MXPA98004689A - Production of baculovirus recombinan - Google Patents

Abstract

The present invention relates to methods that facilitate the production of recombinant baculoviruses that have been engineered by use as biological control agents. More specifically, this invention relates to the regulation of the expression of genes encoded by recombinant baculoviruses in an insect cell or in an insect .

Description

PRODUCTION OF RECOMBINANT BACULOVIRUS BACKGROUND OF THE INVENTION Chemical insecticides are an integral component of modern agriculture, and are an effective means of reducing crop damage by controlling insect pests. However, chemical agents are under constant scrutiny due to potential environmental contamination, selection of resistant populations of agronomic pests, and toxicity to non-target organisms, beneficial insects, aquatic organisms, animals and man. As a result, alternative strategies for insect control are being sought, which are effective and even benign for populations that are not targeted, and the environment. One of these strategies is to use. microorganisms that are pathogens of target insect populations, which occur naturally. There are many endopathogenic candidates that would be control agents -ie promising insects-: - they derive from the properties of classical qui-n-.ccs insecticides, such special host specificity and rapid action. of what the REP: 27392 farmers and others in agribusiness have been accustomed. Viruses from the Baculoviridae family are host-specific and have inert environmental properties, but lack the ability to rapidly neutralize a target population before significant damage to the culture takes a place. Fortunately, modern molecular biology provides the tools to produce recombinant baculoviruses, engineered for use as biological control agents. An attractive attribute of baculoviruses is their close specificity to the host. These viruses infect only arthropods and have relatively narrow host intervals, even within the range. particular insect order. The specificity of the host has been examined by electron microscopy, DNA hybridization and recombinant DNA technology.

(References 1-3). Est: s studies indicate that e. narrow interval to the guest is due, at least er. part, to the inability of the baculoviruses to transfer the viral DNA r.as to the nucleus of cells: e mammal. Baculoviruses are divided into three families, including crosier '/: rs not occluded (NOV), i -f granulosis virus (GV) and nuclear polyhedrosis viruses (NPV) - although certain GV and NOV are have studied carefully, NPVs are the most fully characterized of the baculovirus subfamilies. Examples of NPV include Autographa californica NPV, Spodoptera exigua NPV, Heliothis armigera NPV, Helicoverpa zea NPV, Spodoptera frugiperda NPV, Trichoplusia ni NPV, Mamestra brassicae NPV, Lymantria dispar NPV, Spodoptera litturalis NPV, Syngrapha facifera NPV, Choristoneura fumiferana NPV, Anticarsia gerr-matalis NPV, and Heliothis virescens NPV. Due in part to the availability of efficient cell culture systems or easy cloning vectors, NPVs are used as eukaryotic expression vectors to synthesize desirable heterologous proteins (4,5) - One virus in particular, Autographa caiifórnica NPV (AcNPV) ), is the accepted model virus for the introduction and expression of heterologous genes in baculovirus expression systems, although this virus routinely serves as an important in vitro means to provide high yields of recombinant proteins in a eukaryotic expression system. thus giving appropriate post-transduction modification. -of the expressed proteins, AcNPV is capable of infecting many families of Lepidopteran insects that are important economic pests. Despite the potential practical advantages of baculovirus-based pest control agents, a variety of disadvantages have reduced their use in modern agriculture. The most significant barrier for the most widespread use of these viruses in row crop agriculture is the delay in time between their application and effective control of damage to the crop caused by the host's insects. Different from the rapid effects observed in the application of classical insecticides, baculovirus-mediated, wild-type, significant insect control occurs only after in vivo populations of viruses can reach levels high enough to compromise host activity. This can occur so prolonged c rn several weeks after infection in a cid: -re comprises two types of virions. After 1 -. .reement of insect cells, v ::. -. ^ s treated (BV or extracellular viruses, ECV) are:. : -: in the movement of nucleocapsids has: r i.? plasma membrane. These virions shed - coating derived from the nucleus in the cytoplasm and sprout through the cytoplasmic membrane in the haemoceloma of the host of the insect. This process leads to systemic infection of the host insect. After the infection process, the virions become occluded (occluded virions) by a protein matrix consisting substantially of the polyhedrin protein, thus forming polyhedral inclusion bodies (PIB or occlusion bodies, OB), Occlusion is the orally infectious form of the virus and provides horizontal transmission of the virus among insect hosts (6,7). The uninfected larvae feed on substrates contaminated with virus and ingest the PIB. The protein matrix is solubilized by the action of the basic pH of the insect's midgut found in many lepidapterous larvae. The nucleocapsid = released from the virion, which contains in the genome of the virus, attacks the epithelial cells of the midgut of the larva, typically, the infected insect continues to develop and consume the plant material while the virus is pre a- exponentially within the host. Eventually. Frequently after several weeks or more - which has happened, the infected larvae will become completely compromised and will die. Through the use of recombinant DNA technology, NPVs have been genetically engineered to increase their speed of insect elimination either by introducing genes directly to the expression of insecticidal proteins or by deleting genes from the viral genome ( 8-10). Both strategies produce biological insecticides that exhibit faster insect control than NPVs not handled by engineering, wild type. The most effective recombinant NPVs have been engineered by genetic engineering to express selective neurotoxes to insects (11-18). The expressed toxins accelerate the process of cytotoxicity, resulting in faster insect control. These recombinant viruses eliminate their hosts in 20-30% less time than wild-type NPVs. The baculovir-rs - stir - for use as control agents of:. -. : biological as must be produced in large can :, r. rs Mass production of viruses is possible. with the cell culture systems of: r ••; ' .5, m vitro, normal, c by the production in. "In larvae of infected insects The yields of the viral particles in any system are dependent on sufficient viral replication, which in turn is dependent on the maintenance of the cells or premature cell cytotoxicity or insect death will necessarily limit viral replication, thereby reducing the number of progeny viruses produced.Genetic-engineered baculoviruses, engineered to more rapidly neutralize targeted insects, can result in less viral replication and result in lower viral progeny or viral cell yields (in vitro) or by infected insect (in vivo). In this way, the means used to improve the efficiency of mid-baculovirus insect control agents, making viable alternatives or aids to traditional chemical insecticides, can actually overcome the economic viability of this pest control strategy. Therefore, a method for efficient production is needed: insecticidal baculovirus that overcomes the barrier of premature cytotoxicity and premature elimination-e the host cell or insect.

BRIEF DESCRIPTION OF THE INVENTION This invention relates to methods that facilitate the production of recombinant baculoviruses that have been engineered for use as biological control agents. More specifically, this invention relates to the regulation of the expression of genes encoded by recombinant baculoviruses in an insect cell or in an insect host. In one embodiment, this invention relates to a method for controlling the expression of a gene encoded by the genome and a recombinant baculovirus in the culture of insect cells or viable insects, wherein the insect or insect cells have been handled in genetic engineering to express a protein that regulates the expression of a gene encoded by the baculovirus genome. More particularly, the method for controlling the expression of an insecticidal protein encoded by a chimeric gene in the genome of a recombinant baculovirus that can be exploited for the efficient production of baculoviruses and recombinant insecticides in the culture of insect cells or insects viable, the steps of the method comprising '(a) constructing a recombinant insect cell having a first chimeric gene comprising a first fragment of nucleic acid encoding for a first promoter, the first nucleic acid fragment operably linked to a second nucleic acid fragment encoding a regulatory protein capable of affecting the expression of the gene; (b) construct a recombinant baculovirus expression vector having a second chimeric gene comprising a third nucleic acid fragment that codes for a second promoter that is affected by the regulatory protein of step (a), the third nucleic acid fragment probably linked to a fourth nucleic acid fragment that code to go to the insecticidal protein, (c) enter e. recombinant baculovirus expression vector of (b) which is e-way in the recombinant i secte cell rre a); and (d) maintaining the recombinant insect cell of (a) that contains the baculovirus expression vector, recombinant from (b) under conditions that support baculoviral replication; wherein the expression of the regulatory protein of step (a) affects the expression of the insecticidal protein of step (b), In another embodiment, this invention relates to a method for controlling the expression of an insecticidal protein encoded by a a chimeric gene present in the genome of a recombinant baculovirus, comprising: (a) constructing a recombinant insert cell having the first chimeric rer comprising a nucleic acid fragment coding for: ---. for a first promoter, a prir-e: nucleic acid fragment enl = r. r operably in the second fragment: nucleic acid that codes for. • - protein: e; -. ladora and capable of are '. - the express: in the gen; (b) construct, n baculovirus expression vector. a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter that is affected by the regulatory protein of step (a), the third nucleic acid fragment operably linked to the fourth nucleic acid fragment encoding for an insecticidal protein; (c) enter the expression vector of. baculovirus, recombinant from (b) in the recombinant insect cell of (a); Y (d) maintaining the recombinant insect cell of (a) which contains the recombinant baculovirus expression vector of (b) under conditions that support baculoviral replication wherein the expression of the regulatory protein of step (a) affects the expression of the insecticidal protein of step (b). In another embodiment, this invention relates to the method for controlling the expression of an insecticidal protein encoded by a chimeric gene present in the genome and a recombinant baculovirus, comprising: (a) constructing a recombinant baculovirus expression vector having (1) ) a first chimeric gene comprising the first nucleic acid fragment that encodes for a first conductor, the first nucleic acid fragment operably linked to a second nucleic acid fragment encoding a regulatory protein capable of affect the expression of the gene, and (2) a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter that is affected with the regulatory protein, The third nucleic acid fragment operably linked to the fourth nucleic acid fragment encoding an insecticidal protein. (b) introducing the reecuminant baculovirus expression vector of (a) into an insect cell; and (c) maintain the insect's (bi-low cor .-- .--. -. nes that support the replicator.

Expression of the regulatory protein affects the expression of the insecticidal protein. In another embodiment, the invention relates to a method for the production of recombinant baculovirus, insecticide, comprising: (a) constructing a recombinant insect cell having a first chimeric gene comprising a first nucleic acid fragment encoding a first promoter, the first nucleic acid fragment operably linked to the second nucleic acid fragment encoding a regulatory protein capable of affecting gene expression; (b) constructing a recombinant baculovirus expression vector having a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter that is affected by the regulatory protein step (a), the third acid fragment nucleic acid operably chewed in a fourth nucleic acid fragment encoding an insecticidal protein. (c) introducing the recombinant baculovirus expression vector of (b) into the recombinant insect cell of (a); (d) maintaining the recombinant insect cell of (a) which contains the recombinant baculovirus expression vector of (b) under conditions that support baculoviral replication wherein the expression of the regulatory protein of step (a) affects the expression of the insecticidal protein of step (b); and (e) collect the progeny virus. In another embodiment, this invention relates to a method for the production of recombinant baculoviruses, insecticides, comprising: (a) constructing a recombinant baculovirus expression vector having; i > 'a first zer. chimeric comprising a first nucleic acid encoding d-.r-ir-.te the first promoter, the first fragment of nucleic acid loosely linked or the second nucleic acid fragment coding for a r / regulatory gene capable of affecting the expression of the gene, and (2) a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter that is affected by the regulatory protein, the third linked nucleic acid fragment operably to a fourth fragment of nucleic acid encoding an insecticidal protein; (b) introducing the recombinant baculovirus expression vector of (a) into an insect cell; (c) maintaining the insect cell of (b) under conditions that support baculoviral replication wherein the expression of the regulatory protein affects the expression of the insecticidal protein; and (d) collecting progeny viruses. In another embodiment, this invention relates to methods for the production of recombinant baculovirus, insecticides, wherein the cell is insect containing e-. Recombinant baculovirus cell expression vector is maintained in a cell culture in vitro.

