A method of obtaining a genetically modified baculovirus, the strain of the nuclear polyhedrosis virus, a method of combating insects, insecticidal composition


(57) Abstract:

The use of biotechnology, in particular genetic engineering objects for biological protection of plants from pests. Essence: the proposed group of inventions, which includes a method of obtaining a genetically modified nuclear polyhedrosis virus of Autographa californica, providing the impact on the genome of the virus by inactivation of the gene encoding the enzyme ecdysteroid UDP glucosyl-transferase. The obtained recombinant virus is very effective in insecticidal compositions compared with wild-type viruses or the viruses used in the present time. The next object of the invention in the proposed group is a method of combating insects which comprises the reception of the application of genetically modified viruses to plants. 4 C. and 7 C. p. F.-ly, 14 Il, 3 tables.

The invention was developed with funds of the National Institutes of Health (Grant N GE000918). The United States government may have certain rights in this invention.

The invention relates to methods and compositions for improved biological control of insect pests. More specifically, this invention relates to the use of paloc insects. This invention also relates to genetically modified rod-like viruses and methods for their preparation.

Interest to the biological method of combating insect pests due to the shortcomings of traditional chemical pesticides. Chemical pesticides, as a rule, act as harmful and beneficial species. There is a tendency that insect pests develop resistance to these chemicals, so quickly can develop populations resistant to these pesticides insects. Further, the remnants of the chemicals cause damage to the environment and potentially to human health. Biological control is an alternative way of effects on insects, which reduces dependence on chemical pesticides.

The main strategy of biological control involves the use of naturally occurring organisms that are pathogenic in relation to insects (entomopathogens), and breeding more resistant to insect pests of crops. The approach to the solution of these problems includes the identification and characterization of genes insects or gene products that can serve as a suitable basis for obtaining Agay is modifying existing in the nature of non-pathogenic microorganisms with the aim of turning them against pathogenic to insects), modification and improvement of the currently used entomopathogens, and the establishment of methods of genetic engineering of crops with greater resistance to pests.

To entomopathogens, proposed as biological pesticides, including viruses that cause natural epizootic in populations of insects (A. M. Heimpel Japan seminar on microbial control, 1968, p. 51-62, Biological plant protection, M 1974, 335-345), including Baculoviruses. Rod-shaped viruses (baculoviruses) represent a large group of viruses that infect only arthropods insects (Miller L. K. (1981), in Genetic Engineering in the Plant Sciences, N. Panopoulous, (ed.) Praeger Publ. New York, pp. 203-224; Carstens. (1980) Trends in Biochemical Science 52 107-110; ish Payne (1979) in Advances in Virus Research. v. 25, Lawfer et al. (eds. ) Academic Press N. Y. p. 273-355). Many baculoviruses infect insects, which are pests of commercially important agricultural and forest crops. These baculoviruses potentially represent a valuable biological control agents. The United States Agency for environmental protection (U. S. Environmental Protection Agency) recommended for use as insecticides four rod-shaped virus. One of the advantages of baculoviruses as biological pesticides is their specificially strain rod-shaped virus usually infects only one or a few insect species. Thus, they do not represent any threat to the environment or to humans and can be used without harm to beneficial insects.

The group of baculoviruses include subgroups of nuclear polyhedrosis viruses (JPO), virus nuclear granules (HS) and rod-shaped viruses, not forming a body of inclusion. The occluding forms rod-shaped virus virions (enclosed in the sheath nucleocapsid) are placed inside a crystalline protein matrix. Such a structure, the so-called inclusion body or the body of "embedding" is a form of finding the virus in nature outside of the body, which causes the transmission of the organism to organism. Hallmark virus YAP is that every body inclusions contain a lot of virions. Body enable YAP viruses have a relatively large size (up to 5 microns). Body enable GW are smaller and contain a single virion. Both forms crystalline protein matrix of the inclusion bodies is mainly composed of a single polypeptide from 25,000 to 33,000 daltons called poliaminom or, respectively, granulins. Rod-shaped viruses neochlamisus forms do not produce protein poliakin or granulin and do not form inclusion bodies.

One of the major drawbacks of the use of rod-shaped viruses as pesticides is a prolonged period of time between the moment when the insect swallows a virus, and his death. During this time, the pest continues to feed and damages the crop. In this regard, the determining factor is the reduction of the time feeding insect to a minimum, i.e. the creation of the pesticide, reducing the power of the insect until death. It is also desirable that the death of the insect came faster. Such results can be obtained by treatment of the viral genome, to carry out which in particular allow methods of gene Egan, affect growth, development or behavior of insects. It provides ways of using this gene and the product of this gene, as well as methods of inactivation of this gene or product of this gene to control insects. In a preferred variant embodiment using egt gene, which encodes ecdysteroid UDP glucosyl-transferase (EGT) of AcMNPV virus. The egt gene expression of rod-shaped viruses causes the formation of EGT, which inactivates hormones serving molting insects, preventing them molting or pupation of the larvae. Inaktivirovanie gene egt rod-shaped viruses ensures the normal course of molting and pupation of the larvae.

The present invention includes a number of agents, control of insects using egt genes and products of the functioning of these genes. These agents control or cause a pest of inappropriate protein synthesis EGT or inhibit the normal functioning of, or manifestation of protein EGT, so that the normal development of the insect is disturbed.

Preferably, the organism containing the gene egt, was a site-specific insect virus and to egt gene of the virus was inactivated that would uvelichenie host is associated with a change in its behavior, in particular with decreasing power, and following this decline in the growth of an insect and a more rapid onset of death. The invention also includes a method for the production of improved viral pesticide. It also includes a method of controlling insect pests, which is that insect pests are exposed to enhanced viral pesticide.

