WO1999058705A1 - Recombinant baculovirus-based insecticides - Google Patents
Recombinant baculovirus-based insecticides Download PDFInfo
- Publication number
- WO1999058705A1 WO1999058705A1 PCT/US1999/009914 US9909914W WO9958705A1 WO 1999058705 A1 WO1999058705 A1 WO 1999058705A1 US 9909914 W US9909914 W US 9909914W WO 9958705 A1 WO9958705 A1 WO 9958705A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- recombinant
- baculovirus
- insecticidal
- toxin
- insect
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- polyhedrin protein Late in the infection cycle (12 hours p.i.), polyhedrin protein can be detected in infected cells. It is not until 18-24 hours p.i. that the polyhedrin protein assembles in the nucleus of the infected cell and virus particles become embedded in the proteinaceous occlusions. Viral occlusions accumulate to large numbers over 4-5 days as cells lyse. These polyhedra have no active role in the spread of infection in the larva. BVs in the haemolymph multiply and spread, leading to the death of the larva. When infected larvae die, millions of polyhedra remain in the decomposing tissue, while the BVs are degraded. When other larvae are exposed to the polyhedra by ingestion of, e.g., contaminated plants or other food material, the cycle is repeated.
- An insect that is non-permissive for infection is one that cannot be productively infected at any dose; an insect that is semi-permissive for infection is one that can only be productively infected when exposed to an amount of the virus that is at least one, but more generally two or more, orders of magnitude greater than that required for productive infection in susceptible insects, i.e., permissive hosts.
- an insect that is semi-permissive for infection is one that can only be productively infected when exposed to an amount of the virus that is at least one, but more generally two or more, orders of magnitude greater than that required for productive infection in susceptible insects, i.e., permissive hosts.
- Prior to the present invention no baculovirus of the genus Nucleopolyhedrovirus had been identified for which the diamondback moth, Plutella xylostella, is a permissive host. This insect is an important pest in many vegetable crops and has developed resistance to both chemical and biological insecticides.
- Non-limiting examples of useful heterologous signal peptides include those derived from the pBMHPC-12 signal sequence from Bombyx mori; the adipokinetic hormone signal sequence from Manduca sexta; the apolipophorin signal sequence from Manduca sexta; the chorion signal sequence from Bombyx mori; the cuticle signal sequence from Drosophila melanogaster; the esterase-6 signal sequence from Drosophila melanogaster; and the sex-specific signal sequence from Bombyx mori.
- Figure 3 is a schematic illustration of the structure of pMEV modular expression vectors.
- Figure 5 is a schematic illustration of the cloning of sequences into the modular expression vectors.
- Figure 6 is an illustration of the D. melanogaster hsp70 promoter module in pMEV5 and the amplification primers used to isolate the sequence.
- AalT and the oligonucleotides and amplification primers that were used to synthesize the sequence were used to synthesize the sequence.
- Figure 9 is a schematic illustration of the structure of the pMEV/ADK modular expression vectors.
- Figure 10 is an illustration of a codon-optimized DNA sequence encoding the LqhIT2 toxin and the oligonucleotides and amplification primers that were used to synthesize the sequence.
- PxNPVs isolated, purified recombinant Plutella xylostella baculoviruses
- PxNPV refers to an isolate of baculovirus of the genus Nucleopolyhedrovirus (NPV), the wild-type version of which was isolated from infected Plutella xylostella larvae and 7 which is genetically distinct from other known baculoviruses and exhibits higher infectivity for Plutella xylostella larvae relative to other baculoviruses.
- NPV Nucleopolyhedrovirus
- the genomic sequences of recombinant PxNPVs according to the present invention exhibit at least about 90% sequence identity, and preferably at least about 95% sequence identity, with the genomic sequence of PxNPV deposited as ATCC VR-2607.
- PxNPVs encompassed by the present invention may also be identified by:
- PxNPVs of the invention typically exhibit infectivity for Plutella xylostella larvae that is at least about two orders of magnitude greater than that exhibited by the V8 strain of AcMNPV deposted as ATCC VR-2465.
- PxNPVs of the invention excluding fragments resulting from genetic alterations, exhibit restriction fragments characteristic of PxNPV, i.e. , that are present in PxNPV digests and absent from the digests produced by other baculoviral DNAs.
- the present inventors have discovered that the recombinant PxNPVs of the present invention exhibit superior insecticidal activity on Plutella xylostella larvae relative to wild-type PxNPVs and/or recombinant NPVs derived from other species of baculoviruses.
- Recombinant PxNPVs according to the present invention are PxNPVs in which one or more genetic alterations have been introduced relative to wild-type PxNPV.
- Genetic alteration refers to any change in the sequence of the PxNPV genome including, without limitation, introduction of one or more restriction sites; deletion of one or more restriction sites; modification, deletion, or duplication of one or more viral-encoded genes; and introduction of one or more genes encoding heterologous proteins, i.e., non-virally encoded proteins or proteins encoded by a different virus.
