CN1310767A - Recombinant baculovirus-based insecticides - Google Patents

Abstract

The present invention provides isolated recombinant Plutella xylostella baculoviruses suitable for use as insecticides. Preferably, the recombinant baculovirus of the present invention incorporates a gene encoding an insecticidal toxin into its genome. The present invention also provides pesticidal compositions and products comprising the recombinant diamondback moth baculovirus and methods for killing pests and reducing insect attack on crops.

Description

Sterilant based on recombinant baculovirus

The application requires to enjoy priority according to 35 U.S.C.119, and it is based on the provisional application series No.60/084 that proposed on May 8th, 1998, and 705, it is for referencial use that it whole openly incorporate this paper into.

The present invention relates to improvement small cabbage moth (Plutella xylostella) baculovirus as biotic pesticide.The invention provides recombinant baculovirus, this virus is handled the gene that has mixed the coded insect-killing toxin by genetically engineered.

Baculovirus is to be suitable for the insect viruses that make biotic pesticide.Baculovirus isolate more than 400 kinds has been described.Autographa california (Autographa californica) nuclear polyhedrosis virus (AcMNPV), it is the prototype virus of clover section, be isolating at first from autographa california [a kind of lepidopteran noctuid is commonly called " autographa california " (alfalfa looper)].12 sections in this virus infection lepidopterous insects and more than 30 kinds [Granados etc., baculovirus biology (The Biology of Baculoviruses), I, 99 (1986)].Do not know also whether AcMNPV can infect any kind beyond this order prolifically.

The life cycle of baculovirus (is example with AcMNPV) comprises two stages.Each stage of described life cycle is represented with specific viral form: non-blastogenesis C-type virus C body (BV) that comprises and inclusion body (OB).

In natural insect infection form, find that a lot of virosome are embedded in and be called as " inclusion body " (OB) in the paracrystalline proteic matrix of [it is also referred to as " polygonal inclusion body " (PIB)].This protein virus inclusion body is also referred to as " polyhedron ".Molecular weight is that the polyhedrin of 29kD is the main viral coding structure albumen (U.S. Patent No. 4,745,051) of viral occlusion.

Viral occlusion is the natural baculovirus integral part of life cycle, for the level (insect is to insect) between the susceptible insect kind is provided by the instrument that provides.In environment, susceptible insect (being in larval stage usually) is (for example plant) viral occlusion of ingesting from the contaminated food source.Crystalline inclusion body decomposes in the intestines of susceptible insect and discharges the infectious virus particle.These inclusion body deutero-viruses (ODV) are duplicated in the cell of midgut tissue.

It is believed that virus particle enters cell by endocytosis or fusion, and viral DNA is shelled in nucleopore or nuclear.In six hours, detect dna replication dna.Infected back (p.i.) 10～12 hours, secondary infection is diffused into other insect tissue by the budding (BV) of extracellular virus from cell surface.The BV form of virus causes the cell pair cell diffusion of virus in the infected insect of individuality, and causes infection in cell cultures.

Infectious cycle's later stage (infecting back 12 hours), can detect the polyhedrin in the infected cell.Up to infecting the back 18～24 hours, polyhedrin just assembles in the nucleus of infected cell, so virus particle is embedded in the protein inclusion body.Viral occlusion built up to big quantity in 4～5 days along with cytolysis.These polyhedroies do not have active function in the infection diffusion of larva.BVs breeds in hemolymph and spreads, and causes the death of larva.

When infected larva was dead, countless polyhedroies were retained in the tissue of decomposition, and BVs has then degraded.When other larva by ingesting for example contaminated plant or other food and when contacting, just repeat this circulation with described polyhedron.

In a word, the form that comprises of virus is to cause the reason of insect via the initial infection of enteron aisle, also is the environmental stability reason of virus.When using by injection, ODVs right and wrong basically is infective, but is hyperinfection when per os is ingested.The non-of virus comprises the infection that form (being BV) causes secondary infection and cell pair cell.When using by injection, BVs in the culture or the cell in the insect inner tissue be hyperinfection, but right and wrong are infective when ingesting.