In another embodiment, this invention relates to methods for the production of recombinant baculoviruses, insecticide, wherein the insect cell containing the recombinant baculovirus expression vector is maintained within an intact, intact insect. In another embodiment, this invention relates to a method for the production of recombinant baculoviruses, insecticides, comprising:. (a) constructing a recombinant baculovirus expression vector having (1) a first chimeric gene comprising a first nucleic acid fragment encoding a first promoter, the first nucleic acid fragment operably linked to a second nucleic acid fragment coding for a tetracycline transactivator protein and (2) a second chimeric gene comprising a third nucleic acid fragment encoding one or more tetracycline operator sites operably linked to a minimal promoter sequence, the third fragment operably linked to a quarter nucleic acid fragment encoding an insect-selective neurotoxin; (b) introducing the recombinant baculovirus expression vector of (a) into an insect cell; (c) maintaining the insect cell of (b) in the presence of an effective amount of tetracycline or a tetracycline analog such that the tetracycline transactivating protein is capable of binding to the tetracycline operator sites present in the third fragment of the acid nucleic acid and thus able to induce direct gene expression by the minimal promoter sequence operably linked to the operator sites; and (d) collecting progeny viruses. In another embodiment, this invention relates to a recombinant insect cell containing a chimeric gene comprising the first nucleic acid fragment coding for the first promoter, the first nucleic acid fragment operably linked to a second, c) nucleic acid fragment encoding a regulatory protein capable of affecting the expression of the gene driven by a second promoter. In another embodiment, this invention relates to a transgenic insect comprising one or more recombinant insect cells containing a chimeric gene comprising a first nucleic acid fragment encoding a first promoter, the first nucleic acid fragment operably linked to a second nucleic acid fragment encoding a regulatory protein capable of affecting the expression of the gene driven by a second promoter. In another embodiment, this invention relates to an insect cell or a transgenic insect comprising one or more recombinant insect cells, the insect cells containing a chimeric gene comprising a first nucleic acid fragment encoding a first promoter. , the first nucleic acid fragment operably linked to a second nucleic acid fragment encoding a regulatory protein capable of affecting. - expression of the gene driven by a second promoter, where the regulatory protein is the transactivating tetracyclic protein.

In another embodiment, this invention relates to a recombinant baculovirus expression vector having a chimeric gene comprising a first nucleic acid fragment encoding a promoter operably linked to a second nucleic acid fragment encoding an insecticidal protein, the first promoter affected by a regulatory protein expressed by a recombinant insect cell having a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter, the third nucleic acid fragment operably linked to a fourth fragment of acid nucleic acid that codes for the regulatory protein. In another embodiment, this invention relates to a recombinant baculovirus expression vector having a first chimeric gene comprising first nucleic acid fragment encoding a first promoter operably linked to a second nucelic acid fragment encoding an insecticidal protein, the first promoter affected by a regulatory protein expressed by a recombinant insect cell: - has a second chimeric ger which comprises -..-. third nucleic acid fragment encoding a second promoter, the third nucleic acid fragment operably linked to a fourth nucleic acid fragment encoding the regulatory protein, wherein the first nucleic acid fragment comprises one or more tetracycline operator sites , operably linked to a minimum promoter. In another embodiment, this invention relates to a recombinant baculovirus expression vector having (1) a first chimeric gene comprising the first nucleic acid fragment encoding a first promoter, the first nucleic acid fragment operably linked to a second fragment of nucleic acid encoding a regulatory protein capable of affecting the expression of the gene, and (2) having a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter that is affected by the regulatory protein , the third nucleic acid fragment operably linked to a fourth nucleic acid fragment encoding an insecticidal protein. In another embodiment, this invention relates to a recombinant baculovirus expression vector having (1) a first, chimeric gene comprising - n first fragment of nucleic acid encoding a first promoter, the first nucleic acid fragment linked operably the second nucleic acid fragment encoding a regulatory protein capable of affecting the expression of the gene, and (2) having a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter that is affected by the protein , the third nucleic acid fragment operably linked to a fourth nucleic acid fragment encoding an insecticidal protein, in. wherein the regulatory protein is a transactivating tetracycline protein, wherein the third nucleic acid fragment comprises one or more tetracycline operator sites operably linked to a minimal promoter.

BRIEF DESCRIPTION OF THE DRAWINGS AND LISTS OF SEQUENCE Figure 1. Quantification of recombinant viral and wild type progenies with polyhedrin inclusion bodies (PIB) from larvae of H. virescens. "AcAalT / plO" represents recombinant baculoviruses, wherein the expression of the AaIT toxin is controlled by the late baculoviral promoter plO "AcLqhIT / plO" represents recombinant baculoviruses wherein the expression of the LqhIT2 toxin is controlled by the very late baculoviral plO promoter . "AcLqhlT / IEl" represents recombinant baculoviruses wherein the expression of the LqhIT2 toxin is controlled by the early baculoviral IE1 promoter. "AcNPV" represents wild type baculovirus.

Figure 2. Schematic representation of a controllable system with tetracycline transactivator.

Figure 3. Plasmid map of TV3tTA.

Figure 4 Sequence of the synthetic oligonucleotides used to construct the LqhIT2 gene. Oligonucleotide Lql (SEQ ID NO: 4) codes for the signal peptide of Bombixin. Oligonucleotides Lql (SEQ ID NO: 4) and LqlO (SEQ ID NO: 13) were used as primers for the PCR amplification of the synthetic gene.

Figure 5. Schematic representation of the strategy used to 'prepare LqhIT2. Oligonucleotides Lql (SEC :: NO: 4) and LqlO (SEQ ID NO: 13) (marked with a "-" serve as amplification primers for PCR reactions.) The cleavage sites are indicated by unique restriction enzymes - Figure 6. Nucleotides (SEQ ID NO: 14) and corresponding amino acid sequences of the LqhIT2 gene The lowercase letters in the nucleotide sequence (nucleotides 1-57, which code for amino acids 1-19) indicate nucleotides encoding the peptide of Bombixina signal.

Figure 7. Baculovirus expression vector map TV3tTaTo-LqhIT2.

Figure 8. Amino acid sequence (SEQ ID NO: 21) from TetrlElA. The tetR and IE1A regions of the transactivator are indicated by lowercase and uppercase letters, respectively. The two amino acid residues indicated in bold (positions 208 and 209) have been added as a result of the extra restriction sites present in the sequence used to facilitate the insertion into the structure of tetR and IE1A.

Figure 9. Nucleotide sequence (SEQ ID NO: 23 and 24) of the minimal p35 gene promoter of AcMNPV identifying the positions of the TATA sequence, and the early transcriptional (E) and late (L) transcriptional sites. Restriction sites that are to be used for the future insertion of foreign genes are also indicated.

Figure 10. Nucleotide sequences (SEQ ID NO: 25 and 26) of the minimal, modified AcMNPV p35 gene promoter that identifies the positions of the TATA sequences and the early (E) and late (L) transcriptional start sites. The mutation of the individual base pair in p35m is indicated in bold. The restriction sites that are to be used for the future insertion of foreign genes are also indicated.

Figure 11. Diagram of the region immediately in the 5 'direction of the polyhedrin gene in the transfer vectors pTV3 (M) Iq + and pTV3 (M) Iq-. The relative positions of the tracer gene and a cartridge containing the tet operator sequences are indicated in the 5 'direction of the p35 / p35m and LqhlT minimum promoter, Figure 12. Diagram of the region immediately downstream of the polyhedrin in the transfer vectors ptTA (M) lq + and ptTA (M) lq-. The relative positions of the transactivator gene and a cartridge containing the sequences of the tet operator in the 5 'direction of the minimal promoter p35 / p35m and LqhIT are indicated. The applicant has provided 26 sequence listings according to 37 C.F.R. 1.821-1.825 and Appendices A and B ("Requirements for Descriptions of Requests Containing Nucleotides and / or Amino Acid Sequences") and in accordance with "Rules for the Normal Representation of Nucleotide and Amino Acid Sequences in Patent Applications" and Annexes I and II in the Decision of the President of the EPO, published in Supplement No. 2 to OJ EPO, 12/1992. SEQ ID NO: 1. BglII linker used in the construction of plasmid TV3tTa. SEQ ID NO: 2. Sequence of fragment nucleotides of the restriction sites Xho I to Bam Hl of 407 bp from the pMamid pF43h, which contains the minimal hGH promoter fused in tandem to seven sequences of the racicline te operator.

SEQ ID NO: 3. Notl linker used in the construction of the plasmid ptetophGH. SEC ID NOS: 4-13. Synthetic oligonucleotides used to construct the LqhIT2 gene. Oligonucleotide Lql codes for the signal peptide of Bombixin. Oligonucleotides Lq1 (SEQ ID NO: 4) and Lq10 (SEQ ID NO: 13) are used as primers for the PCR amplification of the synthetic gene. SEQ ID NO: 14. Nucleotide sequence of the synthetic DNA fragment encoding the Bombixin signal peptide followed by the coding region of the LqhIT2 toxin. SEQ ID NOS: 15 and 16. Nucleotide sequences of the PCR primers TETR1 and TETR1 alone to amplify the tetR portion of tTA. SEQ ID NO: 17. Nucleotide sequence of the tetracycline repressor gene (tetR) that was amplified by PCR using the oligonucleotides TETR1 and TETR2. SEQ ID NOS: 18 and 19. Nucleotide sequences of the PCR primers IE1A1 and IE1A2 used to amplify a 454 bp fragment from the pIElH / C plasmid corresponding to the first 145 amino acid residues of IE1.

SEQ ID NO: 20. Nucleotide sequences of the activation domain of IE1 (IE1A) that was amplified by PCR using the oligonucleotides IE1A1 and IE1A2. SEQ ID NO: 21. TetrlElA amino acid sequence. SEQ ID NOS: 22. Nucleotide sequence of the BamHI fragment containing an SV40 polyadenylation signal. SEQ ID NOS: 23 and 24. Nucleotide sequences of two complementary oligonucleotides, P35PR01 (SEQ ID NO: 23) and P35PR02 (SEQ ID NO: 24), which, when tuned, represent the p35 minimal promoter from -8 to -57 bp relative to the translation initiation codon of the p35 gene. SEQ ID NOS: 23 and 26. Nucleotide sequences of two complementary oligonucleotides P35MPR01 (SEQ ID NO: 25) and P35MPR02 (SEQ ID NO: 26), which, when annealed, represent a modified p35 minimum promoter where a total individual change of the base pair has been found in the promoter sequence that eliminates the RNA start site TTAAG, late.

DETAILS OF THE INVENTION A method for suppressing the expression of insecticidal proteins by recombinant NPVs for purposes of in vivo and in vitro production is described. In one embodiment, a gene encoding a regulatory protein in the genome of an insect or insect cell that is a natural host of insect baculovirus is stably integrated. In turn, a regulatory sequence that is responsive to the regulatory protein is stably introduced into the genome of a baculovirus at one or more locations near a promoter and / or enhancer element that directs the expression of a heterologous protein. Upon introduction into the host (i.e., infection of a transgenic insect or transfection of insect cells transgenic with the recombinant baculovirus), the regulatory protein expressed by the transgenic insect and insect cells interact with the regulatory sequence present in the host. Baculovirus genome and affects the expression of the gene or genes that are controlled by that regulatory sequence. Alternatively, both the gene encoding the regulatory protein operably linked to an inducible repressive or viral promoter, and the gene encoding the heterologous gene of interest that is operably linked to the regulatory sequences that are responsive to the regulatory protein. , are present in the genome of recombinant NPV. These recombinant viruses are then propagated in the insect. "Wild types or insect cells In the presence of an appropriate inducer of the gene expression of the regulatory protein, the control of heterologous gene expression will be affected. Structurally encoding the tetracycline repressor protein (tet) that is under the control of a constitutive promoter (e.g., actin promoter) or a viral promoter (preferentially, a promoter derived from a nuclear polyhedrosis virus) can be inserted in a genome of a lepidopteran insect - Regulatory sequences that are sensitive to the tet repressor are introduced into the viral genome close to the promoter and / or enhancer regions that control the expression of an insecticidal protein. The tet repressor binds to the regulatory sequence (s) and prevents transcription of the gene encoding the insecticidal protein. The lack of expression of this. Insecticidal protein will delay the onset of paralysis and death of the infected insect, and will result in an increase in GDP production. Similarly, this strategy can be used to control the expression of cytotoxic proteins or proteins that render unstable viruses, thereby maximizing virus production in insect cell culture systems. In the context of the present description, a number of terms and abbreviations should be used. "NPV" means the Nuclear Polyhedrosis Virus, a baculovirus. "Polyhedrosis" refers to any or several diseases by insect larval virus characterized by the dissolution of tissues and the accumulation of polyhedral granules in the resulting tissue, "PIB" being the polyhedral inclusion bodies. "AcNPV" means the nuclear polyhedrosis virus, Autographa califorica, wild type. "LqhIT2" represents the selective insect neurotoxin derived from Leiurus quinquestriatus hebraeus. "AaIT" represents the selective insect neurotoxin derived from Androctonus australis. "AcLqhIT2" is an abbreviated form that represents? "N? V that has been genetically modified to host the gene coding for LqhIT2 under the control t r. .scr. Of the very late promoter plO of the baculev: r -.s.