- modified PxNPVs whose genomes contain restriction sites not present in wild-type PxNPV or, conversely, are lacking one or more restriction sites present in wild-type PxNPV are encompassed by the present invention.
- restriction site refers to a nucleic acid sequence that is a recognition site for a restriction endonuclease.
- a cloning site not present in wild-type PxNPV i.e., a sequence comprising at least one unique restriction site for incorporation 8 of a heterologous gene into the PxNPV genome.
- recombinant PxNPVs have incorporated within their genome at least one heterologous gene, including without limitation genes encoding an insect-modifying substance such as, e.g. , an insecticidal toxin, a hormone, an enzyme, or a receptor.
- recombinant PxNPVs contain a heterologous gene encoding an insecticidal toxin.
- Suitable insecticidal toxins include without limitation those listed in the Table below:
- the toxin-encoding sequence is operably linked to a promoter, i.e., a promoter sequence is placed upstream of the toxin-encoding sequence so that expression of the toxin is under the control of the promoter.
- the promoter may be a baculovirus-derived promoter, such as, e.g., DA26, 35K, 6.9K, and polyhedrin (polh) promoters (O'Reilly et al. , J. Gen. Virol. 71: 1029 (1990); Friesen et al. , J. Virol. 61:2264, 1987; Wilson et al. , J. Virol. 61:661-666, 1987; Hooft van Iddekinger et al.
- a host cell promoter may be used, such as, e.g. , an insect-derived hsp70 promoter or actin promoter. Any native or synthetic promoter active in promoting transcription in target insect cells may be used.
- the DNA sequence encoding the toxin may comprise the native upstream sequence encoding the signal peptide which, in its cell of origin, directs secretion of the toxin.
- the toxin-encoding sequence may be fused in-frame with an upstream DNA sequence encoding a heterologous signal sequence, i.e.
- a sequence derived from another source including without limitation sequences derived from thepBMHPC-12 signal sequence from Bombyx mori, the adipokinetic hormone signal sequence from Manduca sexta, the apolipophorin signal sequence from Manduca sexta, the chorion signal sequence from Bombyx mori, the cuticle signal sequence from Drosophila melanogaster, the esterase-6 signal sequence from Drosophila melanogaster, and the sex-specific signal sequence from Bombyx mori, all of which are disclosed in U.S. Patent No. 5,547,871.
- the recombinant PxNPVs of the invention have incorporated within their genome a gene encoding wild-type or mutant juvenile hormone esterase (JHE), expression of which can cause irreversible termination of the feeding stage and pupation and thus result in death of the target insect.
- JHE juvenile hormone esterase
- Recombinant PxNPVs may also be produced by genetically altering a wild- type or pre-existing recombinant PxNPV strain in a manner that results in the modification of one or more viral-encoded functions.
- one or more viral genes such as, e.g. , those encoding the viral polyhedrin protein, ecdysteroid glucosyl transferase (EGT), or plO protein may be modified, deleted, or duplicated.
- viral-encoded sequences derived from other baculoviruses may also be introduced, such as, e.g., the region from the V8 strain of AcMNPV which carries a determinant that results in a faster killing phenotype, as disclosed in U.S. Patent No. 5,662,897.
- Non-limiting examples of recombinant PxNPVs according to the invention include those having ATCC deposit numbers VR-2607,VR-2608, and VR-2609.
- the present invention provides methods and compositions for the construction of recombinant PxNPVs. Any method known in the art may be used to construct recombinant PxNPVs. For example, co-infection of an appropriate host cell with two strains of PxNPV may result in homologous recombination in vivo between related sequences, resulting in the formation of a recombinant PxNPV. Similarly, homologous recombination can occur in vivo in cells co-transfected with purified PxNPV viral genomic DNA and a second nucleic acid containing PxNPV sequences. Alternatively, recombinant PxNPVs may be produced by introducing into a cell isolated viral genomic DNA which had been previously modified in vitro. In one series of embodiments, recombinant PxNPVs are formed by the use of direct ligation vectors and modular expression vectors. These components, and methods for using them to form recombinant PxNPVs, are described below.
- Direct ligation virus vectors comprise purified PxNPV viral genomic DNAs which can be used to construct recombinant PxNPV genomes by DNA ligation in vitro. Direct ligation vectors direct the production of recombinant PxNPV virions when introduced into an appropriate host cell.
- PxNPV direct ligation vectors comprise PxNPV genomic DNA that has been modified to incorporate at least one cloning site not present in wild-type PxNPV.
- a cloning site comprises one or more restriction sites which are either absent from wild-type PxNPV genome or are not found within the nucleic acid encoding or regulating an essential PxNPV viral function.
- the direct ligation vectors of the invention are engineered to delete any such restriction sites which lie outside the cloning site, so that the cloning site comprises at least one unique restriction site.