The main inhibition of widespread usage desinsection baculovirus is initial infection of insect and the time lag between its death in agricultural.This time lag can change in the scope of a couple of days to several weeks.During this period, insect continues feed, and plant is caused further infringement.In order to shorten this retardation time, made up recombinant baculovirus, they are expressed control or modify the material of insect, for example toxin, neuropeptide, hormone or enzyme [Tomalski, M.D. etc., nature (Nature), 352:82～85 (1991); Federici, external (In Vitro), 28:50A (1992); Martens etc. use and environmental microbiology (App.﹠amp; Envir.Microbiology), 56:2764～2770 (1990); Menn etc., agricultural food product chemistry (Agric.Food Chem.), 37:271～278 (1989); Eldridge etc., insect biochemistry (Insect Biochem.), 21:341～351 (1992); Hammock etc., nature, 344:458～461 (1990)].

Second main inhibition using the desinsection baculovirus is the limited host range of virus.Though it is safe to non-target biology that the limited host range of baculovirus makes them, mean that also they can not control the open-air various lepidoptera pests that exist.When the lepidopterous insects molectron of needs control comprise for the infection of the concrete desinsection baculovirus of using be non-permission or partly during the insect of permission property, situation is especially true.To the insect that infects non-permission is a kind of insect that all can not effectively be infected at any dosage; To infecting the insect of permission property partly is a kind ofly only to cause the insect that susceptible insect (being permissive host) is just infected when effectively infecting big at least one (but the more normal two or more) order of magnitude of required amount effectively at the amount ratio of contact virus.Before the present invention, do not identify the baculovirus of such Nucleopolyhedrovirus as yet: concerning it, the diamond back moth small cabbage moth is a permissive host.This insect is the important pests in a lot of vegetable crops, has formed the two resistance of chemical insecticide and biotic pesticide.

So, need such biotic pesticide in the art: effectiveness that their performances (ⅰ) improve and shorter infection with cause death between retardation time and (ⅱ) host range improved of performance, make them can control one and organize lepidopterous insects insect (comprising for example small cabbage moth) greatly.

The invention provides reorganization small cabbage moth baculovirus (PxNPVs), this virus shows the insecticidal activity of superior anti-small cabbage moth with respect to other wild-type baculovirus and recombinant baculovirus.

Reorganization PxNPVs of the present invention is the PxNPVs that comprises one or more hereditary changes with respect to wild-type PxNPV.Hereditary change includes but not limited to: the introducing of one or more restriction sites or disappearance; The modification of one or more encoding viral genes, disappearance or repetition; And the introducing of the gene of one or more coding heterologous proteins (that is, the protein of non-encoding viral or by different virus encoded protein matter).

Preferably, reorganization PxNPVs has mixed a kind of nucleotide sequence of the material (for example Pesticidal toxins, peptide hormone, enzyme or acceptor) of modifying insect of encoding in their genome, it by operationally with can activate the target insect in the promotor of transcribing be connected.More preferably, the material of described modification insect is a Pesticidal toxins.Think that the recombinant baculovirus of coded insect-killing toxin provides the desinsection benefit of remarkable improvement under the situation of PxNPV genome background.

The limiting examples of Pesticidal toxins comprises: AaIT, AaHIT1, AaHIT2, LqhIT2, LqqIT1, LqqIT2, BjIT1, BjIT2, LqhP35, Lqh α IT, SmpIT2, SmpCT2, SmpCT3, SmpMT, DK9.2, DK11, DK12, μ-agatoxin, King Kong toxin, Pt6, NPS-326, NPS-331, NPS-373, Tx4 (6-1), TxP-1, ω-atracotoxins, alpha-conotoxin, mu-conotoxin, chlorotoxin and omega-conotoxin.In certain embodiments, used natural secreting signal peptide (that is, relevant peptide) with toxin; In other embodiments, having used the allos signal peptide promotes toxin to secrete from infected insect cell.The limiting examples of the allos signal peptide that is suitable for comprises derived from following those: the pBMHPC-12 signal sequence that derives from silkworm (Bombyx mori); Derive from the adipokinetic hormone signal sequence of maduca sexta (Manduca sexta); Derive from the apolipophorin signal sequence of maduca sexta; Derive from the chorion signal sequence of silkworm; Derive from the stratum corneum signal sequence of fruit bat (Drosophila melanogaster); Derive from esterase-6 signal sequence of fruit bat; And the property specificity signal sequence that derives from silkworm.