"Expression" refers to the transcription and translation of a structural gene to produce the encoded protein. As will be appreciated by those skilled in the art, the levels of expression of the structural gene are agitated by the regulatory sequences (promoter, polyadenylation sites, enhancers, etc.) used and by the host cell in which the structural gene is expressed. . Suitable "regulatory sequences" refer to nucleotide sequences located in the 5 '(5') direction, within, and / or in the 3 '(3') direction of a structural gene. The regulatory sequences control the transcription and / or expression of the coding sequences, potentially or linearly with the biosynthetic protein apparatus of the cell. These regulatory sequences include promoters, operators, enhancer elements, transcription termination sequences, and polyadenylation sequences. "Regulatory proteins" are proteins that recognize and bind to the regulatory sequences and thus affect the transcription and / or expression of the coding sequences. Regulatory proteins may exhibit a relative effect on gene expression (ie, reduce or eliminate the prescription and / or translation) and can therefore be referred to as "repressors". In contrast, certain regulatory proteins may exhibit a positive effect on gene expression (i.e., initiate or increase transcription and / or translation) and therefore may be known as "activators", "transactivators" or "inducers". "Operator" refers to a regulatory sequence that is recognized by a regulatory protein that modifies the rate and initiation of transcription in an adjacent promoter. "Promoter" refers to the nucleotide sequences, generally found at the 5 'end of the structural gene, which directs the initiation of transcription. Promoter sequences are necessary, but not always sufficient, to drive expression in a gene in the 3 'direction. Usually, the promoters drive the transcription preferably in the 3 'direction, although the promotional activity can be demonstrated (at a reduced level in the expression) when the gene is placed in the 5 direction? by the promoter. In this way, in the construction of heterologous promoter / structural combinations, the structural gene is connected under the regulatory control of a promoter such that the expression of the gene is controlled by the promoter sequences. The promoter is preferably placed in the direction 5? to the structural gene of a distance from the transcription start site that approximates the distance between the promoter and the gene, which is controlled in its natural state. As is known in the art, some variation in 1 can be tolerated? distance, without loss of the condition of the promoter. "Minimum promoter" refers to a class II DNA polymerase promoter, generally comprised of a DNA sequence known as a TATA sequence that binds to the RNA polymerase and short stretches of surrounding DNA sequences that, or in the absence of of the additional operator promoter elements, is unable to support the initiation of transcription. The "oblique promoter" refers to a promoter that is active (that is, capable of supporting the transcription of its adjacent gene) in all tissues and organs of the organism. The "inducible promoter" refers to a promoter whose function is controlled by the binding or release of the particular intracellular, transaction factors. The inducible promoters are distinguished from the constitutive promoters that are deregulated and thus are continuously active.

"Non-coding 3 'sequences" refers to the portion of the DNA sequence of a gene that contains a polyadenylation signal and another regulatory signal capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of the polyadenylic acid extensions to the 3 'end of the mRNA precursor. "Gene" refers to the entire portion of DNA comprised in the synthesis of a protein. A gene incorporates the structural or coding portion starting at the 5 'end from the transductional start codon (usually the ATG nucleotides) and extending to the terminator codon (TAG nucleotides)., TGA or TAA) at the 3 'end. It also contains a promoter region, usually located in the 5 'direction to the structural gene, which initiates or regulates the expression of a structural gene. Also included in a gene are the non-coding 3 'sequences. The "chimeric" gene refers to an artificial gene comprising the heterogeneous coding and regulatory sequences. The "heterologous" gene refers to genes or parts of genes normally found in the host organism but which have been introduced with gene transfer. "Nucleic acid fragment" refers to nucleotide sequences that are identified with particular functions within a gene. The "structural gene" is that portion of a gene that comprises the segment of DNA encoding the protein, with the peptide to a portion thereof, and which excludes the 5 'and 3' sequences comprised in the regulatory control and the expression of the gene. The structural gene may be one that is normally found in the cell or one that is not normally found in the cell location where it is introduced, in which case it is called a heterologous gene. A heterologous gene can be derived in whole or in part from any part known to the art, including a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA, DNA or chemically synthesized DNA. A structural gene may contain one or more modifications in either the coding regions or the nc produced which may affect the biological activity and chemical structure of the expression product, the expression vector or the manner of expression control. These modifications include, but are not limited to, mutations, insertions, deletions and substitutions of one or more nucleotides. The structural gene may constitute an uninterrupted coding sequence, or it may include one or more introns that are not expressed and that are joined by the appropriate linkages. The structural gene may be a composite of segments derived from a plurality of sources, which occur naturally or synthetically. The structural gene can also include fusion genes that code for a fusion protein. The "synthetic gene" refers to a DNA sequence of a structural gene that is synthesized chemically in its entirety or throughout most of the qualification region. As exemplified herein, oligonucleotide forming blocks are synthesized using methods known to those skilled in the art and used to form the gene segments and then assembled enzymatically to construct the entire gene. As recognized by those skilled in the art, genes functionally and structurally equivalent to the synthetic genes described herein may be prepared by site-specific mutagenesis or other related methods used in the art. The term "operably linked" refers to nucleic acid sequences in an individual nucleic acid molecule that associates such that the function of one is affected by the other. For example, a promoter is operably linked to a structural gene when it is capable of affecting the expression of that structural gene (ie, that the structural gene is under the transcriptional control of the promoter) - "Transfection" refers to the introduction of stable of a segment of DNA that has a gene in an organism that does not previously contain that gene. "Co-transfection" refers to the simultaneous introduction in addition to a segment of DNA in an organism. The "transgenic insect" refers to an insect comprised of cells that contain one or more transgenes. "Transgenes" are genes that are introduced into the genome of a cell from which a transgenic organism develops and which remain in the mature organism, thereby directing the expression of its products encoded in one or more cell types or tissues of transgenic organisms. "Chemically synthesized", as it relates to a DNA sequence, it means that the component nucleotides are assembled in vitro. Manual chemical synthesis of DNA can be achieved using well-established procedures (19), or automated chemical synthesis can be performed using one of a number of commercially available machines. It is understood that "an insect cell" refers to one or more insect cells maintained in vitro as well as to one or more cells found in a living, intact insect. The "baculovirus" refers to a group of viruses that only infect arthropods, in phylum that includes the genus in Lepidoptera. Baculovirue insecticides have great potential to provide an environmentally benign method for pest control of agricultural insects. However, efficiency improvements are concerned in order to make these agents competitive with pest control agents, chemicals, currents. One approach to making these improvements is through the genetic alteration of the virus. For example, it may be possible to modify the viral genome in order to improve the host's range of the virus, to increase the environmental stability and persistence of the virus, or to improve the infectiousness and transmission of the virus. Furthermore, improving the speed at which the virus acts to engage the infected insect would significantly improve the attractiveness of baculovirus insecticides as adjuvants or replacements of chemical pest control agents. One method to increase the speed with which viruses infect their insect hosts is to introduce into the baculovirus foreign genes that encode proteins that are toxic to the effect where death or incapacitation of the insect is not dependent for a longer time only in the course of viral infection, but instead is helped by the accumulation of toxic levels of the foreign protein. The results are recombinant baculoviruses and insecticides - Many arthropods produce a mixture of proteins and insecticides referred to as venom. These toxic substances are synthesized in specialized glandular tissues, which, when directed by a sharp or penetrating device, are capable of paralyzing the prey of the arthropod. Slow moving or stationary arthropods, small have adapted to a strategy to instantly paralyze their prey by using neurotoxic components of the venom at very low concentrations These components or neurotoxins interfere with the function of the insect's nerve tissues through efficient competition by certain receiving sites. Many of these neurotoxins are polypeptides. These have been divided into different classes based on their host specificity and mode of action (20). For example, neurotoxic peptides isolated from numerous scorpion species have been divided into classes that affect arthropods and classes that affect mammals. Several of the arthropod-specific toxins have been identified, such as insect-selective peptides. For example, the scorpion Buthinae expresses two types of selective insect neurotoxins that differ in their biological effects in the target insects. In the botfly, those classified as classification toxins cause immediate, rapid, irreversible contractive paralysis due to the induction of repetitive triggering of terminal branches of motor neurons (21-23). These toxins are single-chain polypeptides of approximately 70 amino acids and are cross-linked by four disulfide sources. The excitatory effect is attributed to an increase in the activatable action potential, sodium conductance, a voltage-dependent decrease in the closure of the channel. AaIT, a toxin produced from the venom of the scorpion Androctonus australis (24), was the first insect toxin isolated from these organisms that exhibited this excitatory action. A second class of selective insect neurotoxins are depressive toxins, including BJIT2 (25), LqqIT2 (26) and LqhIT2 (27). These toxins are polypeptides of 60 to 65 amino acids that possess primary amino acid sequences that are distinct from the excitatory toxins. These toxins induce progressive, slow paralysis and complete relaxation of the insect's musculature. This activity is the result of the blocking of the action potentials provoked (26, 28), and is attributable to the suppression of the activatable sodium conductance and the depolarization of the axonal membrane. The methods and strategies used for the preparation of recombinant baculoviruses expressing heterologous genes are well known in the art (4, 5, 29). These gene expression methods provide economic preparation of mammalian proteins in a eukaryotic expression vector system, in many cases producing proteins that have achieved their proper tertiary conformation and formed the appropriate bridges from sulfide necessary for the activity.

Commonly, the introduction of heterologous genes in the genome. Baculoviruses occur by homologous recombination between the viral genomic DNA and an appropriate "transfer vector" containing the heterologous gene of interest. These transfer vectors are in general plasmid DNA which are capable of autonomous replication in bacterial hosts, producing easy genetic manipulation. Baculovirus transfer vectors also contain a genetic cartridge comprising a region of the viral genome that has been modified to include the following characteristics (listed in the 5 'or 3' direction) i 1) Viral DNA comprising the 5 'region and a non-essential genomic region; 2) a viral promoter; 3) one or more DNA sequences encoding the restriction enzyme sites facilitating the insertion of heterologous DNA sequences; 4) a transcriptional termination sequence; and 5) viral DNA comprising the 3 'region of a non-essential genomic region. A heterologous gene of interest is inserted into the transfer vector at the restriction site in the 3 'direction of the viral promoter. The resulting cartridge comprises a chimeric gene wherein the heterologous gene is under the transcriptional control of the vial promoter and the transcription termination sequences are present in the transfer vector. In addition, this chimeric gene is flanked by viral DNA sequences that facilitate homologous recombination in an essential region of the viral genome. Recombinant viruses are created by co-transfecting insect cells that are capable of supporting viral replication with viral genomic DNA and the recombinant transfer vector. Homologous recombination between the viral, flanking DNA sequences present in the transfer vector and the homologous sequences in the viral genomic DNA takes place and results in the insertion of the chimeric gene in a region of the viral genome that does not break a function essential viral The infectious recombinant virion consists of the recombinant AD, referred to as the baculovirus expression vector, secondary by a protein coat. In a preferred embodiment, the essential region of the viral genome that is present in the transfer vector comprises the region of the viral DNA responsible for the production of polyhedrin. More preferred is a transfer vector containing the complete polyhedrin gene between the flanking sequences that are comprised in the homologous recombination. The recombination with genomic DNA from viruses that are defective in the production of polyhedrin (due to a defect in the genomic copy of the polyhedrin gene) to result in the restoration of the polyhedrin-positive phenotype. This strategy facilitates the identification and selection of recombinant viruses. In another embodiment, baculoviral genomic DNA can be directly modified by the introduction of a restriction enzyme recognition sequence, unique in an essential region of the viral genome. A chimeric gene comprising the heterologous gene to be expressed by the recombinant virus and operably linked to the regulatory sequences capable of directing gene expression in the insect cells infected with baculovirus, can be constructed and inserted directly into the viral genome in the only restriction site. This strategy eliminates both the need for the construction of transfer vectors and the reliance on homologous recombination for the generation of recombinant viruses. This technology is described by Ernst et al. (30), and in W094 / 28114 (31).