- Digestion of a direct ligation vector using one or more restriction enzymes specified by the cloning site thus produces a DNA preparation into which a heterologous nucleic acid segment can be introduced, usually in a single ligation step, without disrupting an essential viral function.
- the resulting DNA preparation may be introduced into an appropriate host cell for propagation of recombinant PxNPV.
- Direct ligation vectors simplify the production of recombinant PxNPVs by obviating the dependence on in vivo recombination events to form recombinant viruses. See, e.g. , International Patent Application WO 94/28114.
- Cloning sites for use in PxNPV direct ligation vectors are designed by first selecting one or more restriction enzymes that either (i) do not digest PxNPV DNA at all or (ii) recognize a small number of sites that do not lie within the nucleic acid encoding or regulating an essential PxNPV viral function. The selection is performed by (i) searching the PxNPV DNA sequence computationally or (ii) subjecting PxNPV DNA to digestion with the enzyme(s) and detecting the presence or absence of digestion products. If the enzyme recognizes a small number of sites, these sites may be disrupted, using conventional techniques (such as, e.g. , restriction enzyme digestion followed by blunt-ending and re- ligation) to produce PxNPV DNA that lacks the sites but supports viral replication and infectivity.
- restriction enzymes that either (i) do not digest PxNPV DNA at all or (ii) recognize a small number of sites that do not lie within the nucleic acid encoding or regulating an essential PxNPV
- the cloning site is introduced into PxNPV DNA (whether wild-type or modified as described above to inactivate one or more restriction sites) by any appropriate means, including homologous recombination in vivo or ligation in vitro.
- the cloning site includes at least non-overlapping restriction sites to allow (i) directional cloning of an insert nucleic acid and/or (ii) independent insertion of multiple insert nucleic acids.
- direct ligation vectors from PxNPV include other modifications, including without limitation those that result in inactivation of a viral gene, such as, e.g. , that encoding polyhedrin, ecdysteroid glucosyl transferase (EGT) or plO protein.
- a viral gene such as, e.g. , that encoding polyhedrin, ecdysteroid glucosyl transferase (EGT) or plO protein.
- the cloning site is introduced in a location that results in such inactivation. 13
- Modular expression vectors for use in the present invention are plasmid vectors containing an expression cassette which can be excised from the modular expression vector and ligated into the PxNPV direct ligation vectors described above.
- the expression cassette comprises, in a 5' - to - 3' direction: a promoter sequence operably linked to a 5' untranslated region (UTR) which includes the transcription start site; a sequence containing one or more restriction sites to facilitate insertion of a heterologous gene (this sequence is designated an "insertion site"); and a 3' UTR sequence containing at least a site for 3' terminal mRNA processing and polyadenylation.
- the expression cassette is flanked on either end by appropriate restriction sites compatible with a PxNPV direct ligation vector.
- Suitable promoters for use in modular expression vectors include baculovirus promoters and host cell promoters.
- Suitable baculovirus promoters include, without limitation, DA26, 35K, 6.9K, and polyhedrin (polh) promoters.
- Suitable host cell promoters include without limitation hsp70 and actin promoters, preferably derived from an insect species. Sequences "derived from" a promoter sequence encompass modifications, including deletions, insertions, substitutions and duplications, of native promoter sequences. The only requirement is that the final promoter function effectively in a target insect cell to direct the expression of the heterologous gene to which it is operably linked.
- Expression cassettes may also include sequences encoding signal sequences, which direct the secretion of the heterologous protein.
- the signal sequences may be those associated with the heterologous protein or may be derived from a different protein. Suitable signal sequences include, without limitation, those derived from the pBMHPC-12 signal sequence from Bombyx mori; the adipokinetic hormone signal sequence from Manduca sexta; the apolipophorin signal sequence from Manduca sexta; the chorion signal sequence from Bombyx mori; the cuticle signal sequence from Drosophila melanogaster; the esterase-6 signal sequence from Drosophila melanogaster; and the sex-specific signal sequence from Bombyx mori.
- a nucleic acid sequence encoding the signal peptide is inserted between the 5-UTR and the start of the mature heterologous protein.
- the junction between the 3 ' terminus of the signal peptide-encoding sequence and the start of the mature heterologous protein is designed so that insertion of the heterologous sequence results in an 14 in-frame fusion protein between the signal peptide and the heterologous sequence.
- Sequences "derived from" a known signal sequence encompass modifications, including deletions, insertions, and substitutions, of amino acid residues within the signal sequence. The only requirement is that the resulting sequence function effectively in a target insect cell to direct the secretion from the insect cell of the heterologous sequence to which it is linked.
- Heterologous sequences for use in the present invention include, without limitation, those encoding insect-modifying substances, such as, e.g., insecticidal toxins, hormones, enzymes, and receptors.