The present invention also comprises direct connection carrier, designs them so that connect structure reorganization PxNPV genome by external dna fragmentation.The DNA that this connection is produced introduces suitable host cell, and the result produces reorganization PxNPVs.

On the other hand, the invention provides the expression cassette of coded insect-killing toxin.This expression cassette comprises promoter sequence (preferably deriving from fruit bat hsp70 promotor), and it operationally is connected with the nucleotide sequence of toxin-encoding.Expression cassette of the present invention also can be impregnated in plasmid vector (they are called as " module expression vector ").

Aspect another, the invention provides the codon optimization gene of coded insect-killing toxin, for example those shown in Figure 10 and 11 hereinafter.Codon optimization gene is such gene: wherein, specific cryptosystem that exists in the natural toxin encoding sequence has been substituted by the replacement codon that insect cell protein synthesis device more effectively utilizes.

Aspect another, the invention provides insect-killing composition and goods, they comprise at least a above-mentioned reorganization PxNPV and agricultural goes up acceptable carrier.

Aspect another, the invention provides the method for kill insects insect and minimizing insect infestations, this method comprises: insect-killing composition insecticidal effective dose, that comprise PxNPV or goods are used in the place to expectation.

Fig. 1 is the working method synoptic diagram that is used to make up pmd205.1, pmd216.2 and pmd220.2 carrier, and described carrier is used to produce the reorganization PxNPVs of disappearance ecdysteroids Transglucosylase (egt) gene.

Fig. 2 sets forth the working method synoptic diagram that is used to make up the LAB50.2 carrier.

Fig. 3 is the synoptic diagram of setting forth the structure of pMEV module expression vector.

Fig. 4 is the synoptic diagram of setting forth the pMEV carrier that comprises different promoters.

Fig. 5 sets forth the synoptic diagram of sequence clone being gone into the module expression vector.

Fig. 6 has set forth the fruit bat hsp70 promotor module and the amplimer that is used to separate described sequence among the pMEV5.

Fig. 7 has set forth the fruit bat hsp70 promotor module and the amplimer that is used to separate described sequence among the pMEV6.

Fig. 8 has set forth the codon optimization dna sequence dna of coding AaIT and oligonucleotide and the amplimer that is used for synthetic described sequence.

Fig. 9 is the synoptic diagram of setting forth the structure of pMEV/ADK module expression vector.

Figure 10 has set forth the codon optimization dna sequence dna of coding LqhIT2 toxin and oligonucleotide and the amplimer that is used for synthetic described sequence.

Oligonucleotide and amplimer that Figure 11 has set forth the codon optimization dna sequence dna of coding ω-ACTX-HV1 toxin and has been used for synthetic described sequence.

In enforcement of the present invention, can use a lot of technology in molecular biology well known by persons skilled in the art, microbiology, recombinant DNA, protein biochemistry and insect viruses, such as illustrate in detail in the following document those: Sambrook etc., 1989, " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual), second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; " oligonucleotides is synthetic " (Oligonucleotide Synthesis), 1984, (M.L.Gait edits); Ausubel etc., " general scheme in the molecular biology " (Current Protocols in Molecular Biology), 1997 (John Wiley﹠ Sons); The series works, " method in the zymetology " (Methods in Enzymology) (Academic Press, Inc.); " protein purification: principle and experimental implementation " (Protein Purification:Principles and Practice), second edition (Springer-Verlag, N.Y.); Summers etc., " baculovirus vector and insect cell are cultivated the method handbook of operation " (A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures), Texas agricultural experiment centre communique No.1555,1987; And O ' Reilly etc., " rhabdovirus expression vector: laboratory manual " (Baculovirus Expression Vectors:A Laboratory Manual), 1994 (Oxford Univ. Press, N.Y.).