Recombinant baculovirus vectors suitable for the distribution of genetically complicated insect-specific neurotoxins require optimal expression of the toxin gene for maximum efficiency. A number of strategies can be used by those skilled in the art to design and prepare recombinant baculoviruses wherein the expression of the toxin gene results in sufficient quantities of toxin reduced at appropriate times during infection in a functional form and available for binding to target cells within the graft host. A key to optimal mass production of recombinant NPVs is the selection of an inappropriate system to regulate gene expression. This system preferably uses a repressor / operator element that suppresses the transcription of the gene encoding the insecticide activity. Several repressor systems have been described that affect the expression of the gene, including several inducible eukaryotic promoters (32-33). In addition, the regulation of gene expression in eukaryotic cells can be achieved through the use of the ir.d-.et r system of repressor / operator of lac Escherichia coli Three approaches have been described: (i) prevention of the initiation of transcription by appropriately placing lac operators on promoter sites (34-38); (ii) transcription blockade mediated by ARF polymerase II during elongation by a lac repressor / operator complex (39); and (iii) activation of a promoter responsive to a pressure protein comprised of the repressor protein and the activation domain of virion protein 16 (VP16) of the herps simplex virus (40-41). However, due to the slow and inefficient activation by the lac inducer, isopropyl-β-D-thiogalactopyranoside (IPTG), this system has only resulted in moderate regulation (42). The present invention relates specifically to the construction of an adjustable gene expression system employing the tetracycline operator-repressor system (tet). The tetracycline repressor which is a DNA binding protein with high affinity for the operator sequence of tet The tn repressor encoded by TnlO regulates the expression of the genes by binding to the operator sites and overlaying an operator (s) ( 43,44, Tetracycline or a tetracycline analog prevents the repressor from binding to its operator sequence, thereby allowing the RNA polymerase to bind to the promoter sequences and measure RNA transcription. of tet has been used successfully in plants for the effective control of gene expression, for example, Gatz et al. were able to use this system to reduce the activity of a constitutive promoter (promoter of the 35S mosaic virus of the cauliflower; 35S) for 500 times in transgenic plants (45) Specifically, they generated a transgenic tobacco plant that synthesized 1 x 106 tet repressor molecules per cell. tet res in the vicinity of the TATA sequence of the CaMV 35S promoter. Under normal physiological conditions in gene expression was measured in an essential way. However, the addition of tetracycline prevented the repressor from binding to the operator sites, causing complete derepression of the CaMV 35S promoter. More recently, the operator-repressor system of tet has been used to control gene expression in the protozoan parasite Trypanosoma brucei (46). Transgenic trypanosomes expressing the tetracycline repressor of E. coli followed the inducer-dependent expression (tetracycline) of the chromosomatically integrated reporter genes under the control of a promoter having a tet operator. The expression of the indicator could be controlled over a range of four orders of magnitude in response to the concentration of tetracycline. This far exceeds the transcriptional regulation exceeded by other systems based on eukaryotic repression. The present invention controls the expression of an insecticidal protein encoded by a recombinant virus genome. Experimental experience demonstrates a greater than approximately 10-fold decrease in the yield of the GDPs of insects infected with recombinant NPVs, engineered to express an insecticidal protein relative to GDP produced from insects treated with wild type virus ( vei Figure 1). Certain proteins and insecticides may have adverse defects in the insect cells (eg, cytotoxicity) or may induce paralysis and death of an insect host. These effects may result in a reduction in viral progeny and / or an unstable road construction. The suppression of the expression of the insecticide protection would give the insect host cell the ability to withstand the spread of recombinant viruses for productions that reach the productions of wild-type viruses. This invention regulates the expression of the toxin gene by several methods. One method uses a transactivating molecule controlled by tetracycline (also referred to herein as "tetracycline transactivating protein") to control the expression of the promoter address of the toxin gene. Aspects of this hermetically adjustable expression system are described in U.S. Patent No. 5,464,758, incorporated herein by reference, and Gossen & amp;; Bujard (47, 48) and are depicted in Figure 2. The control element used for gene regulation is a fusion protein comprised of the tet repressor protein fused to the activation domain of virus virion 16 protein. simple herpes (tTA). The invention also includes the construction of a transgenic insect that constitutively expresses a repressor protein (or similar to the repressor). When a recombinant NPV containing a chimeric gene (comprised of an operably linked tet operator (s), promoter and toxin structural gene) is being replicated in the transgenic host, the repressor binds to the operator (s) inhibits the expression of the toxin. To construct this conditional regulatory system for the transcription of the gene, two basic genetic manipulations are carried out; i) fragments of the tet operator (s) are inserted in the 5 'direction of the toxin gene, preferably in a region that overlaps with the transcription initiation site and / or the enhancer region; and ii) the repressor gene is suitably engineered into the genome of a eukaryotic cell, preferably an insect or an insect cell line. The repressor can be placed under the control of a constitutive (ie, active) promoter or baculovirus promoter (i.e., IE1, plO or hybrid promoter) in order to drive expression of the host repressor by the circulating virus. Using a baculovirus promoter such as IE1, plO, or polyhedrin, the repressor is expressed only at the time of viral infection. This design provides the appropriate infection of the repressor protein, since the virus provides the machining. i transcriptional necessary for viral replication The site (s) of the tet operator is collated in the viral genome near the transcription initiation site and / or enhancer regions in order to block transcription of the heterologous protein (in the form of preferred, an insecticide protein) driven by the viral promoter. High efficiency vectors for the insertion of foreign genes into the insect genome are required for this strategy. Molecular nucleic acid entities of various types have been identified in different insects (49). The insertion of the mobile elements in the DNA sequences can result in altered or interrupted gene expression that can be identified as mutant phenotypes. Currently, insect transformation is achieved in high efficiency using the transformation vector system based on the P element in Drosophila (50). However, these transformation vectors can not be used for the transformation of other insects (51). Therefore, mobile, alternative genetic elements that have utility in other insects, including high-efficiency transformation in lepidopteran insects, are required. In addition, other methods of transformation have "focused on alternative distribution systems." One such technique is the "maternal microinjection" that involves the injection of DNA into eggs or ovaries within the pregnant female, this technique has been used successfully. to introduce the plasmid DNA sequences into the rapacious mites Metaseiulus occidentalis (52) and Amblyseius finlandicus (53) Recently, electroporation has also been reported to successfully distribute plasmid DNA to insect embryos (Keith Hughes, USDA, Fargo , ND Personal communication) A method to prepare a transgenic insect is to use an almost transposable element (ET) useful for the introduction of heterologous genes into the genome of lepidopteran insects.These genetic elements facilitate the construction of transgenic insects capable of expressing regulatory proteins, which, in turn, control the baculovirus gene expression. it is the marine element that is very widespread in the genomes of the arthropods, and almost oblique in the lepidopteran insects (55). The marine element can be identified and prepared using degenerate primers derived from two conserved regions of the open marine transposase reading structure. Recently, a marine transposable element was isolated from the raptor, Metasieulus occidentalis (55). Using two inverted primers, the complete marine element (Mocl) was sequenced and found to be 1284 bp in length, including inverted terminal repeat sequences of 28 bp. However, the Mocl marine element contains numerous mutations, including changes in temperature, and transposase activity that can not be detected. Based on these operations, the Mocl is considered to be an inactive transposable element. However, an inactive marine element is an attractive tool for the introduction of exogenous DNA by homologous recombination, because it has accumulated numerous mutations, including deletions, insertions and final codons. The insertion of exogenous DNA into this marine site should be advantageous since the transposase gene is not viable for a long time and this exogenous DNA will remain stable. The insertion of the tet repressor gene and a selectable marker (ie, the cyclodiene-Rdl resistance gene); (56,57)) that contains a marine fragment in a marine site in a lepidopteran insect occurs by homologous recommendation. The transformation is carried out by previously described microinjection techniques or newly developed electroporation protocols.

EXAMPLES The present invention is further defined in the following examples. It will be understood that the examples will be by illustration only and the present invention is not limited to the use described in the examples. From the above discussion and the following examples, a person skilled in the art can determine, and without departing from the spirit and scope of it, can make various changes and modification of the invention to adapt it to various uses and conditions. All these uses and modifications are proposed to fall within the scope of the appended claims: Example 1 The regulatory expression of an insecticidal protein can also be achieved by incorporating the tTA and operator sites into the viral genome itself.

In this method, the control elements of the tetracycline resistance operon encoded in E. coli TnlO were inserted into the AcNPV genome in order to regulate the expression of the insecticidal protein (s).

The tTA was placed under the control of the IEL promoter immediately early, which expresses foreign genes immediately after infection for NPV. The baculovirus transfer vector pAcP + IElTV3 (provided by Dr. Donald Jarvis, Texas Agricultural Experiment Station, Texas A &M University, College Station, TX) was used for this construct and is a derivative of de-i transfer vectors Baculovirus early promoter described in U.S. Patent No. 5,162,222. Initially, the tTA was excised from the plasmid pUHD15-l (obtained from Dr Robert Horlic, DuPont-Merck Pharmaceutical Co., Wilmington, DE) by the enzymes EcoRI and BamH1 and inserted into the plasmid Bluescript SK ± R (Stratagene , Menasha, Wl) digested with EcoRI and BamHl. After ligation, the plasmid DNA was transformed into E. coli DH5a (Gibco / BRL1 Gaithersburg, MD), and the presence of the insert was confirmed by restriction enzyme analysis. After confirmation of the tTA insert; an SV40 polyadenylation fragment (SEQ ID NO: 22) (provided by Gary Chun, EI du Pont de Nemours and Company) was inserted with the BamHl restriction sites at the 5 ', and 3' ends, at the restriction site BamHl in Bluescript SK ± R directly in the 3 'direction of the tTA. The insert was confirmed by correct orientation using an internal Hpal site (poly A) and an Xhol site found at the 3 'end of the multiple cloning site of Bluescript SK ± R. The new plasmid containing the tTA and the polyA fragment was digested with EcoRl and filled with Klenow polymerase, and a BglII restriction site was added by the incorporation of the linker BG1II (SEQ ID NO: 1). After ligation, the plasmid DNA was transformed into the DH5a of E. coli. A fragment containing the tTA and the SV40 was then excised from this Bluescript SK ± R plasmid modified with the restriction enzymes BglII and Notl. This fragment was ligated into the pAcP + IElTV3 transfer vector cut with BglII and Notl. This construction resulted in the insertion of the synthetic structural gene encoding tTA and the polyadenylation fragment in the 3 'direction of the baculovirus IE1 promoter and lc. hr enhancer region 5. After ligation, the plasmid DNA was transformed into E. coli DH5a and the resulting transformants were selected for the position of plasmids that contained a unique asymmetric site located at the 3 'end of the tTA gene. The new plasmid was labeled with TV3tTA (Figure 3). The next step to build a regulatory system for a Baculoviru insecticide. recombinant comprised the incorporation of a minimal promoter sequence and the tetracycline operator sequences in the viral genome. The restriction enzymes Xhol and BamHl were used to cleave a minimal promoter (hGH) fused to the sequences of the tandem tetracycline operator (7 tet operators) from the plasmid pf43H (SEQ ID NO: 2, obtained from Dr R. Horlick, DuPont-Merck Pharmaceutical Co.). This fragment was cloned in Bluescript SK ± R that was previously digested with Xhol and BamHl. After the ligation, the plasmid DNA was transformed into the E. coli DH5a and the positive clones were identified by restriction enzyme analysis. This plasmid was then digested with the restriction enzyme Xhol (at the 3 'end of the multiple cloning site), filled with Klenow Polymerase and re-ligated in the presence of a Notl linker (SEQ ID NO: 3). After ligation, the plasmid DNA was transformed into E. coli DH5a and the resulting transformants were selected by digestion with the restriction enzyme NotI. The presence of a fragment of approximately 450 bp after electrophoresis through a 2% agarose gene confirmed the existence of an appropriate construction. This plasmid is known as ptetophGH. Finally, a structural gene coding for an insect selective neurotoxin, LqhIT2, is placed in the 3 'direction of the minimal promoter at the BamHl cloning site. In order to prepare the LqhIT2 gei, ten oligonucleotides were designed (Figure 4; composed of SEQ ID NO: 4-13) and synthesized by the normal phosphoramidite chemistry. These oligonucleotides were phosphorylated using Gibco / BRL (Gaithersburg, MD) kinase, fixed, and ligated using the bound Gibco / BRL following the scheme depicted in Figure 5, and employing the protocols recommended by the manufacturer. The ligated fragments were then amplified using the polymerase chain reaction (PCR) using Perkin-Elmer Cetus AmpliTaqR Polymerase (Norwalk, CT) according to the manufacturer's protocol and the modifications described below. Oligonucleotide Lql (SEQ ID NO: 4) was used as the forward primer and oligonucleotide Lq10 (SEQ ID NO: 13) was given as the inverted primer.

