) and the following reaction conditions: a Mixture of 3 and the profile of those who eratures cycle: 94°C - 30 seconds, 68°C for 30 seconds, 68°C - 30 seconds for 20 cycles. Product extended PCR reactions were cloned into pCR2.1-TOPO (Invitrogen). Fragment BamHI/MluI from a plasmid containing the FR8a, MluI fragment/SacI of product extended PCR pCR2.1, as well as a fragment of the BamHI/SacI of RETA ligated to create FR(1Ac)/pET21a. In the protein FR(1Ac) from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 protein Shua (SEQ ID NO: 70) and amino acids 477-608 protein Cry1Ac (SEQ ID NO: 80).

Example 32. Development of FR(1Ia)

Nucleotide fragment encoding domains I and II protein FR8a, increased by way of PCR from a plasmid containing the FR8a (SEQ ID NO: 3), using primers C2-3A-3 (SEQ ID NO: 95) 1Ia-OL-2 (SEQ ID NO: 119), and a Mixture of 3 and a temperature cycle Profile 3. The second nucleotide fragment encoding domain III protein Cry1Ia (SEQ ID NO: 82), increased by way of PCR from a plasmid containing Shua (SEQ ID NO: 81), using primers 1Ia-OL-1 (SEQ ID NO: 120) and a-3'sac (SEQ ID NO: 121), and a Mixture of 3 and a temperature cycle Profile 3. These two PCR product was used as template in the extended PCR reaction using primers C2-3A-3 (SEQ ID NO: 95) and a-3'sac (SEQ ID NO: 121), and a Mixture of 3 and profile of the temperature cycle: 94°C for 30 seconds, 68°C - 45 seconds for 20 cycles. Product extended PCR cloned into pCR2.1-TOPO (Invitrogen). Fragment BamHI/MluI from a plasmid containing the FR8a, MluI fragment/SacI of product expands the adopted PCR pCR2.1, as well as a fragment of the BamHI/SacI of RETA ligated to create FR(1Ia)/pET21a. In the protein FR(1Ia) from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 protein Shua (SEQ ID NO: 70) and amino acids 513-719 protein Shua (SEQ ID NO: 82).

Example 33. Development of Dm2-3A

Part of the 5' end of the coding sequence increased by way of PCR from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers C2-3A-3 (SEQ ID NO: 95) and FR-1Ab-2 (SEQ ID NO: 122), and a Mixture of 3 and a temperature cycle Profile 2. Nucleotide fragment encoding domain III protein Cry1Ab, increased by way of PCR from a plasmid containing mocry1Ab (SEQ ID NO: 71), using primers FR1Ab-1 (SEQ ID NO: 123) and 1Ab-6-sac (SEQ ID NO: 86), and a Mixture of 3 and a temperature cycle Profile 2. These two PCR product was used as template in the extended PCR reaction using primers C2-3A-3 (SEQ ID NO: 95) and 1Ab-6-sac (SEQ ID NO: 86), a Mixture of 3 and a temperature cycle Profile 2. The resulting amplicon was cloned into pCR2.1-TOPO (Invitrogen). Fragment BamHI/MluI of FR8a and the above PCR product into pCR2.1-TOPO Af1III, FR8a Af1III/SacI ligated into BamHI/SacI RET. Then the entire coding sequence (BamHI/SacI) moved in 1454. In chimeric insecticidal protein DM2-3A from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MTSNGRQCAGIRPYDGRQQHRG (SEQID NO: 127), amino acids 10-451 Cry3A055 protein (SEQ ID NO: 70), the components of the variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 7 amino acids of the conservative block 3, and amino acids 460-648 Cry1Ab protein (SEQ ID NO: 72), comprising 41 C-terminal amino acid of the conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6. Thus, the fold DM2-3A is the position of the crossover between Chua and Cry1Ab located in the conservative block 3 immediately after Ser451 that is to join the domain II and domain III. DM2-3A has activity against the Western corn root beetle, but its activity is less than the bend 8AF and FR8a, as shown in Table 5.

Example 34. Development of T7-8AF.

The fragment of the nucleic acid encoding the N-terminal part of a Cry3A055 protein (SEQ ID NO: 70), increased by way of PCR from a plasmid containing cry3A055 (SEQ ID NO: 69), using primers 5'3A-1-bam (SEQ ID NO: 83) and C3-3A-6 (SEQ ID NO: 84), and a mixture of 1 and a temperature cycle Profile 1.

The fragment of the nucleic acid encoding C-terminal part of the Cry1Ab protein (SEQ ID NO: 72), increased by way of PCR from a plasmid containing mocry1Ab (SEQ ID NO: 71), using primers C3-1b-3 (SEQ ID NO: 85) and 1Ab-6-Sac (SEQ ID NO: 86), and a Mixture of 1 and a temperature cycle Profile 1.

Then these two above is written of PCR product was used as template in the extended PCR reactions using primers 5 A-1-bam (SEQ ID NO: 83) and 1Ab-6-Sac (SEQ ID NO: 86), as well as a Mixture of 2 and a temperature cycle Profile 1.

The resulting amplicon ligated as a blunt fragment to the vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA), cut with SmaI, to form plasmid p8AF/CR2.1. Then the fragment BamHI-SacI from p8AF/CR2.1 ligated to RETA (EMD Biosciences, Inc., San Diego, CA), cut with BamHI-SacI and transformed into E. coli. This open reading frame is indicated T7-8AF (SEQ ID NO: 144) and encodes a hybrid protein T7-8AF (SEQ ID NO: 145). In the hybrid protein of T7-8AF from N-Terminus to C-end are: pipidinny fragment containing the amino acid sequence of MASMTGGQQMGRGS (amino acids 1-14 of SEQ ID NO: 126), amino acids 1-468 Cry3A055 protein (SEQ ID NO: 70), the components of the variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-648 ry1b (SEQ ID NO: 72), the components of the C-terminal 24 amino acids of the conservative block 3, variable plot 4, conservative block 4, variable plot 5, conservative block 5 and the flexible section 6, and 38 amino acids protoxin tail section Cry1Ab protein. The hybrid protein of T7-8AF has very little or no insecticidal activity against the Western corn root of the bug.

Example 35. Development 8AF.