Proposed by the present invention, genetically modified viruses are more effective pesticides compared with till now used by viruses. The rod-shaped viruses such as the nuclear polyhedrosis virus of Autographa californica (AcMNPV) or the nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV), naturally shows egt gene. Expression it increases the period of time which continues infected larva, not suffering from a viral infection. The invention includes inaktivirovanie egt gene in the genome of the virus. Gene egt can inactivate the introduction in its place or in another gene, for example, a gene marker that encodes the synthesis of-galactosidase. It should be clear that violations of the egt gene may be any DNA sequence, because it violates shown the relevant coding segment of DNA or delete or modify the regulatory elements of the genome, controlling gene expression egt. Rod-shaped viruses with deletions that inactivate the gene egt can also be obtained by the sequential transmission of the virus through the culture of insect cells. The viruses with deletions have the following advantage: they do not contain foreign DNA and differ from viruses in wild type only in the absence of a functional egt gene. Such modified baculoviruses more effective as agents of pest control than those used currently. As a simple deletion mutants deletions, and pesticides, genetically engineered, they must be acceptable to regulatory agencies (for example, U. S. EPA), because they do not contain non-homologous DNA. Rod-shaped viruses that do not manifest a functional egt gene, can additionally be modified by introducing different from egt genes that may influence the development of the insect, thereby, increasing the efficiency of such viruses as agents of control insects.

The invention also includes a method of controlling insect pests by infection of insect larvae mutant virus in which the intact gene egt otsustada shedding and pupate, and, therefore, their power is reduced compared with larvae infected with wild type or other currently known viruses. Infected by the mutant virus larvae also die faster than infected wild-type or other currently used by viruses.

The purpose of this invention is to provide a recombinant virus that is more effective compared with wild-type viruses and other forms used at the present time. An example of the present invention is a recombinant AcMNPV virus, referred to as VEGTZ, in which part of the egt gene replaced with the bacterial lacZ gene, encoding education b

galactosidase. As specialists in this field may be replaced by any sequence of DNA, inactivating the gene egt. An example implementation of the present invention is also a recombinant rod-shaped virus, denoted vEGTDEL in which part of the egt gene is deleted. An example implementation of the present invention is a pesticide on the basis of a rod-shaped virus with a remote egt gene. It also includes a rod-shaped viruses that have an inactivation of the gene egt led mutation that occurred in a natural way.

Another object of the invention recombinant viral pesticide, which has no functional egt gene and the second gene is manifested influencing the development of insects, and the natural form of this second gene is not present in the body. The specified second gene encodes the synthesis of the influence of the metamorphoses of the product. This gene product may be the hormone of insects affecting the development of an insect, or an enzyme that inactivates the regulatory metamorphosis hormone. Specific examples include prothoracicotropic hormone, hormone hatching and juvenile hormone esterase. If you want to obtain the agent's control to enter in insect virus encoding these proteins genes, the virus either must not contain egt gene, or egt gene must be inactivated.

Further, the purpose of this invention is to provide insecticidal compositions suitable for use in agriculture. Such compositions comprise, as is known, acceptable in the agricultural sense, the media and insect virus, for example, a rod-shaped virus, pre-genetically modified so that the gene egt specified virus enact heterologous gene, which encodes the hormone insects affecting metamorphosis, or enzyme, inactivating this affect metamorphosis hormone insects. To heterologous genes whose products affect metamorphosis, include, but are not limited to them, prothoracicotropic hormone, hormone hatching and juvenile hormone esterase. In a preferred execution of the insecticidal compositions comprising genetically modified rod-shaped viruses, designed to spray.

Any of the above insecticidal compositions may also include ingredients that stimulate feeding insects. Insecticidal compositions according to this invention the insect pest can swallow when applying it to plants, and insects that are sensitive to the active agent of the composition, will demonstrate reduced power and die.

Also the purpose of this invention is the provision of a modified biological pesticide that inhibits the egt gene expression or function of the product of this gene.

In Fig. 1 schematically shows an AcMNPV genome, indicating the localization of the egt gene. The genome of AcMNPV presented in map units and map-enzyme EcoR1 and Hind III.

In Fig. 3 shows schematically the structure of the gene egt virus AcMNPV (A) and recombinant viruses vEGTZ (B) and vEGTDEL (C). The shaded rectangle represents the lacZ gene.

In Fig. 4 schematically shows an electrophoresis gel of agarose and analysis of spots on Sterno undertaken to identify gene egt rod-shaped virus OpMNPV.

In Fig. 5 schematically represents the restriction map of the viral genome OpMNPV.

In Fig. 6 shows a graph of the profit of weight control uninfected larvae or larvae of the fourth age after virus AcMNPV wild-type or vEGTZ.

In Fig. 7 shows a graph of the mortality of the larvae after infection of larvae of the 4th age AcMNPV viruses wildly the virus AcMNPV wild-type or vEGTZ.

In Fig. 9 shows a graph of the mortality of the larvae after infection with larvae of the 5th age virus wild-type AcMNPV or vEGTZ.

In Fig. 10 shows a graph of the mortality of the larvae after infection with larvae of the first age of viruses of wild-type AcMNPV or vEGTZ concentration of polyhedral inclusion bodies (PTV) 4.8 x 10. PTV/ml.

In Fig. 11 shows a graph of the mortality of the larvae after infection with larvae of the first age of viruses of wild-type AcMNPV or vEGTZ with a concentration of 2.4 x 10 PTV/ml.

Detailed description of embodiments of the invention.

Lepidopteran insects in the process of development from egg to adult undergoing a well-characterized sequence of transformations (for a detailed description see Comprehensive Insect discrimination, Biochemistry and Pharmacology, v. 7, 8, Kerkut and Gilbert (eds), Pergamon Press, Oxford, 1984). After leaving the eggs in the larvae of the insect begins a period of intense power, during which it will be some time shedding that ensures continuous growth. The time intervals between successive linkami called age (larval) stages. Upon completion of the growth period, the larvae pupate and eventually turns into an adult insect. The processes of molting and ocuk is cnyh groups. The initial stimulus is the allocation of certain brain cells prothoracicotropic hormone (PTTH). It stimulates the secretion protogracillin glands of ecdysteroids, which are often referred to as insect molting hormones. The presence of juvenile hormone provides molting larvae, whereas in its absence, the larva will pupate. Hormone hatching, it is also important to carry out some related to the metamorphosis of behavioral changes.

A rod-shaped virus AcMNPV used as a model system for many studies of rod-shaped viruses, unexpectedly intervenes in the above process of development of the insect. Infected with AcMNPV insect larvae are no longer able to fade or to pupate in connection with the fact that AcMNPV directs the synthesis of the enzyme, known as ecdysteroid UDP glucosyltransferase (EGT), which selectively inactivates the ecdysone insect.