- suitable insecticidal toxins include without limitation AalT, AaHITl, AaHIT2, LqhIT2, LqqITI, LqqIT2, BjITl, BJIT2, LqhP35, Lqh ⁇ lT, SmpIT2, SmpCT2, SmpCT3, SmpMT, DK9.2, DKll, DK12, ⁇ -agatoxin, King Kong toxin, Pt6, NPS-326, NPS-331, NPS-373, Tx4(6-1), TxP-1, ⁇ -atracotoxins, ⁇ -conotoxins, ⁇ -conotoxins, chlorotoxin and ⁇ -conotoxins.
- nucleic acid sequences encoding the insect-modifying substances and/or the signal peptides may correspond to the native nucleic acid sequences encoding these peptides.
- sequences may be altered to take into account the optimal codon usage for known genes in either the virus vector (or closely related strains) and/or in the insects that are the targets of the insecticidal viruses of the invention.
- Codon optimized sequences i.e., sequences in which the nucleic acid sequence encoding a particular amino acid has been modified without changing the amino acid encoded at that position, may be designed using methods well-known in the art, such as, for example, by comparing codon usage in known gene sequences in the virus and/or in the target insect and in the nucleic acid sequences encoding the signal peptides and insect-modifying substances of the invention.
- codon usage in the sequences encoding the signal peptides and insect-modifying substances reflects the codon usage of the virus vector or the target insect.
- Recombinant PxNPVs may be produced by either (i) co-transfecting PxNPV DNA and a heterologous sequence into an appropriate host cell, to allow for homologous recombination in vivo or (ii) ligation in vitro of a heterologous sequence into a direct ligation vector, followed by introduction of the construct into an appropriate host cell to 15 allow for viral propagation.
- Appropriate host cells are any cells that support baculovirus replication, including without limitation Sf9 cells, Sf21 cells, and High FiveTM cells (Invitrogen, Carlsbad CA).
- An isolated virus according to the invention is one which has been cloned through plaque purification in tissue culture, for example, or otherwise prepared from a single viral genotype.
- a modular expression vector is constructed to contain an expression cassette in which a suitable promoter sequence is operably linked to a sequence encoding a heterologous protein, i.e. , expression of the heterologous protein is placed under the control of the promoter.
- the expression cassette is excised from the modular expression vector and inserted into a PxNPV direct ligation vector by DNA ligation in vitro.
- the ligation mixture is then transfected into an appropriate host cell.
- Recombinant PxNPVs are recovered from the growth medium and characterized for LC 50 and LT 50 using any conventional method, including without limitation diet overlay assays, diet incorporation assays, and leaf dip assays.
- LC 50 is the concentration of virus at which 50% of infected larvae are dead within the duration of the test period.
- LT 50 is the time after infection when 50% of the infected larvae are dead when exposed to a specified dose of virus.
- PxNPVs according to the invention exhibit an LC 50 of about 1 x 10 5 OBs/ 16cm 2 or less on Plutella xylostella larvae when measured using the standard diet overlay assay as described in Example 6 below.
- Other baculovirus isolates typically exhibit higher LC 50 s on Plutella xylostella larvae, i.e. , they are less efficacious, relative to their infectivity for other insect species.
- the present invention provides insecticidal compositions and formulations that include one or more recombinant PxNPVs.
- the recombinant PxNPVs of the invention kill Plutella xylostella larvae more effectively than wild-type PxNPV or recombinant versions of other baculoviruses (see, e.g., Example 11 below).
- An insecticidal composition according to the invention includes at least one recombinant PxNPV.
- An insecticidal formulation comprises at least one recombinant PxNPV in an insecticidalry effective amount and an agriculturally suitable carrier.
- An insecticidally effective amount is an amount that causes a detectable reduction in the 16 infestation, as manifested in the number or amount of the insect pests in a given area or amount of a crop ; the damage caused by the insect pests ; or any other appropriate parameter of infestation.
- the formulations may be in the form of wettable powders, dispersible granular formulations, granules, suspensions, emulsions, solutions for aerosols, baits, and other conventional insecticide preparations.
- Suitable carriers are, without limitation, water, alcohol, hydrocarbons or other organic solvents, or a mineral, animal, or vegetable oil, or a powder such as talc, clay, silicate, or kieselguhr. Wetting agents, coating agents, UV protectants, dispersants, and sticking agents may also be included. A nutrient such as a sugar may be added to increase feeding behavior and/or attract insects. Flow agents such as, for example, clay-based flow agents, may be added to minimize caking of wettable powders or other dry preparations. The compositions may be formulated as coated particles or as microencapsulated material.
- formulations must be non-phytotoxic and not detrimental to the integrity of the recombinant PxNPV contained therein, nor should any components significantly deter insect feeding or any viral functions.
- Exemplary formulations are disclosed in EP published application 0697 170 Al ; PCT application WA 92/19102; and U.S. Patent No. 4,948,586.
- the insecticidal formulations of the invention may also include one or more chemical insecticides and/or one or more non-PxNPV biological control agents.
- Chemical insecticides include without limitation pyrethroids, pyrazolines, organophosphates, carbamates, formadines, and pyrroles, all of which are well-known in the art. Exemplary compounds are disclosed in PCT applications 96/03048, 96/01055, and 95/95741.