Fragment BamHI-SacI from plasmids p8AF/CR2.1 (Cm. Primer) ligated to the plasmid, containing the constitutive promoter Shoes, which was modified as described in the work Schnepf and others (1985. J. Biol. Chem. 260:6264-6272) to correct the internal start codon ATG, which exists in codon by Schnepf and others, to the codon ATC, which was cut with BamHI-SacI and transformed into E. coli. This reading frame is indicated 8AF (SEQ ID NO: 63) and encodes bend 8AF (SEQ ID NO: 64). This fold 8AF similar bend FR8a, but does not contain the N-terminal peptidnogo fragment. In the bend 8AF from N-Terminus to C-end are: amino acids 1-468 Cry3A055 protein (SEQ ID NO: 70), the components of the variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 24 amino acids of the conservative block 3, and amino acids 477-648 Cry1Ab protein (SEQ ID NO: 72), the components of the 24 C-terminal amino acids of the conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5 and the flexible section 6, and also protoxin tail section of Cry1Ab 38 amino acids. Thus, the fold 8AF is the position of the crossover between Chua and Cry1Ab located in the conservative block 3 immediately after Leu468 sequence SEQ ID NO: 70, which is after the connection of domain II and domain III. This fold 8F has high activity against the Western corn root of the bug.

Example 36. Development-catGAF.

Model was made without site of cathepsin G (Cat G) in order to determine whether the presence of a site Cat G in the domain I bend 8AF required for activity against corn root of the bug. Fragment BamHI/Sa1I size 1359 BP from the plasmid containing mashua (SEQ ID NO: 67), and a fragment of the BamHI/Sall size 3483 BP from the plasmid containing 2OL-8a (SEQ ID NO: 1), ligated to create-catG8AF (SEQ ID NO: 146), which encodes a fold-catG8AF (SEQ ID NO: 147).

This fold-catG8AF very active against Western corn root beetle, this demonstrates that the site of recognition protease cathepsin G in the crook 8AF is not necessary for insecticidal activity.

Example 37. Development 8AFdm3

Described in Example 35 fold 8AF has a point of crossing over between Chua and Cry1Ab located in SW after connecting domains II/III, resulting in domain III bend 8AF has a small N-terminal segment of domain III protein Shua, and the rest of the domain III is a sequence of domain III of the protein Cry1Ab. In order to determine whether a small N-terminal segment of domain III protein Shua for insecticidal activity 8AF, was made another model with a cross between Chua and Cry1Ab located in SW exactly at the junction of domain II and domain III.

The fragment of the nucleic acid that encodes a portion of domain I and domain II of the protein Chua, increased by way of PCR from the plasmid is, containing FR8a (SEQ ID NO: 3), using primers CMS96 (SEQ ID NO: 138) and CMS97 (SEQ ID NO: 139), and a Mixture of 5 and a temperature cycle Profile 5.

The fragment of the nucleic acid encoding domain III protein moCry1Ab, increased by way of PCR from a plasmid containing mocry1Ab (SEQ ID NO: 71), using primers CMS98 (SEQ ID NO: 140) and CMS99 (SEQ ID NO: 141), a Mixture of 5 and a temperature cycle Profile 5.

The resulting two amplicon was used as template in the extended PCR reaction using primers CMS96 (SEQ ID NO: 138) and CMS98 (SEQ ID NO: 140), and a Mixture of 5 and a temperature cycle Profile 6. The resulting amplicon was cloned into pCR4 Blunt (Invitrogen, Carlsbad, CA). The StuI fragment/SacI cloned amplicon size 1633 BP, marked pCR4Blunt-OLWrdm3, and StuI fragment/SacI approximately 3089 BP from the plasmid containing cry3A055 (SEQ ID NO: 69), combined to create 8AFdm3 (SEQ ID NO: 148)that encodes a hybrid protein 8AFdm3 (SEQ ID NO: 149).

In a hybrid protein 8AFdm3 from N-Terminus to C-end are: amino acids 1-454 Cry3A055 protein (SEQ ID NO: 70), comprising domains I and II, including variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 10 amino acids of the conservative block 3, and amino acids 463-610 Cry1Ab protein (SEQ ID NO: 72), comprising the entire domain III, including the C-terminal 38 amino acids of the conservative block 3, areally plot 4, conservative section 4, the flexible section 5, the conservative block 5 and the flexible section 6.

Thus, protein 8AFdm3 is the position of the crossover between Chua and Cry1Ab immediately after Phe454 sequence SEQ ID NO: 70, which is located in the connection domain II - domain III. Protein 8AFdm3 does not have activity against the Western corn root of the bug. This suggests that the 24 amino acid N-terminal segment SV protein Shua or Shua (because they both have the same sequence on this site) are required for activity of the bend 8AF.

Example 38. Development 8AFlongdm3

In order to determine whether the critical position of the crossover in SW between Shua or Shua and Cry1Ab for activity against corn root beetle model was made, in which the position of the crossover is located in SW immediately after amino acid 519 of protein Shua.

The fragment of the nucleic acid that encodes a portion of domain I and the entire domain II, and domain III protein Chua, increased by way of PCR from a plasmid containing Shua (SEQ ID NO: 69), using primers CMS96 (SEQ ID NO: 138) and CMS101 (SEQ ID NO: 143), and a Mixture of 5 and a temperature cycle Profile 5.

The fragment of the nucleic acid that encodes a portion of domain III protein Cry1Ab, increased by way of PCR from a plasmid containing mocrylAb (SEQ ID NO: 71), using primers CMS98 (SEQ ID NO: 10) and CMS100 (SEQ ID NO: 142), and a Mixture of 5 and a temperature cycle Profile 5.

The resulting two amplicon was used as template in the extended PCR reaction using primers CMS96 (SEQ ID NO: 138) and CMS98 (SEQ ID NO: 140), and a Mixture of 5 and a temperature cycle Profile 6. The resulting amplicon was cloned into pCR4 Blunt (Invitrogen, Carlsbad, CA). Sa1I fragment/SacI cloned amplicon size of 460 BP, marked pCR4Blunt-OL8AFlongdm3, and the fragment SalI/SacI size 4265 BP from the plasmid containing 8AFdm3 (SEQ ID NO: 147), has combined to create 8AFlongdm3 (SEQ ID NO: 150), which encodes a hybrid protein 8AFlongdm3 (SEQ ID NO: 151).

In a hybrid protein 8AFlongdm3 from N-Terminus to C-end amino acids are 1-519 protein Shua (SEQ ID NO: 70), comprising domains I and II, which includes variable plot 1, conservative block 1, variable block 2, conservative block 2, variable 3 phase, conservative block 3, variable phase 4 and N-terminal 6 amino acids of the conservative block 4, and amino acids 528-610 Cry1Ab protein (SEQ ID NO: 72), the components of the C-terminal segment of domain III, including the C-terminal 4 amino acids of the conservative block 4, the flexible section 5, the conservative block 5 and the flexible section 6.