The invention provides applicable restructuring diamondback moth baculoviral separation, purifying (PxNPVs) that makes biological insecticides. The PxNPV that this paper uses represents the baculoviral separator that nuclear polyhedrosis virus (NPV) belongs to, its wild type is separated from infected diamondback moth larvae, and, it is different from other known baculoviral at genetic aspect, shows the infectivity higher concerning diamondback moth larvae (with respect to other baculoviral). Restructuring of the present invention PxNPVs genome sequence (not comprising heterologous sequence) shows as at least about 90%, preferably at least about 95% and sequence homogeneity as the genome sequence of the PxNPV of ATCC VR-2607 preservation. The PxNPVs that the present invention includes also can identify by following method:

(ⅰ) mensuration is to the infectivity of diamondback moth larvae. PxNPVs of the present invention generally shows infectivity to diamondback moth larvae and is compared to the height of V8 strain performance of AcMNPV of ATCC VR-2465 preservation at least about two orders of magnitude.

(ⅱ) use Hind III, Xho I and/or Pst I digestion virus genom DNA, the pattern of the restricted fragment pattern of generation with the genomic DNA that derives from PxNPV by digestion and those generations that derive from non-PxNPV baculoviral compared. PxNPVs of the present invention shows (not comprising the fragment that produces from hereditary change) the restricted fragment feature of PxNPV, that is, be present in the PxNPV digestive juice but be not present in the digestive juice that is produced by other baculovirus DNA s.

The inventor has found: restructuring of the present invention PxNPVs shows than wild type PxNPVs and/or derives from the superior insecticidal activity to diamondback moth larvae of restructuring NPVs of the baculoviral of other kind. Restructuring PxNPVs of the present invention is such PxNPVs: wherein, introduced one or more hereditary changes (with respect to wild type PxNPV). Any variation in the genomic sequence of " hereditary change " expression PxNPV includes but not limited to: the introducing of one or more restriction sites; The disappearance of one or more restriction sites; The modification of the gene of one or more encoding virals, disappearance or repetition; And the introducing of the gene of one or more coding heterologous proteins (that is, the protein of non-viral coding or by the protein of different virus coding). For example, the present invention includes the PxNPVs of modification, their genome comprises non-existent restriction site among the wild type PxNPV, perhaps on the contrary, lacks the one or more restriction sites that exist among the wild type PxNPV. Term " restriction site " expression nucleotide sequence, it is the recognition site of restriction endonuclease. Usually, carry out the interpolation of one or more restriction sites and/or disappearance and produce non-existent cloning site among the wild type PxNPV, that is, a kind of sequence, it comprises at least one and is used for heterologous gene is mixed the restriction site of the genomic uniqueness of PxNPV. Preferably, restructuring PxNPVs has mixed at least one heterologous gene in their genome, include but not limited to: coding is modified the gene of the material (for example Pesticidal toxins, hormone, enzyme or acceptor) of insect.

More preferably, restructuring PxNPVs comprises the gene of coded insect-killing toxin. Suitable Pesticidal toxins includes but not limited to those that following table is listed:

Toxin list of references AaIT, AaH ITl, AaH IT2 Darbon etc., international peptide and protein research magazine

                 (Int.J.Peptide Protein Res.)20:

320～330,1982; Loret etc., biochemistry

(Biochem.) 29:1492～1501,1990LqhIT2, Zlotkin etc., biological chemistry 30:4814～4821,

1991; Zlotkin etc., European patent application EP

0374753 A2,1990LqqIT1, LqqIT2 Zlotkin etc., biological chemistry and biophysics

Document (Arch.of Biochem.﹠amp; Biophys.)

240:877～887,1985; Zlotkin etc., Europe

Continent patent application EP 0374753 A2,1990BjIT1, BjIT2 Lester etc., biological chemistry and biophysics

Newspaper (Biochem.Biophys.Acta) 701:

370～387,1982; Zlotkin etc., Europe specially

Sharp application EP 0374753 A2,1990LqhP35 Zlotkin etc., European patent application EP 0374753

A2,1990Lqh α IT Eitan etc., biological chemistry 29:5941～5947,

1990SmpIT2 Zlotkin etc., European patent application EP

0374753 A2,1990SmpCT2 Zlotkin etc., European patent application EP

0374753 A2,1990SmpCT3 Zlotkin etc., European patent application EP

0374753 A2,1990SmpMT Zlotkin etc., European patent application EP 0374753

A2,1990 DK9.2 Krapcho etc., International Patent Application WO

0374753 A2,1990DK11 Krapcho etc., International Patent Application WO 92/

15195,1992DK12 Krapcho etc., International Patent Application WO 92/

15195,1992 μ-agatoxin Skinner etc., journal of biological chemistry (J.Biol.