The authors of the present invention identified encoding EGT gene, it ranges from a high of 8.4 to 9.6 units maps of the genomes of AcMNPV (Fig. 1 and 2). As shown in Fig. 1C containing the egt gene fragments of viral DNA were cloned in plasmids Fig. 19, Bluescript M13+and Bluescript M13the second area of open reading frames. Only frame 2 contains a relatively long open sequence reads, which was identified as the region encoding the gene egt.

In Fig. 12 and 13 shows the nucleotide sequence of the gene egt and derived from it a sequence of 506 amino acids. The coding sequence of the gene egt extends from about nucleotide 149 to nucleotide 1670.

In the preferred embodiment of the present invention egt gene of the baculovirus AcMNPV inactivated by substitution of part of the bacterial sequence encoding the synthesis of b-galactosidase. Such recombinant baculovirus was marked vEGTZ. In the second variant of the preferred embodiment as shown in Fig. 3, part of the egt gene of AcMNPV virus deleted without replacement. Comparison of the proteins synthesized during infection with AcMNPV wild-type and vEGTZ revealed that EGT protein is a peptide chain with a molecular mass of 60 KDa, secreted by infected cells. An alternative mechanism of gene inactivation egt virus insects is the introduction of a gene that encodes the synthesis affecting metamorphosis hormone or inactivating this hormone, enzyme, and which is infected with the indicated virus nasacort egt homologous UDP glucosyltransferase mammals. It was also revealed homology with UDP-glucosyltransferases plants. Comparison of amino acid sequence egt and some of these enzymes are shown in Fig. 14.

Fig. 14 illustrates a comparison of the amino acid sequence egt and number of UDP glycosyltransferase other species. The predicted amino acid sequence egt compared to human (HUMUDPGAT) (Jackson et al. (1987) Biochem. J. 242:581; mouse (MUSUDPGAT) Kimura and Owens (1987) Eur. J. Biochem. 168:515) and rat (RATUDPGAT) (Mackenzie (1987) J. Bicl Chem. 262:9744) UDP-glucosyltransferases corn (ZMAYUDPGT) (Ralston et al. (1988) Genetics 119:185) using the algorithm FASTER (Zipman and Pearson (1985), proposed International Biotechnologies Inc. Upper case letters indicate an exact match: lowercase letters define substitution, common among related proteins (Dayhoff (1978) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, v. 5, Supplement 3, Silver Spring, MD); dots represent rare substitution, the hyphen indicates the gaps in the sequence, the caret is in places deletions of amino acids from the sequence. In vEGTZ and vEGTDEL amino acids egt gene in the interval between the arrows removed. Gomologichnosti AcMNPV gene known transferase UDP-glucose and UDP glucuronosyltransferase confirms surrounding UDP glucuronosyltransferase catalyze the transfer of glucuronic acid to a range of both exogenous and endogenous lipophilic substrates (see review in Glucuronidation of Drugs and Other Compounds, Dutton (ed. ) CRC Press, Boca Raton, Florida, 1986). This reaction accession plays a significant role in detoxification and safe excretion of certain drugs and carcinogens. In addition, through connection with glucuronic acid processes are normal metabolism and placement of various endogenous compounds such as bilirubin and steroid hormones. Existing systems insect data indicate that such reactions proceed sugars include the transfer of glucose and not glucuronic acid (for an overview see Smith (1977) in Drug Metabolism From Microbe to Man, Parke and Smith (eds), Taylor and Francis Ltd, London, p. 219-232). As in mammals, insects such reactions proceed take a variety of exogenous and endogenous compounds.

The authors of this invention have shown that EGT protein of AcMNPV virus is a UDP-glucuronosyltransferase, which selectively connects the glucose ecdysteroids such as Edison, 20-hydroxyecdysone and magistero A (PL.1). No lysates or extracellular environment uninfected cells or infected vEGTZ cells do not modify Edison. A large part of the active ecdysteroid-glucosyltransferase produced AcMNPV infected cells, is secreted in mickleton CLASS="ptx2">

Using the egt gene of AcMNPV virus as a label egt gene has been identified, as shown in Fig. 4, and the other rod-shaped virus, namely, the nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV). For professionals should be clear advantages provided by this discovery that the egt gene of any rod-shaped virus, another virus insects or insect can be identified, characterized and selected in the same way. Genes egt, nucleotide sequence which is at least 70% homologous to the sequence shown in Fig.13, are considered to be equivalent to this sequence, provided that the homologous genes encode the enzyme, representing ecdysteroid-UDP-glucoronosyltransferase.

Functional equivalents of the egt gene are equivalents, which also catalyze the inactivation of ecdysteroids such as Edison, by transferring half of glucose from UDP-glucose to ecdysteroid (ecdysteroids). Such functional egt-equivalents can be identified by methods described herein.

By localization, identification and isolation of gene egt specialist may use the provisions of this opening and known methods, in komago.

Comparing properties vEGTZ properties AcMNPV wild-type (wt), the authors of this invention have shown that the egt gene expression prevents molting or insect pupation. Infected with AcMNPV egt insects do not shed and do not pupate, while insects infected vEGTZ, fade and tend to pupate (PL.2).

Due to the suppression of molting and pupation infection with virus AcMNPV wild-type can really extend the feeding period of larvae. Larvae infected with wild-type virus in the beginning of the fifth age stage (the last age stage), continue to eat until the moment of death, which occurs on the 5th or 6th day after infection. However, uninfected larvae stop feeding, preparing for pupation, 2-3 days after entry into the fifth age stage (Fig. 8). A similar effect is observed when examining the larvae earlier developmental stages. During molting uninfected larvae stop feeding for a period of about 24 h, whereas infected with wt AcMNPV larvae do not shed and, accordingly, continue feeding (Fig. 6).

Recombinant rod-shaped viruses with missing functional egt gene does not increase the duration of power lichen two days after infection and are preparing to pupation (Fig. 8). However, they do not pupate and instead die from viral infection even faster, as shown in Fig. 9 than larvae infected with the wild-type virus. Similarly, larvae infected with the virus in the early fourth age, cease to eat for two days after infection and die then faster than infected wild-type virus (Fig. 6 and 7). What a rod-shaped virus missing functional egt gene kills insects quickly, most clearly seen when the virus vEGTZ infected newly hatched larvae first stages (Fig. 10 and 11). Infected vEGTZ larvae die from viral infection for 3-4 days earlier than those who were infected with wt AcMNPV. Therefore, recombinant rod-shaped viruses with missing functional egt gene is much more effective as control agents of insect development than rod-shaped viruses of wild type. For professionals in this field should be obvious advantages of this discovery lies in the fact that the gene egt can be made non-functional in any rod-shaped virus or virus insects by any known method.