Thus, protein 8AFlongdm3 is the position of the crossover between Chua and Cry1Ab in the conservative block 4 immediately after I1e519 of SEQ ID NO: 70. Hybrid Cry-protein 8AFlongdm3 has no activity is against the Western root of the bug. This suggests that the area critical for activity of the bend Shua-Cry1A against corn root of the bug lies in the region between amino acids, respectively, from amino acid 6 of SWW to 7 amino acids of SV.

Example 39. Development cap8AFdm3

Fragment BamHI/Sa1I approximately 1363 BP from the plasmid containing 8AFdm3 (SEQ ID NO: 148), and a fragment of the BamHI/Sa1I approximately 3362 BP from the plasmid containing FR8a (SEQ ID NO: 3), ligated to create cap8AFdm3 (SEQ ID NO: 152), which encodes bend cap8AFdm3 (SEQ ID NO: 153).

Protein cap8AFdm3 has some activity against the Western corn root beetle, as shown in Table 5. The only difference between the hybrid protein 8AFdm3 non-insecticidal, and bend cap8AFdm3 is in the presence of N-terminal peptidnogo fragment (SEQ ID NO: 127). Thus, the addition of peptidnogo fragment to an inactive hybrid Cry squirrel gave active against corn root beetle hybrid insecticidal protein.

Example 40. Development 8AFdm3T

Fragment Pm1I/SacI approximately 4654 BP from the plasmid containing 8AFdm3 (SEQ ID NO: 148), and the fragment Pm1I/SacI approximately 190 BP of the plasmid containing FR8a (SEQ ID NO: 3), ligated to create 8AFdm3T (SEQ ID NO: 154), which encodes bend 8AFdm3T (SEQ ID NO: 155). In the crook of 8AFdm3T from N-Terminus to C-end are amino acids 1-454 Cry3A055 protein (SEQ ID NO: 70), is leaving the domains I and II, includes variable plot 1, conservative block 1, variable 2, conservative block 2, variable 3 phase and N-terminal 10 amino acids of the conservative block 3, and amino acids 463-610 Cry1Ab protein (SEQ ID NO: 72), comprising the entire domain III, which includes the C-terminal 38 amino acids of the conservative block 3, variable phase 4, the conservative block 4, variable plot 5, conservative block 5, the flexible section 6, and protoxin tail section of Cry1Ab 38 amino acids.

The only difference between the hybrid protein 8AFdm3 and bend 8AFdm3T is the addition of 38 amino acids protoxin tail section Cry1Ab, hence we can conclude that the addition of protoxin tail section can change inactive hybrid Cry protein in the active fold.

Example 41. Development 8AFlongdm3T

Fragment Pm1I/SacI approximately 4693 BP from the plasmid containing 8AFlongdm3 (SEQ ID NO: 150), and a fragment Pm1I/SacI approximately 190 BP of the plasmid containing FR8a (SEQ ID NO: 3), ligated to create 8AFlongdm3T (SEQ ID NO: 156), which encodes a hybrid Cry-protein 8AFlongdmT (SEQ ID NO: 157).

The only difference between hybrid Cry-protein 8AFlongdm3 and hybrid Cry-protein 8AFlongdm3T that do not have activity against the Western corn root beetle, consists in adding a 38 amino acids protoxin tail section Cry1Ab, this svidetel what there about that adding protecting plot itself is not sufficient to impart insecticidal activity of the hybrid Cry-protein 8AFlongdm3. This indicates that to create some folds may be necessary combination of variable regions and conservative blocs as an additive to protocolname tail section and/or N-terminal pateisinamu fragment.

Example 42. Development cap8AFdm3 T

Fragment Pm1I/SacI approximately 4693 BP from the plasmid containing cap8AFdm3 (SEQ ID NO: 152), and a fragment Pm1I/SacI approximately 190 BP of the plasmid containing FR8a (SEQ ID NO: 3), ligated to create cap8AFdm3T (SEQ ID NO: 158), which encodes bend cap8AFdm3T (SEQ ID NO: 159).

Protein cap8AFdm3T has higher activity against the Western corn root beetle than bend cap8AFdm3, as shown in Table 5. The only difference between the bend cap8AFdm3 with specific activity against the Western corn root beetle, and bend cap8AFdm3T is the presence of 38 amino acids protoxin tail section Cry1Ab. Therefore, some hybrid Cry proteins can be made active by adding N-terminal peptidnogo fragment and protoxin tail section.

Example 43. Test hybrid proteins on insecticidal activity.

Western corn root beetle

Hybrid proteins created in the steps is use examples tested for insecticidal activity against the Western corn root beetle in laboratory bioassays. The assay was performed by the method of inclusion in food. The E. coli clones expressing one protein, were grown during the night. 500 ál grown overnight culture was treated with ultrasound and determined the amount of protein for testing. Then the protein solution was mixed with 500 µl of molten artificial feeding, such as those described in the work Marrone and others (1985, J. of Economic Entomology 78:290-293). After hardening power he was placed in a Petri dish, and the food was placed 20 newborn corn root beetles. The temperature of the Petri dishes were maintained at approximately 30°C. Mortality was recorded after 6 days.

The results of the bioassay are shown in Table 5. In column 1 are the names of the hybrid Cry-proteins engineered hybrid insecticidal protein and chimeric insecticidal protein. Column 2 shows the relative levels of activity against the Western corn root beetle ("-"=<40% mortality; "+"=40-49% mortality; "++"=50-59% mortality; "+++"=60-80% mortality; and "++++"=>80% mortality). Column 3 shows the relative levels of the corresponding protein identified by Western-blotting. Column 4 indicates the presence of peptidnogo fragment ("-"=no peptidnogo fragment; #1=SEQ ID NO: 126; #2=SEQ ID NO: 127; #3=SEQ ID NO: 128; #4=SEQ ID NO: 129 #5=SEQ ID NO: 130; #6=SEQ ID NO: 131; #7=SEQ ID NO: 132). Columns 5-7 show the combination and arrangement of the variable regions (V1-V6), conservative blocks (C1-C5) and related domains (domains I-III) of the first Bt SGU-protein or modified SGU-protein and the second Bt SGU-protein, different from the first SGU-protein or modified SGU-protein constituting the core of the hybrid protein and is not active against Western corn root beetle, as well as bend, possessing activity against the Western corn root of the bug. Column 8 shows the number of amino acids in protoxin tail section, if present, and also Cry-protein, which took this tail area ("1b-38"=38 amino acids in pradakshinam the tail of the Cry1Ab; "1 VA-18"=18 amino acids in pradakshinam the tail of Cry1Ba).