Chem.)264:2150～2155,1989；Adams

Deng, journal of biological chemistry 265:861～867,1990King Kong toxin Hillyard etc., biological chemistry 28:358～361,

1989Pt6 Leisy etc., European patent application EP 0556160

A2,1993NPS-326 Krapcho etc., International Patent Application WO 93/

15192,1993NPS-331 Kraphco etc., International Patent Application WO 93/

15192,1993NPS-373 Krapcho etc., International Patent Application WO 93/

15195,1990Tx4 (6-1) Figueriredo etc., toxin (Toxicon) 33:

83～93,1995TxP-1 Tomalski etc., toxin 27:1151～1167,

1989 ω-atracotoxins Atkinson etc., International Patent Application WO 93/

The plain Gray of 15108,1993 α-cone shell etc., journal of biological chemistry 256:4734～

4740,1981; Gray etc., biological chemistry 23:

2796～2802,1984 mu-conotoxin Cruz etc., journal of biological chemistry 260:9280～

9288,1989; Cruz etc., biological chemistry

28:3437～3442,1989chlorotoxin Debin etc., U.S. physiology magazine (Am.J.

Physiol.) 264:361～369,1993 omega-conotoxin Olivera etc., biological chemistry 23:5087

～5090,1984; Rivier etc., biological chemistry

Magazine 262:1194～1198,1987

The toxin encoding sequence operationally is connected with promotor, that is, a kind of promoter sequence is placed the upstream of toxin encoding sequence, so, under the control of promotor, express toxin.Described promotor can be a baculovirus deutero-promotor, for example DA26,35K, 6.9K and polyhedrin (polh) promotor [O ' Reiny etc., general virology magazine (J.Gen.Virol.) 71:1029 (1990); Friesen etc., Journal of Virology (J.Virol.) 61:2264,1987; Wilson etc., Journal of Virology 61:661～666,1987; Hooft van Iddekinger etc., virusology (Virol.) 131:561,1983; And referring to Miller, baculovirus (TheBaculoviruses), Plenum Press, New York, 1997].Also can use the host cell promotor, for example, insect deutero-hsp70 promotor or actin promoter.Activated any natural or synthetic promotor all can be used in the transcribing in promoting the target insect cell.

In addition, the dna sequence dna of toxin-encoding also may comprise the natural upstream sequence of coded signal peptide, and described signal peptide instructs the secretion of toxin in its original cell.The toxin encoding sequence also can with the upstream dna sequence dna of coding allos signal sequence at the frame endomixis, described sequence promptly is the sequence that derives from other source, include but not limited to derived from following sequence: the pBMHPC-12 signal sequence that derives from silkworm, derive from the adipokinetic hormone signal sequence of maduca sexta, derive from the apolipophorin signal sequence of maduca sexta, derive from the chorion signal sequence of silkworm, derive from the stratum corneum signal sequence of fruit bat, derive from esterase-6 signal sequence of fruit bat, and the property specificity signal sequence that derives from silkworm, they all are disclosed U.S. Patent No. 5,547, in 871.

In certain embodiments, reorganization PxNPVs of the present invention has mixed the gene of encoding wild type or mutant juvenile hormone esterase (JHE) in their genome, its expression can cause the irreversible termination of the phase of ingesting and nymphosis, so cause the death of target insect.Referring to for example W094/03588.

Reorganization PxNPVs also can change wild-type in heredity by the mode by the modification that causes one or more encoding viral functions or the reorganization PxNPV strain that is pre-existing in is produced.For example, can modify, lack or duplicate one or more virogenes, the viral polyhedrin of for example encoding, ecdysteroids Transglucosylase (EGT) or p10 proteinic those.In addition, the encoding viral sequence that derives from other baculovirus also can be introduced into, the district of the V8 strain of AcMNPV for example, it carries a determiner, this determiner causes more promptly killing and wounding phenotype, as U.S. Patent No. 5,662, in 897 disclosed like that.

The limiting examples of reorganization PxNPVs of the present invention comprises that the ATCC preserving number is those of VR-2607, VR-2608 and VR-2609.