The description of these protocols are subsequently established in greater detail. Ten separate phosphorylation reactions (one for each oligonucleotide) were carried out. Two hundred fifty pmol of each oligonucleotide (SEQ ID NO: 4-13) were placed in a 1.5 mL microcentrifuge leak tube. Five μL of 10X kinase buffer, 1 μL of 1 mM ATP, and 6 μL of kinase (Gibco / BRL, 10 units / μL), and a sufficient volume of water was added to each tube in order to bring the total volume of reaction at 50 μL. The ten tubes were then incubated at 37 ° C for 1 hour. After incubation, 5 μL of each phosphorylated oligonucleotide (25 pmol) was placed in a single microcentrí leak tube, and the tube was placed in a dry heat block set at 95 ° C. The heat block was then turned off and allowed to cool to room temperature to facilitate fixation of the phosphorylated oligonucleotides. Fifty μL of the mixture of the bound, phosphorylated oligonucleotides were placed in a separate microcentrifuge tube together with 15 μL of 5X ligase buffer, 3 μL of 10 mM ATP, 4 μL of the ligase enzyme (Gibco / BRL, 5 units / μL) and 3 μL of deionized water. This tube was incubated at 37 ° C for 30 minutes and subsequently stored at room temperature overnight. The nucleic acid fragment, synthetic qu. comprises the bound and bound oligonucleotides was exemplified by PCR. Three PCR reactions were performed at varying dilutions of the template DNA (comprising the phosphorylated, bound and bound oligonucleotides). The following reaction mixture was cooled for PCR reactions: '61.5 μL deionized water 10 μL 10X PCR buffer (Perkin-Elmer Cetus) 2 μL of each of dATP, dCTP, dGTP, dTTP (200 μM each) 0.5 μL of AmpliTaqR Polymerase (2.5 units / 100 μL) The template DNA was diluted 1: 100, 1: 1.00 and 1 : 10,000 (v / v) with deionized water: 80 μL of the reaction mixture was placed in each of three 0.5 mL microcentrifuge tubes. Five μL (100 pmol) of the oligonucleotide Lql (SEQ ID NO: 4) and 5 μL (100 pmol) of the oligonucleotide Lq10 (SEQ ID NO: 13), (which serves as the forward and inverted PCR primers, respectively), added to each tube. 10 μL of the appropriately diluted template was diluted in each tube. The PCR reactions were carried out using a Perkin-Elmer DNA Thermocycler® programmed to carry out the following amplification protocol STEP 1 (1 cycle) 96 ° C 3 minutes 75 ° C 3 minutes STEP 2 (25 cycles) 95 ° C 30 seconds 75 ° C 2 minutes STEP 3 (1 cycle) 95 ° C 30 seconds 75 ° C 5 minutes The products resulting from the amplification were analyzed by electrophoresis through a 2% agarose gel. An amplified fragment of approximately 300 base pairs in observed for each reaction. After PCR amplification of the LqhIT2 gene and the flanking regions, the 300 bp band was isolated from a 2.0% agarose gel and purified using the SpinBind® DNA recovery system (FMC, Rockland, ME) according to the manufacturer's protocol. The isolated fragment was digested with BamHI to create the 5 'and 3' cohesive ends of the synthetic oligonucleotide containing the LqhIT2 gene and the signal sequence (Figure 6, SEQ ID NO: 14). The digested fragment was then inserted into the plasmid pTZ-18R (Pharmacia, Piscataway, NJ), at the BamHl cloning site using normal molecular cloning techniques. After transformation of DH5aMCR from E. coli, isolated colonies were chosen and plasmid DNA was prepared. Eight positive clones were identified and sequenced with the commercially available forward and reverse primers of pTz-18R. A clone (No. 16) was found to contain the correct sequence encoding the synthetic gene and the signal sequence. The resulting plasmid (pTZ-18RLq) contained two BamH1 restriction sites: one site near the 5 'end of the toxin gene and the other site after the terminator codon. Plasmid DNA of pTZ: 18RLq was prepared according to the normal protocols, and BamH1 was digested to release the inserted 300 base pair fragment containing the LqhIT2 gene and the signal sequence. This fragment was separated from the vector sequences by electrophoresis through a 1.2% agarose gel, and purified using a SpinBind® DNA recovery system. The isolated fragment was then inserted into the BamH1 cloning site of ptetophGH using normal molecular cloning techniques. After transformation into E. coli DH5aMCR, isolated colonies were selected and plasmid DNA was prepared. This plasmid was then digested with the restriction enzyme NotI, releasing a 750 bp fragment which was isolated from a 2.0% agarose gel and purified using a SpinBind® DNA recovery system. This fragment contains the sites of the tet operator, and minimal hGH promoter and the LqhIT2 / guide gene sequence. The isolated and digested fragment was then inserted into the new baculovirus transfer vectors, TV3tTa (supra), at the Notl cloning site, using normal molecular cloning techniques. After transformation into E. coli DH5aMCR, the isolated colonies were chosen and the plasmid DNA was analyzed by restriction enzyme analysis. Several colonies containing the Notl fragment were isolated, propagated and the plasmid DNA was prepared by co-transfection. The new transfer vector was marked TV3tTaTo-LqhITT (see Figure 7). Spodoptera frugiperda (Sf-9) cells were propagated in the ExCellR 401 medium (JRH Biosciences, Lenexa, KS) supplemented with 3.0% fetal bovine serum, L? Pofectin R (50 μL at 0.1 mg / mL, Gibco / BRL) TV3TaTo-LqhIT2 (500 ng) and AcNPV negative to polyhedrin, linearized (2.5 μg), Baculogold® viral DNA, Pharmigen, San Diego, CA) were added to an aliquot of 50 μL. The Sf-9 cells (50% monolayer) were co-transfected with the viral DNA solution / transfer vector. The supernatant fluid from the co-transfection experiment was collected 5 days after transfection and the recombinant viruses were isolated using normal plaque purification protocols, where only polyhedrin-positive plaques were selected (6). The isolated plates were collected and dispersed in 500 μL of the complete ExCell ™ medium coi. 2.5% fetal bovine serum. Sf-9 cells in 35 mM petri dishes (50% monolayer) were isolated with 100 μL of the viral suspension, and supernatant noises were collected at 5 days after infection. These supernatant fluids were used to inoculate cultures for the large-scale propagation of recombinant virus. The expression of LqhIT2 encoded by the recombinant baculovirus AcTV3TaTo-LqhIT2 is confirmed by immunoblot analysis and bioassay. Sf-9 cells (50 mL) are infected with wild type AcNPV (negative control), AcLqhIT2 (positive control), or AcTV3TaTo-LqhIT2. In the absence of tetracycline or an analog, which expresses the heterologous protein efficiently in cells infected by AcTV3TaTo-LqhIT2. However, in the presence of tetracycline or an analogue, the expression of the toxin is suppressed. Subsequent traditional studies were carried out using different concentrations of tetracycline added to the medium in order to determine the ability of the substrate to effectively control the expression of the heterologous protein. At several intervals after infection, affected cells were collected and the growth medium was removed by centrifugation of the cell suspensions. The spent culture medium was decanted and the remaining cells were harvested with a Branson Sonifier® (Model 450) for 30 seconds at setting 2. Cell debris was then removed by centrifugation at 15,000 rpm for 10 minutes in a refrigerated microcentrifuge (4 ° C). ). Protein concentrations of sound-treated products (cellular, infected were quantified using the BCA Protein Assay (Pierce, Rockford, IL) according to the manufacturer's instructions. A normal curve is prepared based on the known concentrations of the bovine serum albumin samples and the standards are incubated for 30 minutes at 37 ° C, and the absorbance is measured at 562 with a spectrophotometer. The protein concentrations for each sample are determined by linear regression analysis. Individual proteins in quantified samples are prepared by protein electrophoresis. The samples are diluted to 3-4 mg / mL of protein with deionized water. Twenty-five mL of sample cad is added to 75 μL of the electrophoresis sample buffer (3.8 mL of deionized H20, 1.0 mL of 0.5 M Tris-HCl, pH 6.8, 0.8 mL of glycerol, 1.6 mL of 10% SDS (p. / v), 0.4 mL of ß-mercaptoethanol, 0.4 mL of 0.5% bromophenol blue) Molecular weight standards are prepared by diluting 1 μL of the molecular weight standards of biotinylated protein (Gibco) in 100 μL of the buffer Samples and standards are heated at 95 ° C for 2 minutes Samples are loaded (20 μL / well) into 15% Mini-Protean II Tris-HCl Ready Gel® (Bio-Rad, Melville, NY). The electrophoresis run buffer (3 g of Tris base, 14.4 g of glycine, 1.0 g of SDS in 1 L of deionized H20) is added to the assembled electrophoresis gel apparatus and the samples are electrophoresed for approximately 1.5 h 40 mA After the electrophoresis, the separated proteins were transferred from the electrophoretic gel to a nitrocellulose filter by Western transfer. Blotting paper (3 MM, Schleicher and Schuell, Keene, NH) and nitrocellulose (BA-S NC; Schleicher and Schuell) are cut to the approximate dimensions of the gel and soaked in the western blotting buffer (11.6 g of Tris base, pH 8.3, glycine 5.8, SDS 0.74, 400 mL of methanol, 1.6 L of deionized H20). . The blotting paper, nitrocellulose and gel are assembled in the following sequence: 3 sheets of blotting paper, gel, nitrocellulose and 3 pieces of blotting paper. This "sandwich" is placed in the transfer apparatus such that the gel is oriented towards the cathode and the nitrocellulose membrane towards the anode. The proteins are transferred by applying a current to the transfer apparatus for 4 h at 60 mA. After the transfer, the apparatus is disassembled and the nitrocellulose filter is allowed to air dry. Immunoblot analysis of transferred proteins proceeds by the following steps, all of which are performed on a rotary shaker at room temperature. Sites in the nitrocellulose membrane that are not occupied by the transferred proteins are blocked with 3% (w / v) gelatin dissolved in TTBS (20 mM Tris, 500 mM NaCl, pH 7.5, 0.05% Tween 20) by incubation for 30 minutes. The blocking solution is removed and the membrane is rinsed in 100 mL of TTBS for 5 minutes. The rabbit polyclonal antibody specific for LqhIT2 (obtained from Dr. Bruce Hammock, University of California, Davis, CA) is prepared by adding 10 μL of rabbit antibody to 10 mL of TTBS. This primary antibody solution is applied to the blocked nitrocellulose filter which is incubated for 1 hour. After this incubation, the nitrocellulose filter washes in 3 changes of 100 mL of TTBS, each wash in the last 10 minutes. The secondary antibody is prepared by adding 10 μL of conjugated goat anti-rabbit IgG, with peroxidase (Sigma, St. Louis, MO) and 10 μL of streptavidin labeled with peroxidase to 20 mL of TTBS. The nitrocellulose filter is conjugated with this solution for 1 h. After this trituration, the filter is washed in 3 changes of 100 mL of TTBS, each washing lasting 10 minutes. The detection reagent (ECL Detection Equipment, Amersham, Arling Heights, IL) is applied to the nitrocellulose and incubated for 60 seconds. The detection reagent is drained from the filter, and the filter is covered with a Saran wrap Wrap. The signal from the nitrocellulose filter is detected by arranging the membrane processed with the X-ray film (Kodak X-OMAT AR, Rochester, NY) for 2-10 seconds. The isolated products provide a positive response to the immunoblot near 7,000 Mr and are considered positive for the toxin gene. The conditions described above for carrying out the immunoblot procedure were developed using the native LqhIT2 toxin, where the limit of detection was determined to be 15 mg of toxin. In the experiments using sound treated products from infected cells, the LqhIT2 toxin encoded for the recombinant baculovirus AcTV3TaTo-LqhIT2 was not detected, suggesting that 1 'amount of toxin expressed in the infected cells and present in the tested samples was below the limit of the detection of the trial.