Chimeric insecticidal proteins 2OL-8a and FR8a, and bend 2OL-12A tested against various insect species to determine the spectrum of activity. Among insects, took part in the tests were the Western corn root root beetle, Northern corn root beetle (SCI), southern corn root beetle (UCI), Colorado potato beetle (QOL) and European corn borer (UPW). The results of the bioassay are presented in Table 6. The sign "+" indicates the presence of insecticidal activity. The sign "-" indicates the absence of insecticidal asset is barb. Bends 2OL-8a and FR8a was active against CJ, SCJ and QOL. FOLD 2OL-12A was surprisingly active against UPW.

Table 6
Spectrum of activity of the bends
Spectrum of activity
ProteinKGSKJUCIRYWcy
2OL-8a++-+-
FR8a++-+-
2OL-12A-NTNTNT+
Cry3A055++-+-
Shua- --+-
Cry1Ab----+

Example 44. The introduction of genes encoding bends in plants

For transformation into corn plants were selected three genes encoding chimeric insecticidal proteins FR8a, FRCG and FRD3. The expression cassette containing the coding sequence of FR8a, or FRCG, or FRD3, embedded in an appropriate vector for Agrobacterium-mediated transformation into corn. For this example, in experiments on transformation was applied the following vectors: 12207 (Figure 3), 12161 (Figure 4), 12208 (Figure 5), 12274 (6), 12473 (Fig.7) and 12474 (Fig).

Transformation of immature maize embryos was performed generally as described in Negrotto and others, 2000, Plant Cell Reports 19: 798-803. For this experiment, all components of the environment were basically the same as described in the above work Negrotto, etc. But you can use other components known in the industry.

Used for transforming genes cloned in a vector suitable for transformation of maize. Used in this example, the vectors contain a gene isomerase phosphomannose (PMI) for selection of transgenic lines (Negrotto, etc. above).

VCR is CE, the Agrobacterium strain LBA4404 (pSB1)containing plasmid transformation plants were grown on solid medium YEP (yeast extract (5 g/l), peptone (10 g/l), NaC1 (5 g/l), agar (15 g/l), pH 6.8) for 2-4 days at 28°C. Approximately H 109 Agrobacterium suspended in the environment LS-inf, supplemented with 100 μm As (Negrotto, etc. above). Pre-induction of the bacteria in this environment occurred within 30-60 minutes

Immature embryos from A or other suitable genotype removed from the cobs age 8-12 days and placed in a liquid LS-inf+100 uM As. The embryos once rinse fresh infectious environment. Then add a solution of Agrobacterium and subjected to shaking for 30 seconds, then allow to settle with the bacteria for 5 minutes. Then the embryos are transferred by the metanotum up on Wednesday LSAs and cultured in the dark for two or three days. Then from 20 to 25 embryos per Petri dish placed in the environment LSDc with the addition of Cefotaxime (250 mg/l) and silver nitrate (1, 6 mg/l) and cultured in the dark at 28°C for 10 days.

Immature embryos forming embryogenic callus was transferred to medium LSD1M0.5S. Breeding of these crops on the environment was carried out for about 6 weeks with phase subculturing about 3 weeks. Surviving calli were transferred to medium Reg1 with the addition of mannose. After culturing in the light (16 hours light/8 hours dark) the green circle fabric was moved to Wednesday Reg2 without growth regulators and incubated for 1-2 weeks. Shoots were transferred to Magenta boxes GA-7 (Magenta Corp., Chicago, I11.), containing environment Reg3, and were grown in the light. After 2-3 weeks the plants were tested by PCR method for the presence of the pmi gene, and genes FR8a or FRCG. Plants showed positive results of PCR testing, were moved to the greenhouse and tested for resistance to corn root bug.

Example 45. Analysis of transgenic plants corn on the efficacy against corn root beetle: bioassays with removing root

In a typical case, the samples of maize plants take when transplanting from boxes Magenta GA-7 into the soil. This allows to obtain samples of roots from the sterile environment compared to soil conditions. Samples taken by cutting a small piece of root (length approx 2-4 cm) and placed in a small Petri dish with enriched Pitagora (fatagar, 12 g, sucrose, 9 g MS salts, 3 ml, MS vitamins, 3 ml, nystatin (25 mg/ml), 3 ml, Cefotaxime (50 mg/ml, 7 ml, aureomycin (50 mg/ml, 7 ml, streptomycin (50 mg/ml), 7 ml d2, 600 ml). Negative control samples are either transgenic plants that have received a negative result when testing PCR for the presence of gene FR8a or FRCG, from the same experiment in transformation, or not transgenic plants (the same size as the test plants were grown in the phytotron.

Samples of roots so take the e after growing plants in soil. If the roots take samples from the soil, these pieces of roots washed with water to remove residual soil, dip into a solution of nystatin (5 mg/l), removed from the solution, dried with a paper towel and placed in a Cup with Pitagora, as described above.

Samples of roots was carried out by inoculation of the Western corn root beetle, by placing approximately 10 larvae of the first age on the inner surface of the cover each Cup with Pitagora, and then tightly close the lid over the sample root. Larvae bore a thin brush. After inoculation of all cups of the tray with the cups were placed in the dark at room temperature prior to data collection.

Data was collected in approximately 2-4 days after inoculation of roots. Calculate the percentage mortality of larvae, as well as visually assessed the degree of damage to the root. The degree of damage was determined on the basis of the number of detected hollowed out by larvae of the corn root beetle holes (ON) in the sample root, and it was estimated as high, medium, low or zero by assigning categories, respectively, 3, 2 or 1 (category 1 is assigned when a low degree of root damage or no damage). In a typical case, in plants category 1 were from 0 to 2 in the plants category 2 - from 3 to 4, and in plants category 3 - more than 5. Believed that plants ka is of egorie 1 damage - great result, category 2 - average, 3 - weak result. Plants category 1 was selected for further testing in the greenhouse and in the field.

The results in Table 7 show that plants expressing bends FR8a and FRCG, protect the roots from damage by Western corn root beetle. In most cases expressing the chimeric insecticidal protein plants were assigned to category 1, while control plants not expressing the chimeric insecticidal protein, belonged to category 3. Plants expressing bend FRD3, showed comparable levels of struggle against the Western corn root of the bug.