The invention provides the method and composition that is used to make up reorganization PxNPVs.Any method known in the art all can be used to make up reorganization PxNPVs.For example, proper host cell and the coinfection of two strains of PxNPV can cause between the relevant sequence homologous recombination in vivo, generate reorganization PxNPV.Similarly, can in the cell with the PxNPV virus genom DNA of purifying and the second kind of nucleic acid coinfection that comprises the PxNPV sequence homologous recombination take place in vivo.Also can produce reorganization PxNPVs by introducing cell at the isolating virus genom DNA of external modification in advance.

In a series of embodiments, reorganization PxNPVs forms by using direct connection carrier and module expression vector.Hereinafter describe these components and used the method that they form reorganization PxNPVs.The direct connection carrier of PxNPV deutero-

Directly connect the PxNPV virus genom DNA s that virus vector comprises purifying, they can be used to connect structure reorganization PxNPV genome by external DNA.When being introduced into proper host cell, directly connection carrier instructs the production of reorganization PxNPV virion.In certain embodiments, the direct connection carrier of PxNPV comprises the PxNPV genomic dna, and it has been mixed at least one and has not been present in cloning site among the wild-type PxNPV by modification.A cloning site comprises one or more restriction sites, they or be not present in the wild-type PxNPV genome, perhaps do not see the coding or regulate in the nucleic acid of basic PxNPV viral function.Under latter event, direct connection carrier of the present invention is by artificial reconstructed and lack any such restriction site (they are in the outside of cloning site), so cloning site comprises at least one unique restriction site.Use one or more restriction enzymes of representing with cloning site and digest direct connection carrier, so produce the DNA goods, (usually in a step Connection Step) introduced the heterologous nucleic acids sections and do not destroyed basic viral function therein.After heterologous nucleic acids is connected into direct connection carrier, the DNA goods that generate can be introduced proper host cell and are used for breeding reorganization PxNPV.Directly connection carrier is by exempting the reorganization PxNPVs of production simplified to(for) the dependency of recombination event in the body that forms recombinant virus.Referring to for example, International Patent Application WO 94/28114.

The cloning site that is used for the direct connection carrier of PxNPV is design like this: at first select one or more restriction enzymes, this restriction enzyme or (ⅰ) do not digest PxNPV DNA, perhaps (ⅱ) identification is not in coding on a small quantity or regulates site in the nucleic acid of basic PxNPV viral function.Be performed such selection: (ⅰ) seek the PxNPVDNA sequence by calculating, perhaps (ⅱ) with enzymic digestion PxNPV DNA, and the existence that detects digestion product more whether.If described enzyme has been discerned a small amount of site, just can use routine techniques (for example restriction enzyme digestion is then done into flush end and reconnected) and destroy these sites and produce PxNPV DNA (it lacks described site but supports virus replication and infection).

After having selected one or more restriction sites, cloning site is introduced PxNPV DNA (thereby wild-type or by above-mentioned such one or more restriction sites of deactivation of having modified) by any suitable method (comprising homologous recombination or external connection in the body).Preferably, described cloning site comprises non-overlapped at least restriction site and allows (ⅰ) directed cloning to insert fragment nucleic acid and/or (ⅱ) independent insert the fragment nucleic acid that insert more.

In order to form direct connection carrier from PxNPV, can introduce other and modify, include but not limited to cause those of inactivation of virogene, the polyhedrin of for example encoding, ecdysteroids Transglucosylase (EGT) or p10 proteinic those.In certain embodiments, cloning site is introduced the position that causes this inactivation.Module expression vector and expression cassette:

Being used for module expression vector of the present invention is the plasmid vector that comprises an expression cassette, and described expression cassette can be cut and connect into the direct connection carrier of above-mentioned PxNPV from the module expression vector.Usually, described expression cassette 5 ' comprise to 3 ' direction: a promoter sequence, this sequence are operably connected to the 5 ' non-translational region (UTR) that comprises transcription initiation site; Article one, the sequence (this sequence is called as " insertion site ") that comprises the restriction site of one or more promotion heterologous genes insertions; And 3 ' UTR sequence, this sequence comprises at least one site that is used for 3 ' terminal mRNA processing and polyadenylation.Described expression cassette all is connected with the suitable restriction site side compatible with the direct connection carrier of PxNPV at arbitrary end.