EXAMPLE 2 A new transactivator gene hybrid was constructed which contained the tetR gene fused in structure with the activation domain of the AcMNPV gene IE-1. The tetR gene portion of tTA was amplified by PCR using the following oligonucleotide primers.

TETR'l 5'-ATGTCTAGATTAGATAAAAG-3 '(SEQ ID NO: 15) TETR2 5' -AGATCTGGACCCACTTTACATTTAAG-3 '(SEQ ID NO: 16) The DNA template for this reaction was plasmid TV3tTa. The following reaction mixture was used for this PCR reaction 33 μl of deionized H20, sterile 10 μL of 5X Taq buffer (In Vitrogen) 5 μL of 10X dNTP concentrated solution (2.5 mM of each dNTP 1 μL of oligonucleotide TETR1 ( SEQ ID NO: 15) (100 pmol) 1 μL of oligonucleotide TETR2 (SEQ ID NO: 16) (100 pmol) 1 μL of plasmid DNA TV3tTa (10 ng) 0.2 μL of AmpliTaq Polymerase (5 units / μL).

PCR reactions were carried out according to the following amplification protocol. STEP 1 (1 cycle) 95 ° C 1 minute 72 ° C 4 minutes STEP 2 (30 cycles) 95 ° C 30 seconds 45 ° C 30 seconds. 72 ° C 30 seconds STEP 3 (1 cycle) 95 ° C 30 seconds 45 ° C 30 seconds 75 ° C 5 minutes The resulting 622 base pair product (SEQ ID NO: 17) contains the tetR gene of +1 relative to the transductional start codon (ATG) at + 616 base pairs of tTA. An extra BglII restriction site was incorporated at the 5 'end of the TETR2 oligonucleotide for the future insertion of the IE-1 activation domain at this site. Two oligonucleotide primers, IE1A1 5'-CGGGATCCATGACGCAAATTAATTTTAACG-3 '(SEQ ID NO: 18); and IE1A2 5 '-CCAGATCTTTAACCTTGTGAATTGTCCAAGTATTC-3' (SEQ ID NO: 19). To amplify a fragment of 456 base pairs (SEQ ID NO: 20) from the plasmid pIElH / C (58) which corresponds to the first 145 amino acid residues of IE-1 (IE1A). This region has been reported to represent the activation domain of IE-1 (59). A BamHl or BglII site (incorporated in the 5 'end of IE1A1 and IE1A2, respectively) was added at the ends of IE1A during amplification to facilitate insertion into the structure of IE1A with tetR (see below). The conditions for PCR amplification of the activation domain of IE-1 were identical to those described above for the tetR gene. Both PCR amplification products, tetR and IE1A, were cloned directly into the plasmid vector PCRII (Invitrogen, San Diego, CA) using the TA cloning kit (Invitrogen) resulting in the ptetR and pIElA plasmids, respectively. Several potential clones of ptetR were digested with BamHl and BglII to identify those clones in which tetR was cleaved in the 5 'direction by BamHI and in the 3' direction by BglII. The fidelity of both tetR and IE1A were confirmed by the analysis of the sequence. The final hybrid transactivator gene was constructed by digesting pIElA with BamHl and BglII and inserting the 454 bp fragment containing IE1A into linearized ptetR by partial digestion with BglII. Corrected clones in which IE1A was inserted in the same orientation as tetR were identified based on digestions using BamHl and BglII. Only those clones containing tetR inserted in the structure with IE1A generated a fragment of 1080 base pairs. The resulting plasmid, pTERRIElA, contains a hybrid gene consisting of the tetR gene fused in the structure with IE1A (SEQ ID NO: 21, Figure 8). The binding between tetR and IE1A was confirmed by sequence analysis. Also, a BamHI fragment containing the SV40 polyadenylation sequence (SEQ ID NO: 22) was immediately cloned in the 3 'direction of the tetRIElA gene in the BglII site. In addition to the change of the activation domain of VP16 with the analogous domain of IE1, another modification to the system included the substitution of the minimal promoter of human growth hormone (hGH) by the p35 minimal promoter of AcMNPV. This promoter contains both initial and final transcriptional elements but is predominantly an initial promoter (60). Two complementary oligonucleotides (200 pmol), P35PR01 (SEQ ID NO: 23) and P35PR02 (SEQ ID NO: 24) were fixed in 10X ligase buffer (New England Biolabs Inc.) by heating at 100 ° C for 5 minutes and gradual cooling to room temperature over a period of time. of 1 hour. The resulting fixed products represent the p35 minimal promoter from -8 to -57 bp relative to the transduction initiation codon of the p35 gene (Figure 9). The oligonucleotides were designed such that after fixing each other, the ends are compatible with Sstl; however, only the promoter 3 'of the promoter actually regenerated an Sstl site after cloning into an Sstl site. The p35 minimal promoter was cloned into the Sstl site of the plasmid ptetophGH resulting in the substitution of the p35 promoter for the hGH promoter, and placing the p35 minimal promoter under the transcriptional regulation of the tetop sequences. The correct orientation of the p35 promoter relative to the tetop sequences was confirmed by digestion with SstI and sequence analysis. The resulting plasmid was named ptetopp35.

Similarly, a modified p35 minimal promoter was constructed by jointly attaching another set of oligonucleotides, P35MPR01 (SEQ ID NO: 25) and P35MPR02 (SEQ ID NO: 26), to generate a p35 promoter in which it has been incorporated a change of the individual base pair in the promoter sequence that illuminates the final TTAAG RNA start site (Figure 10). The removal of the final start site should lead to the expression of LqhIT2 or other heterologous genes under the total transcriptional control of tetRIElA and reduce the lack of system adjustment that may result from transcription from the final start site in recent times during the infection. The modified p35 minimal promoter was cloned into the SsTI site of ptetophGH resulting in the plasmid ptetopp35m. Both ptetopp35 and ptetopp35m contain the unique Ba Hl and Notl sites for the insertion of DNA into 1? 3 'direction of the p35 / p35m minimum promoters. The LqhIT2 gene and the leader sequence were isolated from pTV3TaTo-LqhIT2 by digestion with Ba Hl and a fragment of 300 base pairs containing the LqhIT2 gene and the leader sequence were subcloned into a single BglII site in both ptetopp35 and ptetopp35m . The resulting plasmids, ptetopp35L and ptetopp35mLq, now contain the LqhIT2 gene / leader sequence while immediately in the 3 'direction of the p35 / p35m promoter. Clones containing LqhIT2 in 1cl correct orientation relative to the p35 / p35m promoter were identified by digestion with Sstl. The tetopp35 / p35mLq cartridge was terminated by inserting a 204 base pair BamHl fragment containing the sequence of the SV40 polyadenylation signal (SEQ ID NO: 22) - An internal Hpal site was used to identify the clones with the sequence of polyA signal in the correct orientation in relation to LqhIT2. The final plasmid, ptetopp35 / p35mLqA, was used to deliver the tetop cartridge for insertion into the final transfer vector. The transfer vectors designed to incorporate tetRIElA and ptetopp35 / p35mLqA into the AcMNPV genome were constructed in two stages. First, the pAcP + IElTV3 transfer vector (provided by Dr. Donald Jarvis, Texas A &M University, College Station, TX) was digested with BglII and Notl (at the multiple cloning site) to linearize the vector. A 1.3 kilobase BamHI / Notl fragment derived from ptetRIElA was directly cloned into these sites in pAcP + IElTV3 by placing the transactivator gene under the transcriptional control of the hr5 / ie-l promoter. Second, the tetopp35LqA and tetopp35mLqA cartridge was inserted into a single Notl site regenerated during insertion of tetRIElA into pAcP + IElTV3. This was achieved by digesting the vector with Notl and using the large fragment of DNA polymerase I (Klenow) for filling in the 5 'overhang to generate the blunt ends. Similarly, ptetopp35 / p35mLqA was digested with Xhol and SstII and a 0.8 kbp fragment containing the tetopp35 / p35mLqA cartridge was blunt-ended using T4 DNA polymerase. This fragment was then inserted into the blunt-ended Notl site. The clones containing each cartridge in both orientations relative to the tetRIElA gene were chosen to generate recombinant viruses to determine the effect of targeting on the expression of the toxin gene. These clones were designated either Lq + or Lq- depending on whether the toxin gene was in the same or opposite orientation as the transactivator, respectively (Figure 11). Consequently, four different transfer plasmids (pTV31q +, pTVlq-, pTV3Mlq + and pTV3Mlq-) were constructed. In addition, transfer plasmids were also constructed in which the tetopp35 / p35mLqA cartridge replaced the tetop-hGH-Lq cartridge in pTV3TaTo-LqhIT2 such that the previous cartridge was now under transcriptional control of the original transactivator tetR-VP16 (tTA) system (Fig. 12). These plasmids were constructed in a similar manner as described for pTV3Lq + and pTV3Lq-, and were used to finally compare the efficacy of alternative transactivators in their ability to be deregulated by tetracycline. Specifically, the blunt-ended Xhol / SstII fragment of 0.8 kbp (see above) was cloned in both directions in pTV3TaTo-LqhIT2 after the vector was digested with NotI (which released the tetop-hGH-LqhIT2 cartridge) and became blunt ends with Klenow. The four resulting transfer plasmids contained tetopp35LqA or tetopp35mLqA in both orientations relative to tTA. These transfer plasmids were ptTAlq +, ptTAlq +, ptTAMlq + and ptTAMlq-. The recombinant viruses were generated by co-transfecting 1 μg of a specific transfer plasmid and 0.5 μg of the BaculoGold ™ viral DNA (Pharmingen, San Diego, CA >; in approximately 1.0 X 10 6 Sf-9 cells using iipofectin (Gibco / BRL, Gaithersburg, MD). Transfection supernatants were harvested after 5-7 days after transfection and used in normal plaque purification protocols. From five to six different plate isolates were selected from the first assay per plate, each plate was purified once additionally, and the virus was initially amplified by infection of approximately 1.0 x 10 6 SF-9 cells in 35 mm culture dishes . The concentrated solution of the resulting virus was used to generate concentrated working solutions in the 75 cm2 culture flasks. The eight recombinant viruses produced in this manner are found in Table 1.

Table 1. Recombinant viruses expressing LqhIT2 under different transactivators.

Virus Characteristics 1 vTV31q + Contains the transactivator repressor -HR5 with the operator cartridge / p35 / lq in the same orientation 5'-3 '. 2 vTV31q + Same as (1) but with op / p35 / lq in the opposite direction.