Example 46. Analysis of the effectiveness of plant transgenic corn against corn root beetle in the field.

The part of plants that have received a positive assessment in the above-described bioassays with the root extract was evaluated on the field. Eighteen plants from each experience learned from field plots and assessed the degree of damage to their roots. The degree of damage to the roots was estimated on the basis adopted in the state of Iowa for the linear scale of the damage to the roots from 0 to 3 (Oleson, J.D., and others, 2005. J. Econ Entomol. 98(1): 1-8), where 0, 00=no damage (lowest possible level); 1,00=one node (circle roots) or the equivalent of an entire node, Yaderny approximately 11 inches of the stem line of the soil on the 7th node); 2,00=two full jigger node; 3,00 three or more pin nodes (highest possible level); and damage, not expressed in a large number of nodes, marked as the share of the missing node, that is, 1,50=1 1/2 eaten node, a 0.25=1/4 one node eaten, etc.

The results of field trials against Western and Northern corn root of the bug is shown in Table 8, and against Mexican corn root beetle in Table 9. All transgenic corn expressing the chimeric insecticidal protein FR8a, showed results better than standard commercial chemical insecticide against Western, Northern and Mexican corn root of the bug.

Table 8
The results of field trials in Western and Northern corn root beetle
ExperiencePlasmidThe degree of damage to the root
112161 (ubi:FR8a)0,08
2121610,05
3121610,09
4121610,04
512274 (cmp:FR8a)0,04
6122740,08
7122740,05
Chemical0,15
Control negative0,87

Table 9
The results of field trials on Mexican corn root beetle
ExperiencePlasmidThe degree of damage to the root
112161 (ubi:FR8a)0,04
512274 (cmp:FR8a)0,22
6122740,05
Chemical0,15
To the negative control 1,04

1. Engineered hybrid insecticidal protein comprising, from N-Terminus to C-end of the N-terminal site of the protein Cry3A, fused with a C-terminal site of the protein Cry1Ab, and the position of the crossover Cry3A protein and the protein Cry1Ab is conservative in block 2, in the conservative block 3, or conservative block 4, and this protein is optional includes:
(a) protoxin tail plot of Bt Cry, located at the s-end; or
(b) N-terminal pipidinny fragment; or
(c) both (a)and (b),
moreover, the specified engineered hybrid insecticidal protein includes an amino acid sequence having at least 80% identity with SEQ ID NO:64, and has activity against the Western corn root of the bug.

2. Engineered hybrid insecticidal protein according to claim 1, characterized in that the Cry3A is Shua or modified Shua.

3. Engineered hybrid insecticidal protein according to claim 2, characterized in that
(a) Shua includes SEQ ID NO:68 or SEQ ID NO:135, or
(b) modified Shua includes SEQ ID NO:70, and in which Cry1Ab includes SEQ ID NO:72.

4. Engineered hybrid insecticidal protein according to claim 3, characterized in that the position of the crossover between the Cry3A and Cry1Ab or modified Cry3A and Cry1Ab located in the IU what do amino acids, the corresponding amino acid 6 of the conservative block 3 and the amino acid 7 of the conservative block 4.

5. Engineered hybrid insecticidal protein according to claim 4, characterized in that the position of the crossover between the Cry3A and Cry1A or modified Cry3A and Cry1A is conservative block 3 immediately after the amino acid corresponding to Ser451, Phe454, or Leu468 sequence SEQ ID NO:70.

6. Engineered hybrid insecticidal protein according to claim 5, characterized in that the position of the crossover is located in the conservative block 3 immediately after the amino acid corresponding to Ser451, Phe454, or Leu468 sequence SEQ ID NO:70.

7. Engineered hybrid insecticidal protein according to claim 1, characterized in that it includes at least two positions of the crossover between the amino acid sequence Cry3A protein and the amino acid sequence of the protein Cry1Ab.

8. Engineered hybrid insecticidal protein according to claim 7, characterized in that
(a) the first position of the crossover is located in the conservative block 2, and the second position of the crossover is located in the conservative block 3; or
(b) the first position of the crossover is located in the conservative block 3, and the second position of the crossover is located in the conservative block 4.

9. Engineered hybrid insecticidal protein according to claim 8, characterized in that
(a) first the position of the crossover between the Cry3A and Cry1Ab or between the modified Cry3A and Cry1Ab is conservative block 2 immediately after amino acid, the corresponding Asp232 sequence SEQ ID NO:70, and a second position crossover Cry1A and Cry3A or Cry1A and modified Cry3A is conservative block 3 immediately after the amino acid corresponding to Leu476 sequence SEQ ID NO:72; or
(b) the first position of the crossover between the Cry3A and Cry1Ab or modified Cry3A and Cry1Ab is conservative block 3 immediately after the amino acid corresponding to Leu468 sequence SEQ ID NO:70 and the second position of the crossover between Cry1Ab and Cry3A or between Cry1Ab and modified Cry3A is conservative unit 4 immediately after the amino acid corresponding to Il527 in the sequence SEQ ID NO:72.

10. Engineered hybrid insecticidal protein according to claim 9, in which Cry3A is Shua or modified Shua, characterized in that
(a) the first position of the crossover between Shua and Cry1Ab or modified Shua and Cry1Ab is conservative block 2 immediately after Asp232 sequence SEQ ID NO:70, and a second position crossover Cry1Ab and Shua or Cry1Ab and modified Shua is conservative block 3 immediately after Leu476 sequence SEQ ID NO:72; or
(b) the first position of the crossover Shua and Cry1Ab or modified Shua and Cry1Ab is conservative block 3 immediately after Leu468 sequence SEQ ID NO:70 and a second position crossover Cry1Ab and Shua or Cry1Ab and modificirovana what about Shua is conservative unit 4 immediately behind Ile527 sequence SEQ ID NO:72.

11. Engineered hybrid insecticidal protein according to claim 1, characterized in that the insecticidal protein has additional activity against Northern corn root beetle or Mexican corn root of the bug.

12. Engineered hybrid insecticidal protein according to claim 1, characterized in that it comprises the amino acid sequence selected from the group comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:147, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:159 and SEQ ID NO:160.

13. Selected molecule of nucleic acids encoding engineered hybrid insecticidal protein according to any one of claims 1 to 12.

14. The selected nucleic acid molecule according to item 13, which comprises a nucleotide sequence selected from the group comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:146, SEQ ID NO:152, SEQ ID NO:154 and SEQ ID NO:158.