- Biological control agents include, e.g. , non-PxNPV baculoviruses (native or recombinant); Bacillus thuringiensis; Nosema polyvora; M.
- the present invention also provides methods for killing insect pests.
- the methods comprise contacting the insects with an insecticide-effective amount of the compositions or formulations of the invention.
- the invention also provides methods for reducing insect infestation of, e.g. , a crop, which comprise administering to a desired locus an insecticidally effective amount of the compositions or formulations of the invention.
- the insecticidal formulations are administered using conventional techniques, such as, e.g. , spraying or dusting crops. Typically, the formulations are administered at dosages of between about 2.4 X 10 8 and about 2.4 X 10 12 OBs/hectare (OBs are occlusion bodies). 17
- Effective dosages depend on, for example, the insect target, the recombinant PxNPV used, and the plant crop being treated.
- the dosages comprising an insecticidally effective amount can be determined by those of ordinary skill in the art using conventional methods.
- Example 1 Construction of Egt-Deleted ⁇ -Galactosidase-Containing Recombinant PxNPV The following experiments were performed to produce a recombinant
- a vector comprising the ⁇ -gal cassette, designated pmd 216.1, was constructed as follows.
- a Bam Hl-to-Xba I fragment containing the ⁇ -gal gene under control of the Drosophila melanogaster hsp 70 promoter was isolated from pAcDzl (Zuidema et al. , J. Gen. Virol. 71:2201 (1990)).
- oligonucleotides were synthesized to form the Bsu-Sse linker, having the sequences: 5 '- CCTCAGGGCAGCTTAAGGCAGCGGACCGGCAGCCTGCAGG -3 ' (Oligo 32) and 5'- CCTGCAGGCTGCCGGTCCGCTGCCTTAAGCTGCCCTGAGG -3' (Oligo 33) 19
- the 8.8 kb DNA band was purified by electrophoresis in a 1 % low melt preparative grade agarose gel (BioRad, Richmond VA).
- the gel slice containing the 8.8 kb linear DNA was melted at 65°C and an in-gel ligation using approximately 1/10 of the gel slice was used to recircularize the DNA, which was then used to transform E. coli DH5 ⁇ cells.
- the resulting plasmids were screened using polymerase chain reaction (PCR) to determine the orientation of the Bsu-Sse linker relative to the egt gene.
- Oligos 32 and 33 w ere s eparately paired w ith oligomer EGT 1 (5 ' - GCGGCCAATATATTGGCCGTGTTT-S'), which is specific for the region of the egt gene 5 ' to the deletion.
- the orientation of the Bsu-Sse linker in LAB 50.2 is indicated in Figure 2.
- the polylinker module is designed to allow placement of a protein coding region immediately adjacent to the 5 ' UTR of the promoter module without the introduction of extraneous linker sequences.
- the polylinker contains an Esp3 I site which is positioned so that digestion of the vector with Esp3 I cuts between positions -4 and -5 in the top strand of the promoter module and at the junction between the promoter and polylinker modules in the bottom strand.
- Treatment of Esp31-digested DNA with DNA polymerase in the presence of the four standard 2 ' -deoxynucleoside triphosphates (dNTPs) creates a linearized vector that is blunted at the exact 3 ' terminus of the promoter 21 module.
- This segment can be joined to a 5' blunt-ended fragment whose sequence begins with the ATG initiation codon of the desired protein coding region.
- the 3' terminus of the fragment is constructed so that it contains a recognition site for one of the enzymes that cleave within the polylinker module (illustrated with BamH I in Figure 5) and both the protein coding fragment and vector are digested with this enzyme prior to ligation.
- pMEV5 and pMEV6 Two vectors, pMEV5 and pMEV6, were constructed to incorporate a D. melanogaster hsp70 (major heat shock) gene promoter.
- pMEV5 contains a 724 bp segment of the D. melanogaster hsp70 promoter/5' UTR (designated hsp70Bam in Figure 4) extending from position -493 to position +231 with respect to the transcription start site of the hsp70 gene.
- pMEV6 contains a 475 bp segment of the same promoter/5' UTR (designated hsp70Xba in Figure PD2), extending from position -244 to position +231.
- each primer contains sequences homologous to the phcHSP70PL template and defines one of the boundaries of the hps70- specific sequences in each module. 22
- the Pst I/Xho I fragment representing the 5' segment of the hsp70Bam/ADK module was combined with the Xho I/Xba I fragment representing the 3' segment of the hsp70Bam/ADK module and ligated into a Pst I/Xba I vector fragment prepared from pMEVl.
- the DNA was digested with Xma I and the 5' -blunt/3 '- Xma I tox34 coding region fragments (designated tox34, tox34L or tox34S) were purified and cloned into a MEVS vector prepared as described in Example 3, except that the polylinker was cleaved with Xma I (which cleaves at the Sma I site) rather than with BamH I.