The effect described above and in the following note will still occur only in carefully controlled laboratory conditions. In the absence of strict control of temperature and lighting, and many larvae cannot complete their moult. These insects do not begin again to eat and soon after die.

Despite the fact that shortens the length of time during which the progeny virus can accumulate in the larva infected rod-shaped virus that does not have a functional egt gene, and the power of an infected insect decreases, there is a significant production subsidiary of the virus. The amount of virus per cylinder, obtained after infection vEGTZ larvae late developmental stages is approximately half the received upon infection with wild-type virus. This is sufficient to ensure transfer of the virus in the field and effective in terms of cost of producing large amounts of viral particles.

In another variant embodiment of the present invention insect virus with no functional egt gene altered through genetic engineering in such a way that its effectiveness as a biological control agent is increased due to the introduction of the second gene product which affects insect development.

which in this case is quite high, to influence the processes of molting. Infected this virus larvae in highly disturbed hormonal control of development. These insects quickly get sick, which leads to defective development and growth, reducing power and hasten death.

Hormone hatching is also a small peptide hormone, a gene which may be introduced by known methods in the viral genome with non-functional egt gene. Due to the fact that hormone hatching defines many associated with metamorphosis behavioral changes, the insect is infected with a virus Egt-producing this hormone in large enough quantities, will exhibit abnormal behavior and/or development, for example, reduced power.

The primary regulator of the activity of juvenile hormone in insect enzyme is an esterase, inactivating juvenile hormone. Recombinant virus lacking a functional egt and producing high levels of juvenile hormone esterase, can have adverse effects on behavior and/or insect development.

It is important to note that while all of the aforementioned genes can be introduced into the genome of the viruses dicosolo the egt gene expression by wild-type viruses inactivate ecdysteroid molting hormones and this prevents, despite the production of other hormones, the normal course of metamorphosis. Thus, a successful strategy must include, as described in this invention, the generation of viruses that are designed to change the course of metamorphosis, depending on the previous inaktivirovanie egt gene.

In this invention, the agent control over the growth and development of insects is a composition or an active ingredient of the composition, which has an adverse effect on insect pests. Reaction to the agent pest control is the reduction of power is disrupted normal metamorphosis, resulting in death of the insect becomes inevitable. The active ingredient of the composition according to this invention is an insect virus, in particular baculovirus, genetically modified to inactivate the gene encoding the synthesis, causing modification of ecdysteroid enzyme or insect virus, subjected to further genetic changes that result in expression of the heterologous gene affecting metamorphosis.

Insecticidal compositions suitable for application to plants to control insect damage insecticidal compositions of this invention can protect plants from insect pests by reducing power and death of susceptible insects.

Experts know how to choose the active principle of the composition, namely the virus, suitable for a particular insect pest.

For professionals it is clear that insect pests can be subjected to a controlling agent of the present invention by any conventional method, including swallowing, inhalation or direct contact.

Described in this invention are subjected to genetic changes in rod-shaped viruses will find primary use as components of agricultural compositions intended for application on plants with biological control of insect pests of plants. Known various methods of cooking such acceptable for agriculture compositions.

The concentration of the main ingredient, which is required to obtain effective insecticidal compositions used in agriculture to protect plants depends on the type of organism used mutation and composition formulas. As it is clear to the specialist, the effective value of the concentration controlling agent in the insecticidal composition can be easily determined experimentally. For example, the concentration of the virus, doskonalenia composition should be suitable for use in agriculture and good dispergirujutsja in the field. In General, the components of the composition should not be phyto-toxic and harm the whole community of the occluding viruses. Drawing on the leaves should not bring harm to leaves, or damage them. In addition to the relevant firm or that is preferred liquid carriers, agricultural compositions may include components that enhance lipocell or adhesion, emulsifiers or moisturizers, but not components, reducing food insects or the functioning of the virus. It may be also desirable introduction components, protecting controlling agent from inactivation by ultraviolet radiation. Agricultural composition for controlling insect pests may also include agents that stimulate feeding insects.

There are reports describing the methods of application of biological control agents of insects and their application in agriculture. See, e.g., Couch and Ignoffo (1981) Microbial Control of Pests and Plant Disease 1970-1980, Burges (ed) chapter 34, p. 621-634; Corke and Rishbeth, ibid, chapter 39, p. 717-732; Brockwell (1980) in Methods for Evaluating Nitrogen Fixation. Bergersen (ed) p. 417-488; Burton (1982) in Biological Nitrogen Fixation Technology for Tropical Agriculture, Graham and Harris (eds) p. century. pp. 105 -- 114; and Roughley (1982) ibid., p. 115-127; The Biology of Baculoviruses, v. II Supra.

Dunn is limiting the scope of this invention. It is clear that professionals can be represented in different ways to specific embodiments, modifications and equivalents of the invention, not beyond the ideas of the present invention and/or the accompanying claims.

Example 1.