15. Expression cassette, which comprises a heterologous promoter sequence functionally linked with the nucleic acid molecule according to item 13.

16. The recombinant vector containing the expression cassette, for 15 to ensure the expression or production of hybrid insecticidal protein according to any one of claims 1 to 12, respectively.

17. Transgenic CL is TKA-master, containing the expression cassette according to item 15, to ensure the expression or production of hybrid insecticidal protein according to any one of claims 1 to 12, respectively.

18. Transgenic a host cell according to 17, which represents a bacterial cell.

19. Transgenic a host cell according to 17, representing the plant cell.

20. Transgenic plant comprising the expression cassette according to item 15, and this plant produces insecticidal protein according to any one of claims 1 to 12.

21. Transgenic plant according to claim 20, and this plant is a plant of maize.

22. Seeds obtained from the transgenic plant according to claim 20, moreover, these transgenic seeds include a nucleic acid molecule according PP or 14.

23. Seeds obtained from the transgenic plants of maize under item 21, and these transgenic seeds include a nucleic acid molecule according PP or 14.

24. Transgenic plant that is resistant to Western corn root beetle, including:
(a) a nucleic acid molecule comprising the nucleotide sequence encoding engineered insecticidal protein selected from the group comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:147, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:159 and SEQ ID NO:160; or
(b) a nucleic acid molecule to the slots, comprising the nucleotide sequence selected from the group comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:146, SEQ ID NO:152, SEQ ID NO:154 and SEQ ID NO:158.

25. Insecticidal composition for the fight against the Western corn root beetle, containing the engineered hybrid insecticidal protein according to any one of claims 1 to 12, and conventional excipients.

26. The method of obtaining engineered hybrid insecticidal protein according to claim 1, active against insects, comprising:
(a) obtaining a transgenic host cell according to 17; and
(b) growing the transgenic host cell under conditions allowing expression of the owner constructed a hybrid insecticidal protein effective against insects.

27. The method of obtaining insect-resistant transgenic plant, comprising transforming the plant cell with the expression cassette according to § 15 and regeneration of transgenic plants from the transformed plant cell, wherein the nucleic acid encoding engineered hybrid insecticidal protein according to claim 1, is expressed in the transgenic plant in an amount effective to control insects.

28. The method according to item 27, in which insects are beetles insects.

29. The method according to p, when what oterom beetles represent the Northern corn root beetle, Mexican corn root beetle, or the Colorado potato beetle.

30. A method of combating insects which comprises obtaining the insects an effective amount of engineered hybrid insecticidal protein according to claim 1.

31. The method of obtaining engineered hybrid insecticidal protein according to any one of claims 1 to 12, which includes:
(a) obtaining a first nucleic acid that encodes a protein Cry3A;
(b) obtaining a second nucleic acid that encodes a protein Cry1Ab;
(c) the connection in the direction from 5' to 3' 5' part of the first nucleic acid, which encodes the N-terminal site of the Cry3A protein and the 3' part of the second nucleic acid, which encodes the C-terminal site of the protein Cry1Ab, and the position of the crossover Cry3A protein and the protein Cry1Ab is conservative block 2, conservative block 3, variable area 4 or conservative unit 4 to obtain nucleic acids encoding engineered insecticidal protein having activity against the Western corn root beetle; and optionally merge with the 5' end sequence of the hybrid nucleic acid, which encodes pipidinny plot that leads to 5' elongation or merge with the 3' end of the hybrid nucleic acid sequence that encodes protoxin tail protein site Bt Cry, which leads to 3' elongation, or both t and more;
(d) embedding in the expression cassette of the hybrid nucleic acid having one or both of these 5' extension 3' extension, or not having any of these movements;
(e) transformation of the expression cassette into a cell of the host, resulting in above-mentioned a host cell produces engineered hybrid insecticidal protein.

32. The method according to p, characterized in that the Cry3A protein is Shua or modified Shua.

33. The method according to p, wherein the engineered hybrid insecticidal protein includes
(a) prototoxin tail protein site Bt Cry, located at the s-end; or
(b) N-terminal pipidinny fragment comprising at least 9 amino acids; or
(C) both (a)and (b).

34. The method according to p at which the specified prototoxin tail section comes from Cry1Aa or CryAb.

35. The method according to clause 34, characterized in that the above-mentioned prototoxin tail section contains 38 amino acids.

36. The method according to p, characterized in that the specified prototoxin tail section includes an amino acid sequence that corresponds to amino acids 611-648 sequence SEQ ID NO:72.

37. The method according to p, characterized in that prototoxin tail section includes the amino acid sequence 611-648 sequence of SEQ D NO:72.

38. The method according to clause 34, wherein pipidinny the site comprises the amino acid sequence of YDGRQQHRG (SEQ ID NO:132) or TSNGRQCAGIRP (SEQ ID NO:133).

39. The method according to § 38, characterized in that pipidinny plot selected from the group consisting of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131 and SEQ ID NO:132.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: method is characterised in that the DNA of the structure RNAb indicated on Figure 1, which encodes the fused protein of three parts, where N-terminal position is green fluorescent protein GFP, central - peptide of 73 amino acid residues with the amino acid sequence of SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and C-terminal - light chain of double-stranded protein Kunitz-type inhibitor from potato tubers (PKPI-BI), are introduced into cells of E. coli. The cells transformed by this construction are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifugation. The product of expression in the form of inclusion bodies is solubilised with the denaturant. Chromatography is carried out under denaturing conditions. The resulting product is used for detection of specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.

EFFECT: invention enables to obtain the recombinant antigen G2 of Hantavirus Dobrava with increased yield.

6 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce a polypeptide, including cell cultivation, which intensely expresses a bicarbonate carrier and has a transferred DNA, which codes the desired polypeptide.

EFFECT: invention makes it possible for the cell to produce the specified polypeptide and the appropriate cell.

12 cl, 15 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to chemical engineering and techniques for producing veterinary, medical and pharmaceutical preparations. The method of producing a novel antiviral substance based on 2,5-dihydroxybenzoic acid and gelatine includes oxidising 2,5-dihydroxybenzoic acid with laccase enzyme to intermediate phyenoxy radicals and semiquinones, which are then copolymerised with gelatine, and separating the obtained copolymer from low-molecular weight components by dialysis; optimum concentrations of components of the reaction mixture are as follows: 2,5-dihydroxybenzoic acid - 15-80 mM, gelatine - 1-13 mg/ml reaction mixture, laccase - 0.5-10 units of activity/ml reaction mixture.