The suitable promotor that is applied in the module expression vector comprises bacilliform virus promoter and host cell promotor.Suitable bacilliform virus promoter includes but not limited to: DA26,35K, 6.9K and polyhedrin (polh) promotor.The proper host cell promotor includes but not limited to: hsp 70 and actin promoter (preferably deriving from the insect kind).The sequence that " derives from " promoter sequence comprises the modification of natural promoter sequence (it comprises disappearance, insertion, substitutes and repeats).Unique requirement is, final promotor works effectively in the target insect cell and instructs the expression of heterologous genes that it operationally is attached thereto.

Expression cassette also can comprise the sequence of coded signal sequence, the secretion of this sequence-directed heterologous protein.Described signal sequence can be and those of heterologous protein qualitative correlation, perhaps can derive from different protein.The appropriate signal sequence includes but not limited to derive from following those: the pBMHPC-12 signal sequence that derives from silkworm; Derive from the adipokinetic hormone signal sequence of maduca sexta; Derive from the apolipophorin signal sequence of maduca sexta; Derive from the chorion signal sequence of silkworm; Derive from the stratum corneum signal sequence of fruit bat; Derive from esterase-6 signal sequence of fruit bat; And the property specificity signal sequence that derives from silkworm.Article one, the nucleotide sequence of coded signal peptide is inserted between the starting point of 5-UTR and ripe heterologous protein.Joint between the starting point of 3 of modelled signal peptide-coding sequence ' end and ripe heterologous protein, thus the insertion of heterologous sequence produces the in-frame fusion rotein that is between signal peptide and the heterologous sequence.The sequence that " derives from " known signal sequence comprises the modification of amino-acid residue in the described signal sequence (it comprises disappearance, inserts and substitutes).Unique requirement is, the sequence of generation works effectively in the target insect cell and instructs the secretion from insect cell of heterologous sequence that it is attached thereto.

Be applied to heterologous sequence of the present invention include but not limited to encode those of the material (for example Pesticidal toxins, hormone, enzyme and acceptor) of modifying insect.Suitable Pesticidal toxins includes but not limited to: AaIT, AaHIT1, AaHIT2, LqhIT2, LqqIT1, LqqIT2, BjIT1, BjIT2, LqhP35, Lqh α IT, SmpIT2, SmpCT2, SmpCT3, SmpMT, DK9.2, DK11, DK12, μ-agatoxin, King Kong toxin, Pt6, NPS-326, NPS-331, NPS-373, Tx4 (6-1), TxP-1, ω-atracotoxins, alpha-conotoxin, mu-conotoxin, chlorotoxin and omega-conotoxin.

The material of coding modification insect and/or the nucleotide sequence of signal peptide can be consistent with the natural acid sequence of these peptides of coding.Also can change described sequence, consider the suitableeest codon purposes for known in the virus vector (or closely-related strain) and/or in the insect (they are targets of desinsection virus of the present invention).Can use the sub-optimizing sequence of method pin design well known in the art (is such sequence, wherein, the nucleotide sequence of coding specific amino acids has been modified but has not been changed amino acids coding on described position), for example, by password comparison purposes in the known sequence and purposes in the nucleotide sequence of described signal peptide and modification insect material of the present invention of encoding in virus and/or in the target insect.Preferably, the purposes reflection codon of codon in the sequence of described signal peptide of coding and modification insect material is in the purposes aspect virus vector or the target insect.The example of codon optimizing toxin sequence is shown in Figure 10 and 11.The production of reorganization PxNPVs

Reorganization PxNPVs can produce like this: perhaps (ⅰ) goes into proper host cell with PxNPV DNA and heterologous sequence cotransfection, make it homologous recombination in vivo, perhaps (ⅱ) connects into direct connection carrier external with heterologous sequence, and then construction is introduced proper host cell is used for viral proliferation.Proper host cell is to support the arbitrary cell of shaft-like virus replication, includes but not limited to: Sf9 cell, Sf21 cell and High Five ^TMCell (Invitrogen, CarlsbadCA).The isolating virus of the present invention is such virus: for example, it has been cloned by plaque purification in tissue culture, not so from single virogene type preparation.