The position of the egt gene in the genome of AcMNPV shown in Fig. 1, above the map of the genome of AcMNPV shows the scale in map units. To determine the nucleotide sequence of this type and of the adjacent areas, which would make subsequent genetic manipulation, it is necessary first to clone some of the DNA fragments flanking this region, plasmid vectors (standard methods cloning, see: T. Maniatis et al. (1982) in Molecular Cloning: A Laboratory Manual, Cold Sprihg Harbor Laboratory, Cold Spring Harbor, N. Y.). Panel A of Fig. 1 shows a linear map of the genome of AcMNPV after splitting his restrictase Eco RI and Hind III. Panel B presents in an enlarged scale the region of the genome from a 7.6 to 11.1 map units showing the position of the egt gene. As the source AcMNPV was used strain L1 (Lee and Miller (1978) J. Virol. 27:754). Exposed cloning of DNA fragments and names the resulting plasmid is shown in Fig. 1. Panel C. Fragment 1, which occupies the area from site > PST, located at point 7, 6,.E. up to 8,at 65. E.) to SalI (10,5 have.E.), respectively] are cloned into the vector Bluescript M13+and Bluescript M13-(Stratagene, San Diego, California). Fragment 5 [is from Bst EII site (8,35.E.) to Bst EI website (8,7 have.e.)] cloned in Bluescript M13+< / BR>
It then generates a large number of plasmid subclones BCPSE and BCES (Henikoff (1984) Gene 28:351). These podklady differ consistently increasing deletions of viral inserts, so they contain different amounts of viral DNA from less than 50 base pairs to complete viral fragment. Many of these subclones and plasmids BCB then increasing (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463), so in both directions recovers the sequence of the whole gene egt. Then the obtained nucleotide sequence is analyzed for the presence of open reading frames that could encode a protein with the help of computer programs Pustell and Kefatas (1984), Nucl. Acids Res 12:643-655 and Devereaux et al. (1984) Nucl. Acids Res 12:387-396. This analysis shows that the egt gene encodes a protein consisting of 506 amino acids. The nucleotide sequence egt gene and predicted amino acid sequence of the product of this gene is shown in Fig.13.

Example 2

In order to construct recombinant viruses, which in example 1 clones plasmids. Plasmid pUCBCPsB digested with restriction endonucleases Eco RI and Xba I restriction sites within a gene (egt, see Fig. 3) and discard the small piece. The gene of Escherichia coli lacZ, derived from pSKS 104 (Casadaban et al. (1983) Methods Enzymbol. 100:293-303) using restricted Eco RI and Aha III, is introduced between sites Eco RI and Xba I, after the free ends of Xba I filled in using DNA polymerase of phage T4. The obtained plasmid denote pEGTZ. In this plasmid is introduced gene lac Z is within the scope together with the previous coding sequence egt. Plasmid pEGTDEL, opposite, simple design binding sites Eco RI and Xba I (after all both sites are filled) without inserting between them any nucleotide sequence. All viruses are derived from the original strain L-I AcMNPV (Lee and Miller (1978) Supra), are cleared through plaques and multiply in the culture cell line Spodoptera frugiperda IPLBST 21 (SF cells) Vaughn et al. (1977) in vitro 13:213-217) using previously described methods (Lee and Miller (1978); Miller et al. (1986) Genetic Engineering, Principles and Methods, v. 8 (eds. J. Sitlow and A. Hollaender), Plenum Press, N. Y. 277-298, 1986).

Then plasmid pEGTZ transferred together with DNA AcMNPV wild-type SF-cells, as described in Miller et al. (1986) Supra. This procedure ensures that homologous recombination between the IDA egt - lac z As the remaining coding sequence egt is reading frame together with the sequence of the lac Z such recombinant virus will produce a protein that represents the first 84 amino acids egt, United with b-galactosidase. The recombinant virus, designated vEGTZ, difficult of identification, as the expression of b-galactosidase leads to the formation of blue viral plaques in the presence of a chromogenic indicator, such as 5-bromo-4-chloro-3-indolyl - b-D-galactopyranoside (X-gal). Chart egt gene of the virus vEGTZ shown in panel B of Fig. 2.

Recombinant virus vEGTDEL is produced by transfer of plasmids pEGTDEL and DNA from the virus vEGTZ in SF cells. Homologous recombination leads to the substitution in vEGTZ gene ligament egt-lac Z gene with a deletion of the egt. Recombinant virus vEGTDEL identify by its inability to form blue plaques in the presence of X-gal. The structure of the egt gene of the virus vEGTDEL shown in Fig. 3, C.

Example 3.

The egt gene product identified by comparing the proteins synthesized virus AcMNPV wild-type (which produces EGT) and viruses vEGTZ or vEGTDEL (unable to produce EGT). SF-cells infect or AcMNPV wild-type or vEGTZ with the plurality of dice is infected cells incubated for 1 h in the presence of [35 S]-methionine for the purpose of making radioactive labels in all proteins. The cells are then subjected to lysis and separate proteins by electrophoresis in a gel SDS-polyacrylamide (SDS-PAGE)(Laemmli et al. (1970) Nature 227:680-685). All secreted by cells proteins are also collected and analyzed. After carrying out SDS-PAGE radiolabelled proteins are determined using autoradiography. Infected with wt AcMNPV cells secretively protein with a molecular mass of 60 KDA, which did not happen in the case of infected vEGTZ or uninfected cells. This protein, however, not detected in lysates of cells infected with wild type AcMNPV, which proves its secretion by the cells. These data show that the egt gene product is synthesized 60 KDA protein, which is in good agreement with the data in the sequence of nucleotides and amino acids.

Example 4

Fermenting activity EGT protein was determined by comparison of SF cells infected with wt AcMNPV or vEGTZ.

SF cells infected with wt AcMNPV or vEGTZ described in example 3. After 12 h pi cells and the extracellular medium was collected and processed separately. Concurrently treated uninfected cells. Cell lysate or intercellular environment incubated in the presence of 1 mm UDP-glucose and 0.25 m Ci [-3H] ecdysone, as described by O'reilly and Miller (1989) Science 245:1110-1112.

The substrates are incubated in the presence of the environment obtained from an appropriately infected cells and 0.05 m CiUDP-[U-14C]-glucose. The number of migrated glucose is calculated based on the number of pulses from the respective areas of the chromatographic plates.

To further control was introduced UDP-[U-14C]-glucuronic acid in separate reactions with environments derived from virus infected wild-type cells, in order to demonstrate that glucuronic acid in these reactions is not per hydroxyecdysone and magistero A (all ecdysteroids). In environments extracted from false-positive or infected vEGTZ cells, the conjugates were not detected, which confirms the dependence of the observed activity by expression of egt. When using UDP-[U-14C]-glucuronic acid conjugates are not observed, which is a demonstration that glucuronic acid is not migrated by EGT.

Example 5.