EFFECT: obtained copolymer has antiviral activity on herpesvirus, particularly Aujeszky's disease virus.

2 tbl, 1 dwg, 3 ex

FIELD: biotechnology.

SUBSTANCE: method includes cultivation of previously prepared culture of the recombinant strain B. anthracis 55ΔTPA-1Spo-. The cell mass is separated using the filtration module with a membrane having a pore diameter of 0.2 mcm. Protein EA1 is extracted from the washed cell mass using a buffer with 1% sodium dodecyl sulfate, and purified by diafiltration using membrane filters and two-stage ion-exchange chromatography on hydroxyapatite. The protective antigen is isolated from the culture filtrate and purified by successive steps of concentration and diafiltration.

EFFECT: use of the invention enables to obtain in one processing chain the highly purified antigens of anthrax microbe - protective antigen and protein EA1 needed to create chemical vaccines.

3 dwg, 5 ex

FIELD: biotechnology.

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EFFECT: invention enables to accelerate preparation of bacteriocin and to increase the degree of purification of the resulting product in a yield of 90% of the total activity in the culture fluid.

1 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. There are presented versions (aCt1 and aCt2) of one-domain antibodies specifically binding the Chlamydia trachomatis antigen. There are described versions of the method of inhibiting an infection caused by Chlamydia wherein the method involves the preparation of elementary bodies C.trachomatis by a therapeutically effective amount of the nanoantibody aCt1 or aCt2 before being attached to infected target cells.

EFFECT: use of the invention provides the antibodies to detect and block the infections Chlamydia trachomatis that can find application in medicine.

6 cl, 4 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of preparing a pharmaceutical composition, CHO cell to obtain the desired protein, the CHO cell - DNA recipient encoding the desired polypeptide, the method of production of the desired polypeptide. The method of production of the desired polypeptide comprises cultivation of CHO cell which is transformed DNA encoding alanine aminotransferase and DNA encoding the desired polypeptide. In the particular case the CHO cell is cultured in α-ketoglutarat-containing medium. The method of preparing of the pharmaceutical composition comprises preparing of the desired polypeptide with the method described above. The polypeptide is mixed with pharmaceutically acceptable carriers or additives. The preparation is prepared. The CHO cell for preparing of the desired protein has DNA transferred into it, encoding alanine aminotransferase, and DNA transferred into it, encoding the desired polypeptide.

EFFECT: invention enables to prepare a desired polypeptide with a high yield.

10 cl, 22 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant host cell Pichia pastoris includes a cascade of fucosylation reactions, where ferments make up the specified cascade of reactions. The specified host cell Pichia pastoris includes nucleic acids, which code GDP-mannose-dehydratase (GMD), GDP-ketodesoxymannose-epimerase/GDP-ketodesoxygalactose-reductase (FX), a carrier of GDP-fucose (GFTr) and fused protein, which contains a catalytic domain of α1,6-fucosyltransferase EC 2,4.1.68, fused with amino acids 1-36 Mnn2 S.cerevisiae. A hybrid vector includes regulatory elements of DNA, which are functional in the host cell and which are functionally connected with a coding DNA sequence, which codes the fused protein, including amino acids 1-36 Mnn2 S.cerevisiae, fused with the catalytic domain of α1,6-fucosyltransferase EC 2.4.1.68. The specified vector is used to transform the host cell Pichia pastoris.

EFFECT: invention makes it possible to create a host cell Pichia pastoris, which will produce glycoproteins with fucosylated N-glycans.

8 cl, 6 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: protein of flu virus hemagglutinin is disclosed, as well as a molecule of nucleic acid, which codes such protein, a vaccine for treatment or prevention of infections, which are mediated by a flu virus, a set and also methods to produce protein and vaccines. The protein of hemagglutinin H5 of the flu virus is characterised by the fact that in the position 223 of the series it is substituted with asparagine, and the second lysine is built into the position 328.

EFFECT: modified protein has improved immunogenic properties.

14 cl, 1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to feed production, particularly a method for microbiological production of feed yeast from grain wastes. The method involves preparation of grain material by grinding to 120-160 mcm particles. Further, the ground grain material is used to prepare an aqueous suspension containing 15-25% dry substances. The obtained suspension is saccharified with α-amylase and glucoamylase to 6-10% glucose content in the suspension. Nitrogen and phosphorus sources are added to the obtained suspension such that 90 mg of nitrogen and 45 mg of phosphorus are required for synthesis of 1 g of biomass. A culture is added to the obtained nutrient medium, said culture being a producer of the Saccharomyces cerevisiae yeast strain VKPM U-3585, having amylase activity, which is deposited in the Russian National Collection of Industrial Microorganisms (VKPM) and can be used in producing feed protein. The Saccharomyces cerevisiae yeast strain VKPM U-3585 is continuously grown in a multiple-section apparatus while feeding the nutrient medium simultaneously into several sections, followed by concentration of the suspension by vacuum evaporation and drying. Moisture freed at the vacuum evaporation and drying steps is used to prepare the aqueous suspension of grain material.

EFFECT: invention increases output of crude protein and true protein.

5 ex

FIELD: biology.

SUBSTANCE: plant cell is transformed by exogenous polynucleotide, expression of which provides cell with enhanced tolerance to abiotic stress, with further cultivation of grown plant from the cell. Transformation is performed by introduction of construction with constitutive or abiotic stress-induced promoter to the cell. Either the plant is infected by avirulent virus including indicated exogenous polynucleotide which, when expressed in plant, provides plant with tolerance to abiotic stress.

EFFECT: resistance to abiotic stress, such as soil salinity, water deficiency, low or high temperature etc.

37 cl, 7 dwg, 6 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: method of obtaining a continuous cell line capable of maintaining growth of cell-associated alpha herpes viruses involves the infection or transfection of a cell with the nucleic acid or fragment of the herpes virus, the selection of cells expressing the said nucleic acid or its fragment, and the cultivation of the said infected or transfected cell or its progeny in conditions suitable for the expression of the said nucleic acid and the multiplication of the said cell or its progeny. The said nucleic acid includes the herpes virus glycoprotein gene gE or its functional fragment. The expression of the said gE gene or its functional fragment takes place in a sustainable manner. The cell of the continuous cell line capable of maintaining growth of cell-associated alpha herpes viruses includes the herpes virus nucleic acid or its fragment. The said nucleic acid includes the herpes virus glycoprotein gene gE or its functional fragment, the expression of the said gE gene or its functional fragment takes place in a sustainable manner. The said cell can go through at least 10 passages. The invention group includes the application of the said cell for obtaining a vaccine capable of inducing protection against disease in vertebrates, and for obtaining and/or maintaining and/or isolation of a hypervirulent and/or very virulent Marek's disease virus strain, as well as for obtaining the Marek's disease diagnostic antigen. The invention group also includes a method of obtaining and/or isolation and/or maintaining the Marek's disease virus strain. The method involves the infection of the above cell with the said strain and the cultivation of the said cell in conditions suitable for the multiplication of the said cell and obtaining, maintaining, and isolation of the Marek's disease virus.