Virus Characteristics 3 vTV3Mlq + Same as (1) but with a modified p35 promoter with the final expression removed. 4 vTV3Mlq- Same as (3) but with the cartridge lq in the opposite direction. 5 vtTAlq + Contains the tet-transactivator repressor VP16 (cartridge lq in the same orientation 5 '-3'). 6 vtTAlq- Same as (5) but with the cartridge lq in the opposite direction. 7 vtTAMlq + Same as (5) but with the modified p35 promoter. vtTAMlq- Same as (7) but with the cartridge lq in the opposite direction.

The in vivo activity of isolated products selected from a few of these recombinant viruses was evaluated in feeding trials using 3rd instar Heliothis virescens larvae to determine if the active toxin was expressed during the infection and if so, if the The presence of tetracycline or an analogue (such as doxycycline or aureomycin) would increase the LT5o of the virus relative to untreated controls. In preliminary studies, several different treatments were evaluated to determine if the antibiotic was efficiently absorbed in the midgut of the insect, and if not, the ingredients in the route affected its absorption, and if the absorption could be improved by the injection. direct the antibiotic in the insect or by the topical application of the antibiotic. The larvae of H. virescens 3a chrysalis were allowed to consume a diet plug containing 2000 PIB of vTV3Mlq + for 24 hours either in the presence or absence of 0.3% doxycycline ("De", Sigma Chemical Co ", Cat. No D9891). After 24 hours, the individual larvae were transferred to the corresponding uncontaminated diet until 40 hours after infection when the larvae were treated in the following manner: A. maintained in the diet (either at 0% or 0.3%); B. injected with 4 μl of a 0.3% solution in De in H; 0. C. maintained in agar at 2% (w / v) + or - At 0.3%; or D. Topical application of 4 μL of De to 0.3% in acetone.

In treatments A, B and D, the larvae were returned to their corresponding diet after treatment. The insects were killed several times after infection by paralysis symptoms. The result of this test is summarized in Table 2 and provides evidence to suggest that the LT5o of the test virus was affected by De and that this effect is dependent on the route by which the De is introduced into the insect.

Table 2. Effect of doxycycline in LT50 of larvae of Heliothis virescens infected with vTV3Mlq + TREATMENT LT50 () A1 maintained at 0% diet 84-92 injected with De to p / 0.3% 92-108 maintained on agar p / o From 84-92 topical application 0.3% to 92-108 B2 maintained in 0.3% of diet 92 injected with De to p / 0.3% 116-135 maintained in agar + From to 0.3% 135 Topical application of From to 0.3% 108-116 1 Insects infected and maintained on a diet of 0% to 0% 2 Insects infected and maintained on a diet of 0.3%. Insects infected with vTV3Mlq + and maintained e; The presence of De either by injection or feeding on agar died more slowly than control insects not exposed to De. This effect was especially strong in agar treatments where the LT50 was increased by 45 to 60%. In vivo activity in selected vtTAlq +, vtTAMlq- and vTV3Mlq + viruses was also determined using bioassays by diet pills. The larvae of H. virescens from third chrysalis did feed with diet pills (volume of 50 μL) contaminated with 2000 PIB (representing approximately LC5o 20x) from vtTalq +, vtTAMlq-, vTV3Mlq +, CG201, or AcMNPV (C6) during 24 hours before these insects that had completely consumed their diet were transferred to an untreated diet (medium for soybean meal / wheat germ) (Bioserv, Cat. No. 9967)) containing tetracycline at 0.3 (p. / v) >Sigma, Cat. No. T3258) ,. doxycycline 0.3% (w / v) Ísigma, Cat. No. D9891), or aureomycin 1% (w / v) Bieserve). The antibiotics were added to the molten diet once the temperature of the diet was below 50 ° C. the amount of antibiotic used in the diet was selected to maximize exposure to the antibiotic without having an adverse effect on the insects (ie, atrophy). The insects were incubated at 28 ° C and were marked by symptoms of viral intoxication / infection at various times after infection and the LT50 of each virus was determined for each antibiotic treatment (Table 3) using the VISTAT / TIME analysis, of the Time Response Data Program (Boyce thomson Institute at Cornell University 1990). CG201 is a recombinant virus that expresses LqhIT2 under the control of the hr5 / ie-l promoter without the transactivator system of tetracycline.

Table 3. LT50 of recombinant transactivator viruses exposed to different tetracycline analogues Viras Treatment LT50 (-) VtTAlq + 3 Not treated 78.4 Aureomycin 95.0 Tetracycline 97.4 Doxycycline 88.5 VtTAlq + .4 Not treated 88.2 Aureomycin 99.9 Tetracycline 96.9 Doxycycline 105.4 VtTAlq +6 Not treated 66.6 Aureomycin 78.3 Tetracycline 87.4 Doxycycline 91.6 VtTAMq + 2 Not treated 89.5 Aureomycin 113.7 Tetracycline 117.2 Doxycycline 113.8 VtTV3Mlq + .l Not treated 77.3 Aureomycin 85.3 Tetracycline 79.3 Doxycycline 81.9 VtTV3 q + .5 Not treated 76.9 Aureomycin 82.3 Tetracycline 80.0 Doxycycline 75.9 CG201 No treated 52.6 Aureomycin 59.6 Tetracycline 58.4 Doxycycline 62.7 AcMNPV (C6) Not treated 97.2 Aureomycin 107.7 Tetracycline 104.7 Doxycycline 97.5 All antibiotic treatments were performed 0-3% (w / v) except Aureomycin (1%).

In general, antibiotic treatments increased the LT50 of all viruses, however, the effects of these antibiotics on vtTAlq + .6 and vtTAMlq-2 were significantly more dramatic than CG201 and AcMNPV. Specifically, these two viruses exhibited an increase of about 30 to 40% at the time of death in the presence of tetracycline or doxycycline relative to untreated control insects. This difference represented an increase of 2 to 3 times over the difference observed in insects infected with CG201. Other vtTAlq + viral isolates exhibited less dramatic increases in LT50 values relative to CG201. This variability between the products isolated from the same recombinant virus is again a result of the subtle differences in the expression of both the transactivator and the toxin for each isolated product and is a normal phenomenon observed by people using the expression vector system of baculovirus. For this reason, several silver isolated products, different for each recombinant were tested. The recombines: n-rs that perform poorer were those containing LqhIT2 under modified trans-activator transcriptional control, tetRIElA (vTV3Mlq +). The significant decrease in LT50 of these recombinants relative to wild-type AcMNPV suggests that while tetRIElA was able to transactivate the expression of LqhIT2, the expression is not repressed in the presence of the antibiotic. This is due to conformational changes not anticipated by the tet portion of the transactivator protein when the activation domain of VP16 is replaced with the activation domain of IE1 which comprises the ability of the transactivator to interact with tetracycline or an analogue. In summary, the results of the assay with the selected AcMNPV recombinants expressing LqhIT2 under the control of tetracycline-sensitive transactivator indicate that LT50 of the recombinant viruses can be considerably extended when the infection occurs in the presence of an antibiotic that can interact with the transactivator to reduce / delay the expression of a selective insect toxin.