In order to obtain evidence and other baculovirus genes, homologous substantially gene AcMNPV egt, separated DNA virus OpMNPV and subjected individually to the impact of the restriction endonucleases Eco RI, Bam HI and Hind III. These enzymes break down the viral DNA into several segments of different size, the position of which in the OpMNPV genome is already known (Leisy et al. (1984) J. Virol. 52:699). Then spent hybridization on Southern described in T. Maniatis et al. (1982) Supra way. Cut out the inner segment of the egt gene of AcMNPV plasmids BCES using enzymes Eco RI and Xba I (Fig. 1). This fragment was labeled with radioactive isotope32P and used as a means of identifying any related sequences in the genome OpMNPV. It is clear that in appropriate circumstances, the DNA fragment will attach another fragment, which contains analogichnymi AcMNPV egt to any DNA fragment OpMNPV, contains a cognate DNA sequence. The place of attachment of the label can be visualized using the exposure of the membrane to x-rays. First hybridization tag egt nylon membrane in soft conditions (1 M sodium chloride, 0.3 M sodium citrate, 5% dextran sulfate, 5 x solution Denhardt'a, and 0.25% SDS at 37oC). This allows the hybridization of the tag with a relatively distant related sequences. Then gradually increase the rigidity of hybridization, raising the temperature of the hybridization or entering into a solution hybridization formamid, until and unless there is specific hybridization of communication. Hybridization shown in Fig. 4, occurs in the following conditions: 1 M sodium chloride, 0.3 M sodium citrate, 5% dextran sulfate, 5 x solution Denhardt'a, and 0.25% SDS; temperature 68oC, duration 15 hours and Then the membrane is washed twice, each time for 15 min, a solution of 0.3 M sodium chloride, 0.1 M sodium citrate and 0.1% SDS at 68oC. In Fig. 4 shows that the label AcMNPV egt joins certain OpMNPV DNA fragments, namely, to fragment B Eco RI fragment of A Bam HI and faces N and S Hind III. Note that the egt gene is in the OpMNPV genome the same relative position as the genes. It is clear that to confirm EGT activity in the case of highly divergent sequences will need a method restriction described in example 4. Then to highlight the gene encoding the enzyme EGT will require molecular genetic analysis, the methodology of which is known.

For detection of the gene encoding EGT in other organisms is used, in contrast to the above, the specific method of testing the activity of UDP-glucosyltransferase described in example 4. This test is used to determine the enzyme during the purification process, the standard biological methods. After cleaning is determined partial amino acid sequence of the protein EGT. The obtained information is used to generate oligonucleotide tags, which determine the position of the EGT gene in the genome.

Example 6.

The titer ecdysteroid in hemolymph is changed cyclically by adjusting the transformation of the larva-the larva, and the larva-pupa, and, since it is assumed that the accession of glucose inactivates ecdysteroids (Warren et al. (1986) J. Liq. Chromatogr. 9: 1759; Thompson et al. (1987) Arch. Insect. Biochem. Physiol. 4:1; Thompson et al. (1988) Arch. Insect. Biochem. Physiol. 7:157), it is possible the relationship disruption of normal on edusim way: just undergoing molting larvae of the fourth age S. frugiperda infected by viruses wt AcMNPV or vEGTZ by injection and daily observed for any changes in their development. For negative control, one group of larvae of the introduced liquid tissue culture. The results of the experiment are given in table. 2. All larvae, which introduced tissue culture (false infection), as expected, has fledged into larvae of the fifth age. Of the sixteen larvae infected with wild-type virus, such transformation was observed only in one. On the contrary, all larvae infected with the mutant virus vEGTZ, fledged from the fourth to the fifth age stage. Thus, the egt gene expression of wt virus AcMNPV clearly and specific inhibits shedding of the insect host. Subsequently, both infected groups of larvae died from a viral infection, which proves that the dysfunction of the egt virus vEGTZ does not prevent him to kill the master.

The larvae of S. frugiperda fourth age introduced 1105pfu wt AcMNPV or vEGTZ in 5 ml of False-positive larvae were injected with 5 l of liquid tissue culture. Each group consisted of 16 larvae that were contained in the following conditions: artificial diet (R. L. Burton (1969) ARS publication, p. 33-134), the temperature of the 28oC cycle "light: dark 14:10 h the ri injection of the virus larvae early fifth stages obtained similar results. None of the newly molted larvae of the fifth age, infected with wild-type virus did not find any signs of pupation, whereas the majority of infected vEGTZ larvae showed some behavioural abnormalities (termination power, anxiety and weaving), which is an indicator of the proximity of the transformation from larva into pupa. However, all larvae, which was introduced viral infection, died before pupation. These data indicate that infection with AcMNPV prevents shedding or pupation of the larvae. They also show that such violation of insect development due to the egt gene expression.

Example 7.

The in vivo bioanalysis AcMNPV wild-type (wt) and vEGTZ revealed that the expression of the gene egt increases the time during which the larva feeds, and that the dysfunction of the egt improves the characteristics of the virus as a pesticide. When conducting these studies, the larvae of S. frugiperda were injected with either wt AcMNPV or vEGTZ at an early stage 4. For comparison, the control larvae were injected Wednesday tissue culture, which did not contain the virus. The larvae every day to check profit weight, signs of molting or pupation and mortality. In Fig. 6 and 7, respectively, given what erwich two days showed moderate growth. During the second day, all they fledged in the fifth stage. Next, the larvae have risen sharply over the two days, and then stopped to eat and got ready for pupation. Only one of the 16 larvae infected with wt AcMNPV, fledged, instead of what the rest within three days after infection, larvae were growing. At this stage they were sick, but up to 5 days none of the larvae did not die. By the 6th day killed all infected with wt AcMNPV larvae. On the contrary, all larvae infected vEGTZ, moved from the fourth moult in the fifth age stage and during this period of time they would not eat. This explains the lack of gain from the 1st to the 2nd day. After molting they resume eating, but by the 3rd day begin to show signs of the disease. Four days after infection they begin to die and by the 5th day all die. Larvae infected derived from AcMNPV virus that does not have a functional egt gene, eat less and die after infection faster than larvae infected with wild type AcMNPV.

During infection with larvae of the 5th age, these phenomena are observed even more clearly (Fig. 8 and 9). As expected, the control larvae for two days increased significantly, then PI, infected with wt AcMNPV, showed no signs of stopping power, they continued to eat and to gain weight for two more days, and then began to show signs of the disease. Mortality until the seventh day after infection was not complete.

You can make the following conclusion. Larvae infected vEGTZ as control, eat only during the first two days after infection. Then observed as they prepare for pupation, sudden weight loss. However, none of these larvae do not pupate; the third day is observed symptoms of the disease, and six days after infection, all of them perish.