- the desired product was isolated from this mixture by PCR, using 0.5 ⁇ l of the ligation reaction as a source of template and oligonucleotides LqhIT2 PCRF (phosphorylated at its 5' terminus) 28 and LqhIT2 PCRR as primers ( Figure 10). Amplification was carried out for 25 cycles of 1 min at 94°C, 1.5 min at 55°C and 3 min at 72°C, as described in Example 3.
- the DNA was cleaved with BamH I, which cuts in the linker segment adjacent to the termination codon, and the desired 118 bp 5 ' -blunt/3 ' -BamH I fragment was purified by gel electrophoresis and cloned into MEVS vectors 29 pMEVl/ADK, pMEV2/ADK, pMEV5/ADK and pMEV6/ADK.
- the sequence of the ⁇ - ACTX-HV1 coding region in each vector was confirmed by DNA sequencing.
- Example 1 The culture was refed with fresh medium 24 hours after transfection. After an additional 72 hours, the culture supernatant was harvested, diluted, and used to re-infect
- the data indicate that the increased efficacy of recombinant PxNPVs is not limited to AalT-expressing constructs.
- the tox34 expressing PxNPVs exhibit as low an
- LT 50 and LC 50 as the AalT-expressing recombinant.
- LC 50 and LC 50 as the AalT-expressing recombinant.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Insects & Arthropods (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Pest Control & Pesticides (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Agronomy & Crop Science (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Description
Claims
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99921724A EP1076717A1 (en) | 1998-05-08 | 1999-05-07 | Recombinant baculovirus-based insecticides |
BR9910515-2A BR9910515A (en) | 1998-05-08 | 1999-05-07 | Recombinant baculovirus, recombinant baculovirus insecticides, direct binding vector and method for killing insects. |
IL13940199A IL139401A0 (en) | 1998-05-08 | 1999-05-07 | Recombinant baculovirus-based insecticides |
CA002331853A CA2331853A1 (en) | 1998-05-08 | 1999-05-07 | Recombinant baculovirus-based insecticides |
AU38856/99A AU753930B2 (en) | 1998-05-08 | 1999-05-07 | Recombinant baculovirus-based insecticides |
KR1020007012790A KR20010043627A (en) | 1998-05-08 | 1999-05-07 | Recombinant Baculovirus-Based Insecticides |
NZ507918A NZ507918A (en) | 1998-05-08 | 1999-05-07 | Recombinant baculovirus-based insecticides |
JP2000548496A JP2002514435A (en) | 1998-05-08 | 1999-05-07 | Insecticides based on recombinant baculovirus |
HU0101684A HUP0101684A3 (en) | 1998-05-08 | 1999-05-07 | Recombinant baculovirus-based insecticides |
SK1685-2000A SK16852000A3 (en) | 1998-05-08 | 1999-05-07 | Recombinant baculovirus-based insecticides |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US8470598P | 1998-05-08 | 1998-05-08 | |
US60/084,705 | 1998-05-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999058705A1 true WO1999058705A1 (en) | 1999-11-18 |
Family
ID=22186700
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/009914 WO1999058705A1 (en) | 1998-05-08 | 1999-05-07 | Recombinant baculovirus-based insecticides |
Country Status (16)
Country | Link |
---|---|
EP (1) | EP1076717A1 (en) |
JP (1) | JP2002514435A (en) |
KR (1) | KR20010043627A (en) |
CN (1) | CN1310767A (en) |
AU (1) | AU753930B2 (en) |
BR (1) | BR9910515A (en) |
CA (1) | CA2331853A1 (en) |
HU (1) | HUP0101684A3 (en) |
ID (1) | ID28101A (en) |
IL (1) | IL139401A0 (en) |
NZ (1) | NZ507918A (en) |
PL (1) | PL345242A1 (en) |
SK (1) | SK16852000A3 (en) |
TR (1) | TR200003245T2 (en) |
WO (1) | WO1999058705A1 (en) |
ZA (1) | ZA200006301B (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001059146A1 (en) * | 2000-02-10 | 2001-08-16 | University Of Connecticut | Insecticidal compounds and methods for selection thereof |
JP2005531576A (en) * | 2002-05-28 | 2005-10-20 | エムディー バイオアルファ カンパニー リミテッド | Fractions with anticancer and antimetastatic activity from carrot leaves and stems |
WO2007035382A2 (en) * | 2005-09-16 | 2007-03-29 | University Of Connecticut | Acaricidal compositions and methods of use thereof |
US7279547B2 (en) | 2000-02-10 | 2007-10-09 | University Of Connecticut | Insecticidal compounds and methods for selection thereof |
WO2011099541A1 (en) | 2010-02-12 | 2011-08-18 | 大塚製薬株式会社 | Method for producing recombinant virus |
CN103409430A (en) * | 2013-08-19 | 2013-11-27 | 南京师范大学 | Diamondback moth ubiquitin gene UBL40 and applications thereof in treating deltamethrin resistance of diamondback moth |
US20140366227A1 (en) * | 2011-03-31 | 2014-12-11 | The Food and Environment Research Agency (FERA), representing the Sec.of State for Environment, etc | Pesticides |