EFFECT: invention group provides a method of obtaining a vaccine capable of inducing protection against the disease; allows to satisfy the need in new cell systems allowing to maintain new properties acquired by the cell.

35 cl, 6 dwg, 6 ex.

FIELD: cell engineering.

SUBSTANCE: breast cells are isolated and cultivated on substrate for 2-3 days. Then cells are treated directly on substrate with Hoechst 33342 colorant and tested with microscope in ultraviolet light with wave length of 350 nm.

EFFECT: method for effective determination in vitro of breast stem cells followed by evaluation of morphological characteristics thereof.

1 tbl, 2 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a humanized human monoclonal CD19 antibody prepared of an HB12B antibody, or a fragment thereof characterised by amino acid sequences of variable domains. Also, there are presented nucleic acids coding polypeptides having the sequences of the variable domains, and a cell expressing the antibody under the invention, and a pharmaceutical composition, and a method for treating a B-cell diseases or disorders in a human.

EFFECT: invention can find further application in treating various CD19-associated diseases, including autoimmune diseases, and preventing or treating the graft-versus-host disease (GVHD), and the humoral rejection and post-transplantation lymphoproliferative disorder in a human graft recipient.

21 cl, 45 dwg, 40 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant nucleic acid expresses one or several polypeptides of interest, a vector of expression and bacteria, which contain this recombinant nucleic acid. The recombinant nucleic acid contains a natural promotor of a gene of HU-like DNA-binding protein (PhilA) of Lactococcus type with the sequence SEQ TD NO:28, or its homological or functional version, which at least by 95% identical to the promotor with sequence SEQ ID NO:28, functionally linked with one or several open reading frames, heterological for the promotor RhIIA, where the promotor RhIIA is located above one or several open reading frames. The expression vector contains the above recombinant nucleic acid, preferably, the specified vector is produced from pTINX. A bacterium contains the above recombinant nucleic acid or the above vector.

EFFECT: proposed invention makes it possible to increase level of expression of polypeptide genes of interest and therefore produce sufficient number of expressed proteins.

19 cl, 26 dwg, 12 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biochemistry and immunology. What is considered is an isolated antibody molecule which binds an alpha chain of human granulocyte/macrophage colony stimulating factor receptor (GM-CSFRα) and inhibits GM-CSF binding to human GM-CSFRα. There are presented: a composition for GM-CSFRα inhibition or neutralisation and a composition for treating rheumatoid arthritis, asthma, chronic obstructive pulmonary disease or myeloid leukaemia, containing the antibody under the invention; the isolated nucleic acid molecule coding the antibody under the invention, a host cell and a method for preparing the antibody under the invention, as well a method for inhibiting or neutralising GM-CSFRα activity and a method of treating rheumatoid arthritis, asthma, chronic obstructive pulmonary disease or myeloid leukaemia in a patient.

EFFECT: invention can find further application in therapy of the GM-CSFRα-mediated diseases.

41 cl, 5 tbl, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers biotechnology and medicine. What is described is an immunogen for inducing the IgE immune response. The immunogen contains at least one IgE antigen peptide bound to an immunogenic carrier. The immunogenic carriers may be presented by virus-like particles specified in a group consisting of HBcAg, HBsAg and Qbeta VLP There are also disclosed compositions and method for preventing, relieving or treating an IgE-associated disorder in an individual with using the above immunogen.

EFFECT: group of inventions may be used in medicine for treating the allergic diseases.

20 cl, 2 dwg, 13 tbl, 14 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce a polypeptide, including cell cultivation, which intensely expresses a bicarbonate carrier and has a transferred DNA, which codes the desired polypeptide.

EFFECT: invention makes it possible for the cell to produce the specified polypeptide and the appropriate cell.

12 cl, 15 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: by the recombinant method a line of cells CHO[V3D] is produced, which are transformed with plasmid pV3D, coding the human vessel endothelium growth factor, isoform A165, with C-terminal 3xDED-epitope (SEQ ID NO: 1), which is able to produce a biologically active recombinant protein VEGF-A165 of humans with C-terminal 3xDED-epitope in the amount of at least 0.2 mg 1 l of medium for 72 hours with 1 ml of growth medium per 5 cm2 of single-layer culture. The cell line is deposited in the All-Russian Collection of Industrial Microorganisms VKPM under the registration number VKPM H-123.

EFFECT: no impact at biological activity of protein and possibility to do protein treatment.

4 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of immunology. Claimed are versions of isolated anti-CD30 antibody, in fact free of fucosyl residues. Claimed antibody is characterised by the fact that it contains in one version 3 CDR from heavy chain and three CDR from light chain, in the other version it is characterised by presence of variable region of heavy chain and variable region of light chain. Described is host cell, producing said antibody, in fact free of fucosyltransferase, described is method of inhibiting growth of cells CD30+ and method of inhibiting growth of tumour cells expressing CD30 with application of antibody. Described are versions of antibody application for obtaining chimeric or humanised version of antibody.

EFFECT: antibodies by claimed invention demonstrate increased ADCC cell cytotoxicity with respect to lines of cells of Hodgkin human lymphoma: L428, L540, L1236 and line of T-cell human lymphoma: KARPAS, expressing CD30, which are not lysed by fucolysed form of antibodies.

24 cl, 15 dwg, 4 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of immunology. Claimed is fully human monoclonal antibody, which binds insulin-like growth factor II (IGF-II) and has cross-reactivity to IGF-I, as well as its antigen-binding fragment. Described is molecule of nucleic acid, encoding antibody by the invention, vector and host cell for expression of antibody by the invention. Described is pharmaceutical composition, as well as conjugates for treatment and diagnostics of malignant tumour, application of antibody by the invention in medication manufacturing and method of determining level 1GF-II and IGF-I in patient's sample.

EFFECT: invention can find further application in cancer therapy.

55 cl, 27 ex, 18 tbl, 3 dwg

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