Example 8.

Compares the effects of infection vEGTZ and wt AcMNPV on newly hatched larvae of S. frugiperda first age. Newborn larvae of S. frugiperda give food that contains various quantities pole - shape body enable vEGTZ or wt AcMNPV, and see daily mortality. In Fig. 10 and 11 shows the results obtained for the two different doses. You can see that in both cases the infected vEGTZ larvae considerable degree of mortality occurs much earlier than larvae infected with wild-type virus. In General, larvae, Sara aetsa additional evidence, that baculoviruses containing inactivated genes egt, better act as biological pesticides than wild-type baculoviruses with intact genes egt.

Example 9.

To construct a recombinant virus, expression of prothoracicotropic hormone (PTTH), PTTH gene clone in plasmid vector transfer pEV mod XIV, which is described in U. S. Patent Application Serial N 07/353,847 dated may 17, 1989, the application is incorporated herein by reference. This plasmid contains the ascending and descending sequences of AcMNPV polyhedrin gene, which is mediated homologous recombination expressed PTTH gene from the genome of the virus EGT-. The multiple cloning site is located at the usual site of initiation of translation polyhedrin, down from LS XIV modified polyhedrin promoter (Rankin et al. Gene 70:39 (1988). PTTH gene Bombix mori stands out from the plasmid pBc22k-C19 (Kawakami et al. Science 247:1333 (1990) using the restriction enzyme Hind III. Connecting the ends of the Hind III filled in using DNA polymerase of phage T4, and then the plasmids were cleaved with Eco RI. Plasmid pEV mod XIV digested with kpni restriction sites. "Sticky" end Kpn I remove the DNA polymerase of phage T4 and digest the plasmid with Eco RI. The fragment, which contains the PTTH gene, clone through the memory after LS XIV modified polyhedrin promoter.

Recombinant viruses with the expression of PTTH produced by contrastable pEVPTTH in SF cells with DNA viruses wt AcMNPV or vEGTDEL. Recombination between sequences flanking poliakin into viral DNA, and sequences flanking the PTTH gene in the plasmid pEVPTTH, leads to the replacement of the polyhedrin gene-for-gene PTTH. Viruses, the genome of which took place this recombination, identify negative for occlusion phenotype (Miller et al. in Genetic Engineering, Principles and Methods. v. 8, J. Setlow and A. Hollaender, eds. Plenum Press, N. Y. 1986, p. 277-298). The corresponding recombinant viruses are indicated vEGTPTTH and vWTPTTH.

Similarly receive recombinant viruses with an expression of juvenile hormone esterase. First cut out the gene of juvenile hormone esterase Heliothis virescens Hanzlik et al. (1989) J. Biol Chem. 264:12419) from plasmids pCHE16B (Hammock et al. (1990) Nature 344:458) by cleavage with Eco RI and Kpn I. the Plasmid transfer pEV mod XIV also otscheplaut by Eco RI and Kpn I and the fragment containing the gene of juvenile hormone esterase introduce between these sites. Thus, pEVJHE includes the polyhedrin gene sequence flanking the gene of juvenile hormone esterase. Recombinant viruses EGT-JHE and vWTJHE get contrastedly pEVJHE with DNA vEGTZ or wt AcMNPV, respectively, and it hatch cut out the gene hormone hatching Manduca sexta from the plasmid pF 5-3 (Horodyski et al. (1989) Proc. Natl. Acad. Sci. USA 86:8123) by cleavage with Eco RI and Hpal.pEV mod XIV digested with kpni restriction sites, the free end of the kpni restriction sites are removed by DNA polymerase of phage T4, and then digested with Eco RI. The introduction of the fragment containing the gene for the hormone hatching in plasmid move gives recombinant pEVEH, in which the gene hormone hatching is located down from the promoter LS XIV modification polyhedrin. Cotransfected this plasmid with DNA vEGTZ or vDA26Z allows you to select respectively recombinant viruses vEGT-EH and vDA26Z EH. vDA26Z is a recombinant virus to insert a gene lac Z E. coli within a gene DA26 (O'reilly et al. J. Gene Virol. in Press). The function of the gene DA26 unknown, and the phenotype, as for metamorphosis, vDA26Z represents the wild type. Because of the presence of the gene lac Z derivatives vDA26Z give blue plaques in the presence of X-gal.

Example 10.

To evaluate in vivo the effect of such recombinant viruses, the larvae of S. frugiperda introduced 2105pfu virus vEGTDEL, vEGT-PTTH, vwTPTTH or simultaneously vEGT-PTTH and vEGT-JHE (1105pfu each); the larvae were in the late 3rd age stage or in the early 4th. With the introduction of all EGT--virus infected insects moved moult and survived the 4th stage, and by the 3rd day found the T-PTTH or jointly vEGT-PTTH and vEGT-JHE expression of PTTH leads to rapid disease insects, and by the 4th day, there was a significant mortality. In contrast, insects, infected vEGTDEL, until the 5th day did not find a significant mortality. As expected, insects infected with the virus vWPTTH, never show signs of the transition to the 5th stage. The head capsule slippage does not occur until the 6th day is not observed significant mortality. Thus, expression of PTTH in the absence of EGT brings on day 1 compared with infection vEGTDEL dates of the deaths of insects as a result of infection. These data demonstrate improvement pesticide properties of baculoviruses due to the expression of genes that affect the metamorphoses of insects, and show that the effectiveness of this strategy is only possible when inaktivirovanie egt gene (table. 3).

Example 11.

Generating positive on the basis of the formation of inclusion bodies viruses expressing heterologous proteins influencing the development of insects, can be carried out according to the following scheme. First, we construct a negative sign occlusion of the virus with the expression of-galactosidase. Gene lac Z E. coli that encodes the enzyme b-galactoside
includes sequences that are located in the genome of AcMNPV in both the ascending and descending from the polyhedrin gene and a multiple cloning site is located directly downstream from synthetic modified polyhedrin promoter. Gene lac Z cut out from the plasmid PSKS 105 (Casadaban et al. 1983) supra by splitting Pst I. Protruding end is removed using nucleases mung bean, and the plasmids were cleaved by Sst II. pSyn VI-split across sites Eco RV and Sst II and clone the gene lac Z in these sites. The resulting plasmid is designated pSyn VI-a gal in her gene lac Z is located directly downstream from synthetic modified polyhedrin promoter. pSyn VI-gal is transferred to the SF-cells with virus DNA vEGTDEL, then separated negative sign of occlusion plaque with the expression of b-galactosidase, which is a virus vEGT-Syn VI-gal.