US11198711B2 (en) | 2013-12-11 | 2021-12-14 | University Of Durham | Pesticidal fusion protein improvements |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1313604C (en) * | 2004-09-22 | 2007-05-02 | 山西大学 | Recombined rhabdovirus containing double valence insect resisting gene |
CN105441469B (en) * | 2016-01-15 | 2018-08-07 | 西南大学 | Recombinate insect moulting hormones inactivated gene Bbsp::Egt and its disinsection fungal agent |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5461032A (en) * | 1991-03-01 | 1995-10-24 | Fmc Corporation | Insecticidally effective peptides |
US5674485A (en) * | 1988-11-01 | 1997-10-07 | The Regents Of The University Of California | Insect diagnostic and control compositions with truncated JHE |
US5695959A (en) * | 1993-07-07 | 1997-12-09 | Fmc Corporation | Recombinant expression of insecticidally effective spider toxin |
US5741669A (en) * | 1992-01-24 | 1998-04-21 | Fmc Corporation | Insecticidally effective peptides |
-
1999
- 1999-05-07 PL PL99345242A patent/PL345242A1/en unknown
- 1999-05-07 SK SK1685-2000A patent/SK16852000A3/en unknown
- 1999-05-07 JP JP2000548496A patent/JP2002514435A/en active Pending
- 1999-05-07 HU HU0101684A patent/HUP0101684A3/en unknown
- 1999-05-07 TR TR2000/03245T patent/TR200003245T2/en unknown
- 1999-05-07 NZ NZ507918A patent/NZ507918A/en unknown
- 1999-05-07 ID IDW20002404A patent/ID28101A/en unknown
- 1999-05-07 CA CA002331853A patent/CA2331853A1/en not_active Abandoned
- 1999-05-07 BR BR9910515-2A patent/BR9910515A/en not_active IP Right Cessation
- 1999-05-07 AU AU38856/99A patent/AU753930B2/en not_active Ceased
- 1999-05-07 CN CN99807162A patent/CN1310767A/en active Pending
- 1999-05-07 KR KR1020007012790A patent/KR20010043627A/en not_active Application Discontinuation
- 1999-05-07 WO PCT/US1999/009914 patent/WO1999058705A1/en not_active Application Discontinuation
- 1999-05-07 IL IL13940199A patent/IL139401A0/en unknown
- 1999-05-07 EP EP99921724A patent/EP1076717A1/en not_active Withdrawn
-
2000
- 2000-11-03 ZA ZA200006301A patent/ZA200006301B/en unknown
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5674485A (en) * | 1988-11-01 | 1997-10-07 | The Regents Of The University Of California | Insect diagnostic and control compositions with truncated JHE |
US5461032A (en) * | 1991-03-01 | 1995-10-24 | Fmc Corporation | Insecticidally effective peptides |
US5741669A (en) * | 1992-01-24 | 1998-04-21 | Fmc Corporation | Insecticidally effective peptides |
US5695959A (en) * | 1993-07-07 | 1997-12-09 | Fmc Corporation | Recombinant expression of insecticidally effective spider toxin |
Non-Patent Citations (1)
Title |
---|
PADMAVATHAMMA K, VEERESH G K: "EFFECT OF LARVAL AGE AND DOSAGE OF NUCLEAR POLYHEDROSIS VIRUS ON THE SUSCEPTIBILITY OF DIAMONDBACK MOTH, PLUTELLA XYLOSTELLA", ENTOMOLOGIA EXPERIMENTALIS ET APPLICATA., KLUWER ACADEMIC PUBLISHERS, DORDRECHT., NL, vol. 60, 1 January 1991 (1991-01-01), NL, pages 39 - 42, XP002919483, ISSN: 0013-8703, DOI: 10.1007/BF00164957 * |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8362201B2 (en) | 2000-02-10 | 2013-01-29 | University Of Connecticut | Insecticidal compounds and methods for selection thereof |
US7951929B2 (en) | 2000-02-10 | 2011-05-31 | Vestaron Corporation | Insecticidal compounds and methods for selection thereof |
US7173106B2 (en) | 2000-02-10 | 2007-02-06 | University Of Connecticut | Insecticidal compounds and methods for selection thereof |
WO2001059146A1 (en) * | 2000-02-10 | 2001-08-16 | University Of Connecticut | Insecticidal compounds and methods for selection thereof |
US7279547B2 (en) | 2000-02-10 | 2007-10-09 | University Of Connecticut | Insecticidal compounds and methods for selection thereof |
US6583264B2 (en) | 2000-02-10 | 2003-06-24 | University Of Connecticut | Insecticidal compounds and methods for selection thereof |
JP2005531576A (en) * | 2002-05-28 | 2005-10-20 | エムディー バイオアルファ カンパニー リミテッド | Fractions with anticancer and antimetastatic activity from carrot leaves and stems |
WO2007035382A3 (en) * | 2005-09-16 | 2007-12-06 | Univ Connecticut | Acaricidal compositions and methods of use thereof |
WO2007035382A2 (en) * | 2005-09-16 | 2007-03-29 | University Of Connecticut | Acaricidal compositions and methods of use thereof |
WO2011099541A1 (en) | 2010-02-12 | 2011-08-18 | 大塚製薬株式会社 | Method for producing recombinant virus |
US20140366227A1 (en) * | 2011-03-31 | 2014-12-11 | The Food and Environment Research Agency (FERA), representing the Sec.of State for Environment, etc | Pesticides |