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LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: (A) NAME: E.l. DUPONT DE NEMOURS AND COMPANY (B) STREET: 1007 MARKET STREET (C) CITY: WILMINGTON (D) STATE: DELAWARE (E) COUNTRY: USA (F) POSTAL CODE: 19898 (G) TELEE: 302-892-8112 (H) TELEFAX: 302- 773-0164 (I) TELEX: 6717325 (ii) TITLE OF THE INVENTION: PRODUCTION OF RECOMBINANT BACULOVIRUS (iii) SEQUENCE NUMBER: 26 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: 3.5 INCH DISK (B) COMPUTER: CUMPATIBLE WITH IBM PC (C) OPERATING SYSTEM: MICROSOFT WINDOWS 3.1 (D) PROGRAM: MICROSOFT WORD 6.0A (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (vi) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: 60 / 009,120 (B) DATE OF SUBMISSION: 22-DEC-1995 (viii) INFORMATION OF AGENT / LAWYER (A) NAME: FLOYD, LINDA AXAMETHY (B) REGISTRATION NUMBER: 33,692 (C) ORDER NUMBER / REFERENCE: BA-9078-A ORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear ii) TYPE OF MOLECULE: DNA (genomic; (xi) DESCRI PTION FOR SEQ. ID No: 1: CAGATCTG 8 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 407 base pairs (B) TYPE: nucleic acid _ (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 2: CTCGAGTTTA CCACTCCCTA TCAGTGATAG AGAAAAGTGA AAGTCGAGT? TACCACTCCC SO TATCAGTGAT AGAGAAAAGT GAAAGTCGAG TTTACCACTC CCTATCAGTG ATAGAGAAAA 120 GTGAAAGTCG AGTTTACCAC TCCCTATCAG TGATAGAGAA AAGTGAAAGT CGAGTTTACC 180 ACTCCCTA7C AGTGATAGAG AAAAGTGAAA GTCGAGTTTA CCACTCCCTA TCAGTGATAG_240_AGAAAAGTGA AAGTCGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCGAG 300 CTCAACAGTG GGAGAGAAGG GGCCAGGGTA TAAAAAGGGC CCACAAGAGA CCAGCTCAAG 360 GATTCCAAGG CCGCGGCCCC GAATTCGAGC TCGGTACCCG GGGATCC 407 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 3: AGCGGCCFCT 10 2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 75 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: AND (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 4: ACGATGAATT CGGATCCTAT GAAGATCCTC CTTGCTATTG CCCTTATGCT TAGCACCGTG 60 ATGTGGGTGA GCACC 75 2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 5: GACGGCTACA TCAAACGCCG CGACGGCTGC AAAGTGßCCT GCCTTATCGG C 51 ) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: ADNC (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 6: AACGAGGGCT GCGACAAAGA GTGCAAAGCC TACGGCGGCA GCTACGGCTA C 51 ) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRI PTION FOR THE SEQUENCE: SEQ. ID No: 7: TGCTGGACCT GGGGCCTCGC ATGCTGGTGC GAGGGCCCCCC CCGACGACAA A 51 INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic! (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: ACCTGGAAAA GCGAGACCAA CACCTGCGCC rAACGATCCT CTAGASTC) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 9; CACCCACATC ACGGTGCTAA GCATAAGGGC AATAGCAAGG AGCATCTTCA TAGGATCCGA 50 ATTCATCGT 69 ) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 10 AAGGCAGGCC CTTTGCAGC CGTCGCGGCG TTTGATSGAG CCGTCGGTGC 51 ) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 11 GTAGCTGCCG CCGTAGGCTT TGCAeTCTTT GTCGCAGCCC? CS5? 6CCGA T 51 ) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRI PTION FOR THE SEQUENCE: SEQ ID No: 12: GTCGGG € AGG CCCTCGCACC AGCATGCGAG GCCCCAGGTC CAGCAGTAGC G 51 ) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRI PTION FOR THE SEQUENCE: SEQ. ID No: 13: GACTCTAGAG GATCCTTAGC CGCAGGTGTT GGTCTCGCTT TTCCAGGTTT TGTC 54 ) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 243 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (i i) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 14: A? GAAGATCC TCCTTGCTAT TGCCCTTATG CTTAGCACCG TSATSTSSST - AGGACCS & SO SGCTACATCA AACGCCSCGA eGeeTGOU - STSGCC§CC C SGTGC 120 GACAAAGAGT GCAAGGCCTA CGGCGGCAGC TACGGCTACT GCTßGACE-TG SGGCCTCGCA 180 TGCTGGTGCG AGGGCCTCCC CGACGACAAA ACCTßGAAAA GCGAAACeAA CACCTSCGGC 240 ? Aft 243 ) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 15: ATGTCTAGAT TAGATAAAAG 20 ) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 16: AGATCTGGAC CCACTTTACA TTTAAG 26 INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 623 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic:PTION FOR THE SEQUENCE: SEQ. ID No: 17 ATGTCTAGA? TAGATAAAAG TGATTAACAG CGCATTAGAG CTGCTTAATG AGGTCGGAAT 60 CGAAGGTTTA ACAACCCGTA AACTCGCCCA GAAGCTAGGT GTAGAGCAGC CTACATGTA 120 TTGGCATGTA AAAAATAAGC GGGCTTTGCT CGACGCCTTA GCCATTGAGA TGTTAGATAG_110_GCACCATACT CACTTTTGCC CTTTAGAAGG GGAAAGCTGG CAAGATTTTT TACGTAATAA 240 CGCTAAAAGT TTTAGATGTG CTTTACTAAG TCATCGCGAT GGAGCAAAAG TACATTTAGG 300 TACACGGCCT ACAGAAAAAC AGTATSAAAC TCTCGAAAAT CAATTAGCCT TTTTATGCCA 3S0 ACAAGGTTTT TCACTAGAGA ATGCATTATA TGCACTCAGC GCTGTGGGGC ATTTTACTTT 420 AGSTTGCGTA TTGfiAAGATC AASAGCATCA AGTCGCTAAA GAAGAAA € GG AAACAX? AC 490 TACTGATAGT ATGCCGCCAT TATTACGACA AGCTATCGAA TTATTTGATC ACCAA6GTGC 540 AGAGCCAGCC TTCTTATTCG GCCTTGAATT GATCATATGC GGATTAGAAA AAGAACTTAA (SQQ S TGTSAAAG GßßTCGASA TCT 623 ) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 18 CGGGATCCAT GACGCAAATT AATTTTAACG 3C ) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRI PTION FOR THE SEQUENCE: SEQ. ID No: 19: CCAGATCTTT AACCTTGTGA ATTGTCCAAS TATTC 35 INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 455 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 20: CGGGATCCAT GACGCAAATT AATTTTAACG CGTCGTACAC CAGCGCTTCG ACGCCGTCCC 60 GAGCGTCGTT CGACAACAGC TATTCAGAGT TTTGTGATAA ACAACCCAAC GACTATTTAA 120 3TTATTATAA CCATCCCACC CCGGATGCAG CCGACACGGT GATATCTGAC AGCGAGACTG 180 CGGCACGTTC AAACTTTTTG GCAAGCGTCA ACTCGTTAAC TGATAATßAT TTAGTGGAAT 240 GTTTGCTCAA GACCACTGAT AATCTCGAAG AAGCAGTTAG TTCTGCTTAT TATTCGGAAT 300 CCCTTS? GCA GCCTSTT6TG GAGCAAGCAX CGCCCAGTTC TGCTTATCAT GCGGAATCTT 360 TTGASCATTC TGCTßGTGTG AACCAACCAT CGGCAACTGG AACTAAACGG AAGCTGGACG 420 AATACTTGGA CAATTCACAA GGTTAAAGAT ACTGG 455 INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 354 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 21: Met Ser Arg Leu Aßp Lys Ser Lyß Val He Asn Ser Ala Leu Glu Leu 1 5 10 15 Leu Asn Giu Val Gly He Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln twenty-. 9 «i \ 3a0 Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr? Gp His Vll Lys As-, Lys 35 40« 5 Arg Ala Leu Leu Asp Ala Leu Ala He Glu Het Leu Asp Ar? His His 50 55 60 Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gs- Asp Phe Leu Axg 65 70 75 80 Asn Asn Ala Lys Ser Phe Arg Cys Ale Leu Leu Ser His Arg Asp Gly 85 90 95 Aia Lys Val His Leu Gly Thr Arg Pro Thr Glu Lyn Gln Tyr Glu Thr 100 IOS 110 Leu Glu Asn Gin Leu Wing Phe Leu Cys Gln Gln Gly Phe Ssr Leu Glu 115 120 125. Asn Ala Leu Tyr Ala Leu Ser Wing Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Giu Ase Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu The 145 '150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Wing He Glu Leu 165 170 175 Phe Asp His Gln Giy Aia Glu Pro Wing Phe Leu Phe Gly Leu Glu Leu 180 185 190 He lie Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser Arg 195 200 205 Ser Met Thr Gln He Asn Phe Asn Wing Ser Tyr Thr Ser Wing Ser Thr 210 215 220 Pro Ser Arg Ala Ser Phe Asp Asn Ser Tyr Ser Glu Phe Cys Aap Lys 225 230 235 240 Gln Pro Aan Aap Tyr Leu Sez Tyr Tyr? An Hia Pro Thr Pro Asp Gly 245 250 255 Wing Asp Thr Val He Sar Asp Ser Glu Thr Ala Ale Arg Ser As-. ? h * 260 265 270 Leu Ala Ser Val Asn Ser Leu Thr Asp Asn Aßp Leu Val Glu Cys Leu 275 280 285 Leu Lys Thr Thr Asp Asn Leu Glu Glu Wing Val Ser Be Wing 290 295 300 Ser Glu Ser Leu Glu Gln Pro Val Val Glu Gis. Pro Ser Pro Ser Ser 305 310 315 320 Ala Tyr His Wing Glu Ser Phe Glu Hiß Ser til Gly Val Asn Gln Pro 325 330 335 Ser Aia Thr Gly Thr Lys Arg Lys Leu Asp Glu Tyr Leu Asp Aßn Ser 340 345 350 Gln Gly ) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 205 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 22: GGATCCAGAC CATGASAACA TACATTGATG AGTTTGGACA AACCACAACT AGAATGCAGT 60 GAAAAAAATG CTTTATTTGT GAAATTTGTG ATGCTATTGC TTTATTTGTA ACCATTATAA 120 GCTGCAATAA ACAAGTTAAC AAC &ACAA? T 3C? TTCATTT TATGTTTCAG GTTCATTTTT 180 A6GTGTG6GA GGTTTTTTCG GATCC 205 ) INFORMATION FOR SEQ I D NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE: acid r .-- cie? co (C) TYPE OF RIGHT FLEECE (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 23: CTATAAATAT TCAACGTTGC TTGTATTAAG TGAGCATTTG AGCTTTACCA AGGATCCGCG 60 GCCGCAGCT 69 ) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 24: GCGGCCGCGG ATCCTTS6TA AASC CAAAT SCTCACTTAA TAjEftASCSAC STTSAATATT SO TATAGAGeT S9 ) INFORMATION FOR SEQ I D NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 25: CTATAAATA? TCAACGTTGC TTGTATTCAS TGAGCATTTG AGCTTTACCA AGGATCCGCG 60 GCCGCAGCT 69 INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 26: GCG3CCGCGG ATCCTTGGTA AAGCTCAAAT GCTCACTGAA TACAAGCAAC GTTGAATATT 60 TATAGAGCT 69 It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention.

Having described the invention as above, the contents of the following are claimed as property:

Claims (8)

1. A method for controlling the expression of an insecticidal protein, encoded by a chimeric gene, present in the genome of a recombinant baculovirus, characterized in that it comprises: (a) constructing a recombinant insect cell having a first chimeric gene comprising a first fragment of nucleic acid encoding a first promoter, the first nucleic acid fragment operably linked to a second nucleic acid fragment encoding a regulatory protein capable of affecting gene expression; (b) constructing a recombinant baculovirus expression vector having a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter that is affected by the regulatory protein encoded by the second nucleic acid fragment, the third fragment of nucleic acid bound probably? a fourth fragment of nucleic acid encoding an insecticidal protein; (c) introducing the recombinant baculovirus expression vector constructed in step (b) stably in the recombinant insect cell constructed in step (a); and (d) maintaining the recombinant insect cell created in step (c) under the conditions that support baculovirus replication, wherein the expression of the regulatory protein encoded by the second nucleic acid fragment affects the expression of the insecticidal protein .

2. A method for controlling the expression of an insecticidal protein encoded by a chimeric ger present in the genome of a recombinant baculovirus, characterized in that it comprises: (a) constructing a recombinant baculovirus expression vector having (1) a first ger. chimeric comprising a first nucleic acid fragment encoding a first promoter, the first nucleic acid fragment operably linked to a second nucleic acid fragment encoding a regulatory protein capable of affecting the expression of the gene and (2) a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter 10 which is affected by the regulatory protein, the third nucleic acid fragment operably linked to the fourth nucleic acid fragment qur encodes an insecticidal protein; (B) introducing the expression vector of the baculovirus, recombinant from step (a) stably into an insect cell; and (c) maintain the insect cell created in Step (b) under conditions that support the reflectance of the baculovirus where the expression of the regulatory protein affects the expression of the insecticidal protein. 25

3. A method for the production of insecticidal recombinant baculoviruses, characterized in that it comprises: (a) constructing a recombinant insect cell having a first chimeric gene comprising a first nucleic acid fragment encoding a first promoter, the first nucleic acid fragment linked "operably to a second nucleic acid fragment encoding a regulatory protein capable of affecting the expression of the gene; (b) constructing a recombinant baculovirus expression vector having a second chimeric gene comprising a third nucleic acid fragment that encodes for a second promoter that is affected by the regulatory protein encoded by the second nucleic acid fragment, the third nucleic acid fragment operably linked to the fourth nucleic acid fragment encoding an insecticidal protein; (c) introducing the recombinant baculovirus expression vector of step (b) into the recombinant insect cell of step (a); (d) maintaining the recombinant insect cell created in step (c) either in a cell culture in vitro or with a living insect, intact under conditions that support baculovirus replication wherein the expression of the regulatory protein encoded by the second nucleic acid fragment affects the expression of the insecticidal protein; and (e) collecting progeny viruses.

4. The method for the production of the recombinant baculovirus, insecticide, characterized in that it comprises: (a) constructing a recombinant baculovirus expression vector having (1) a first chimeric gene comprising a first nucleic acid fragment encoding a first promoter, the first nucleic acid fragment operably linked to a second nucleic acid fragment encoding a regulatory protein capable of affecting the expression of the gene, and (2) a second chimeric gene comprising a third nucleic acid fragment encoding a second promoter that is affected by the regulatory protein encoded by the second fragment of 10 nucleic acid, the third nucleic acid fragment operably linked to a fourth nucleic acid fragment encoding an insecticidal protein; 15 (b) introducing the expression vector of the recombinant baculovirus of (a) into an insect cell; (c) maintaining the insect cell produced by step (b) either in a 20 cell culture in vitro or within a living insect, intact under conditions that support the replication of the baculovirus where the expression of the regulatory protein encoded by the The second nucleic acid fragment affects the expression of the insecticidal protein encoded by the fourth nucleic acid fragment; and (d) collecting progeny viruses.

5. The method according to claim 4, characterized in that in step (a): (i) the first nucleic acid fragment operably linked to the second nucleic acid fragment encoding the regulatory protein is a tetracycline transactivating protein; (ii) the second promoter encoded by the third nucleic acid fragment comprises one or more sites of the tetracyclic operator operably linked to a minimal promoter sequence, (iii) the insecticidal protein encoded by the fourth nucleic acid nucleus is a Selective neurotoxam of insect, and where, step (c) conditions that support. The replication of the rn-r baculovirus comprises the presence of an effective amount of tetracycline to a tetracycline analog such that the tetracycline transactivator protein is unable to bind to the sites of the tetracycline operator comprising the third nucleic acid fragment and this mode is unable to induce gene expression directed by the minimal promoter sequence operably linked to the sites of the tetracycline operator.

6. A transgenic insect, characterized in that it comprises one or more recombinant insect cell, containing a chimeric gene comprising a first nucleic acid fragment encoding a first promoter, the first nucleic acid fragment operably linked to a second nucleic acid fragment that codes for a regulatory protein capable of affecting the expression of. gene directed by the second promoter.

7. The recombinant baculovirus expression vector, characterized in that it has a chimeric gene comprising a first nucleic acid fragment encoding a promoter that is affected by the regulatory protein expressed by the recombinant insect cell of claim 6, the first fragment of nucleic acid operably linked to the second nucleic acid fragment encoding an insecticidal protein.

8. The recombinant baculovirus expression vector according to claim 9, characterized in that the first nucleic acid fragment comprises one or more sites of the tetracycline operator operably linked to a minimal promoter.