To build positive on the basis of the formation of inclusion bodies of recombinant virus, which is the expression of PTTH, PTTH gene clone in the vector transfer pSp XIVVI+X3.

When designing pSp XIVVI+X3 first built intermediate plasmid pSp XIVVI+. Plasmid pSp LSXIVVI+CAT (described in application for U.S. patent is automatically written in the patent application U.S. serial number 07/353,847 dated may 17, 1989) is cut by EcoRV and SacI and small EcoRV fragment SacI clean. Fragments of two plasmids link and select pSp XIVVI+CAT. Plasmid pSp XIVVI + identical plasmid pSpLSVIVVI+CAT except that the multiple cloning site between the BgLII site to site SacI coincides with pSynVI+wtp. To construct the multiple cloning site V3 (described in patent application U.S. serial number 07/353,847 dated may 17, 1989) between sites EcoRV and Sacl plasmids pSp XIVVI+ entered polylinker. The sequence of the multiple cloning site in pSp XIVVI+X3 from the BgLII site to the SacI site is AGATCATC GAATTCTCGAG CTGCAGATCT GTCGACCCGGG AATAAA GAGCTC.

EcoRV/Bgl EcoRI-XhoI > PST -BglI SalI-SmaI poly A SacI.

This plasmid contains an intact polyhedrin gene under control of the polyhedrin promoter wild type. Synthetic modified polyhedrin promoter is located upward from the polyhedrin gene and with the opposite orientation. The position of the multiple cloning site provides the possibility of introducing genes, the expression of which should be under the control of the synthetic modified polyhedrin promoter. PTTH gene cut out from the plasmid pBc22k (Kawasaki et al. (1990) supra) by splitting by Hind III. The free end of the Hind III fill in the DNA polymerase of phage T4 and digest the plasmid by EcoRI. pSp XIVVI+X3 cleaved by EcoRI and SmaI sinteticheskogo promoter modified polyhedrin, but in the neighborhood and with the opposite orientation relative to the polyhedrin promoter wild-type and coding sequences. Both genes, gene PTTH and polyhedrin, and paired with ascending and descending sequences of the polyhedrin gene of AcMNPV genome.

pSp PTTH is transferred to the SF-cells together with DNA vEGT-SynVI-gal. Recombination between ascending and descending sequences in the viral polyhedrin DNA sequences flanking genes PTTH and polyhedrin in pSp PTTH, leads to the replacement of the gene lac Z EGT-SynVI-gal genes PTTH and polyhedrin from pSp PTTH. Recombinant virus EGT-SpPTTH identify the phenotype, a negative sign of b-galactosidase and positive on the basis of occlusion.

To construct positive on the basis of occlusion virus expression system exterity juvenile hormone carved gene of juvenile hormone esterase from the plasmid pJHE16B (Hammock et al. (1990) supra) by splitting along the kpni restriction sites, removal of the free end by DNA polymerase of phage T4 and rassekaya by EcoRI. Then cloned gene exterity juvenile hormone in plasmid pSp XIVVI+X3, which was digested by EcoRI and SmaI. The resulting plasmid, pSp JHE expose cotransfected the AI virus expression of juvenile hormone esterase is under the control of a synthetic promoter modified polyhedrin.

Similarly, cut out the gene hormone hatching from the plasmid pF 5-3 (Horodyski et al. (1990) supra) by rassekaya by EcoRI and Hpal and cloning in pSp XIVVI+X3, which is then cleaved by EcoRI and SmaI. As a result of cotransfection obtained plasmids pSpEH with DNA vEGT-SynVI-gal get recombinant virus vEGT-pEH.

Positive on the basis of the formation of inclusion bodies EGT-viruses are subjected to further genetic changes that cause the expression of a protein that affects metamorphosis insects (peptide hormone insects or enzyme, inactivating the hormone insects), as a result they reduce the power and hasten the death of the infected insect larvae more effectively compared with wild-type baculoviruses or baculoviruses, genetic changes which caused only inaktivirovanie egt gene.

As regulators of growth and development of insects of example 11 to form a body of incorporation, these viruses can be introduced in an effective insecticidal acceptable to agricultural compositions which can be applied to infected crops. Swallowing such occluded viral particles insects will lead to the spread of viruses through the fields and samyeli from insect pests.

It should be understood that the foregoing relates only to a particular, preferred examples of embodiment of the present invention, and that numerous modifications or alterations may correspond to the idea and scope of the invention, as postulated in the attached claims.

1. A method of obtaining a genetically modified baculovirus, wherein spend inactivation of virus gene encoding the enzyme ecdysteroid UD-glucosyltransferase.

2. The method according to p. 1, wherein said baculovirus is a nuclear polyhedrosis virus.

3. The method according to p. 2, characterized in that the nuclear polyhedrosis virus is a nuclear polyhedrosis virus of Autographa californica (AcMNPV).

4. The method according to p. 1, characterized in that the genome of the virus additionally embed heterologous gene that determines protein synthesis, affecting the metamorphoses of insects.

5. The strain of the nuclear polyhedrosis virus of Autographa californica was ATSS N VR 2244 possessing insecticidal activity.

6. A method of combating insects, providing for the application of the virus to the plant, characterized in that the use of genetically modified baculovirus, a gene which encodes EN zymes is TBE use of baculovirus nuclear polyhedrosis virus.

8. The method according to p. 7, characterized in that as nuclear polyhedrosis virus using a strain of the nuclear polyhedrosis virus of Autographa californica was ATSS N VR 2244.

9. Insecticidal composition containing the active beginning of the virus and agronomically acceptable carrier, characterized in that the active agent it contains genetically modified baculovirus with inaktivirovannye gene coding for the enzyme ecdysteroid UD-glucosyltransferase, in an effective amount.

10. The composition according to p. 9, characterized in that as baculovirus it contains a nuclear polyhedrosis virus.

11. The composition according to p. 10, characterized in that as nuclear polyhedrosis virus it contains a strain of the nuclear polyhedrosis virus of Autographa californica ATS N VR 2244.


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