US10117433B2 (en) * | 2011-03-31 | 2018-11-06 | University Of Durham | Pesticides |
CN103409430A (en) * | 2013-08-19 | 2013-11-27 | 南京师范大学 | Diamondback moth ubiquitin gene UBL40 and applications thereof in treating deltamethrin resistance of diamondback moth |
US11198711B2 (en) | 2013-12-11 | 2021-12-14 | University Of Durham | Pesticidal fusion protein improvements |
Also Published As
Publication number | Publication date |
---|---|
CN1310767A (en) | 2001-08-29 |
ZA200006301B (en) | 2001-06-06 |
NZ507918A (en) | 2002-07-26 |
HUP0101684A3 (en) | 2003-08-28 |
TR200003245T2 (en) | 2001-03-21 |
EP1076717A1 (en) | 2001-02-21 |
KR20010043627A (en) | 2001-05-25 |
HUP0101684A2 (en) | 2001-08-28 |
PL345242A1 (en) | 2001-12-03 |
ID28101A (en) | 2001-05-03 |
AU3885699A (en) | 1999-11-29 |
CA2331853A1 (en) | 1999-11-18 |
AU753930B2 (en) | 2002-10-31 |
IL139401A0 (en) | 2001-11-25 |
BR9910515A (en) | 2003-04-15 |
SK16852000A3 (en) | 2001-10-08 |
JP2002514435A (en) | 2002-05-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Szewczyk et al. | Baculoviruses—re-emerging biopesticides | |
US5266317A (en) | Insect-specific paralytic neurotoxin genes for use in biological insect control: methods and compositions | |
AU692290B2 (en) | Insect viruses, sequences, insecticidal compositions and methods of use | |
KR19990079361A (en) | Recombinant baculovirus, a method for producing the same, and microbial pesticides containing the same | |
US5547871A (en) | Heterologous signal sequences for secretion of insect controlling proteins | |
AU753930B2 (en) | Recombinant baculovirus-based insecticides | |
US6235278B1 (en) | Biological insect control agents expressing insect-specific toxin genes, methods and compositions | |
Haase et al. | Genetic engineering of baculoviruses | |
EP0621337A1 (en) | Codon optimized DNA sequence for insect toxin AaIT | |
AU743526B2 (en) | Transgenic virus | |
US6130074A (en) | Recombinant insect virus with reduced capacity for host-to-host transmission in the environment and methods to produce said virus | |
AU675939B2 (en) | Recombinant insect virus with reduced capacity for host-to-host transmission in the environment and methods to produce said virus | |
US6087165A (en) | Recombinant baculovirus and its use as a biocontrol agent for crop pests | |
MXPA00010981A (en) | Recombinant baculovirus-based insecticides | |
CZ20004156A3 (en) | Recombinant baculovirus-based insecticidal agents | |
US6355240B1 (en) | Enhanced insecticidal insect virus through the expression of heterologous proteins with early promoters | |
AU720082B2 (en) | Heterologous signal sequences for secretion of insect controlling toxins | |
Palli et al. | Biological control of forest pests: a biotechnological perspective |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 99807162.5 Country of ref document: CN |
|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 139401 Country of ref document: IL Ref document number: 507918 Country of ref document: NZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2000/03245 Country of ref document: TR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 200006301 Country of ref document: ZA |
|
ENP | Entry into the national phase |
Ref document number: 2331853 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 16802000 Country of ref document: SK Ref document number: 1999921724 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2000 548496 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: PA/a/2000/010981 Country of ref document: MX Ref document number: 16852000 Country of ref document: SK Ref document number: PV2000-4156 Country of ref document: CZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: 38856/99 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1020007012790 Country of ref document: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: IN/PCT/2000/00361/DE Country of ref document: IN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1200001119 Country of ref document: VN |
|
WWP | Wipo information: published in national office |
Ref document number: 1999921724 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: 1020007012790 Country of ref document: KR |
|
WWP | Wipo information: published in national office |
Ref document number: PV2000-4156 Country of ref document: CZ |
|
WWG | Wipo information: grant in national office |
Ref document number: 38856/99 Country of ref document: AU |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1999921724 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1020007012790 Country of ref document: KR |