Usually, make up the module expression vector and comprise an expression cassette, wherein, suitable promoter sequence operationally is connected with the sequence of coding heterologous protein, that is, the expression of heterologous protein is placed under the control of promotor.From module expression vector excision expression cassette, connect direct connection carrier with its insertion PxNPV by external DNA.Then, should connect mixture and be transfected into proper host cell.Reclaim reorganization PxNPVs from growth medium, use any ordinary method and characterize LC ₅₀And LT ₅₀, described ordinary method includes but not limited to: food covers checking method (diet overlay assays), food blending checking method (diet incorporationassays) and blade dipping checking method (leaf dip assays).

LC ₅₀Refer to such virus concentration: under this concentration, at duration of test, 50% infected larva death.LT ₅₀When referring to be exposed in the virus of given dose, the time after the infection when 50% infected larva death.

Preferably, the standard food of describing in using embodiment 6 hereinafter covers checking method when measuring, and PxNPVs of the present invention shows the LC to diamondback moth larvae ₅₀Be about 1x10 ⁵OBs/16cm ²Or it is littler.Other baculovirus isolate generally shows the LC50s higher to diamondback moth larvae, that is, concerning the infectivity of other insect kind, their efficient is lower with respect to them.Insect-killing composition and goods

The invention provides the insect-killing composition and the goods that comprise one or more reorganization PxNPVs.Preferably, reorganization of the present invention PxNPVs more effectively kills diamondback moth larvae (referring to for example hereinafter embodiment 11) than the reorganization translation of wild-type PxNPV or other baculovirus.

Insect-killing composition of the present invention comprises at least a reorganization PxNPV.Reorganization PxNPV and agricultural that insect killing product comprises at least a insecticidal effective dose go up suitable carriers." insecticidal effective dose " refers to certain amount, and it causes the detectable minimizing of infection, by in the given area or the number of insect pest in a certain amount of crop or amount, the insect pest loss or other any suitable infection parametric representation that cause.Described goods can be following form: wettable powder, dispersed granular product, particle, suspension, emulsion, aerosol solution, bait and other conventional insect killing product.Suitable carriers has but is not limited to: water, alcohol, hydrocarbon or other organic solvent, perhaps mineral oil, animal oil or vegetables oil, perhaps powder (for example talcum powder, clay, silicate or diatomite).Also can comprise wetting agent, coating agent, ultraviolet protective agent, dispersion agent and tackiness agent.Can add nutrition (for example sugar) to strengthen the feeding behavior and/or to lure insect.Can add the flowing agent flowing agent of clay (for example based on) and make the caking of wettable powder or other dry products reach minimum.Described composition can be used as the dressing particle or prepares as the micro encapsulation material.Described goods are poisonous plant and do not damage the integrity of the reorganization PxNPV that is contained in wherein not, and any component all can not hinder insect to ingest or any viral function.Exemplary goods are disclosed the application 0679170A1 that EP announces; PCT applies for WA92/19102; With U.S. Patent No. 4,948, in 586.

Insect killing product of the present invention also can comprise one or more chemical insecticides and/or one or more non-PxNPV biocontrol agents.Chemical insecticide includes but not limited to: pyrethroid, pyrazolines, organophosphate, amino formate, formadines and pyroles, they all are well known in the art.Exemplary compound is disclosed in the PCT application 96/03048,96/01055 and 95/95741.Biocontrol agent for example comprises: non-PxNPV baculovirus (natural or reorganization); Bacillus thuringiensis (Bacillus thuringiensis); Nosemapo1yvora; M.grandis; Bracon mellitor ', the entomopathogenic fungi; And nematode.

The present invention also provides the method for kill insects insect.This method comprises: insect and insecticidal effective dose composition of the present invention or goods are contacted.The present invention also provides and has reduced the method that insect infestations is for example attacked crop, and this method comprises: insecticidal effective dose composition of the present invention or goods are used in the place to expectation.Described insect killing product is used routine techniques (for example crop being sprayed or dusting) and is used.Usually, with about 2.4X10 ⁸～about 2.4X10 ¹²Dosage between the OBs/ hectare (OBs is an inclusion body) is used.Effective dose depends on the reorganization PxNPV of for example insect target, application and the plant crop of processing.The dosage that comprises insecticidal effective dose can be used ordinary method by those of ordinary skills and determine.

The description of embodiment preferred

Following embodiment has set forth the present invention without limitation.The structure of embodiment 1:Egt reorganization PxNPV disappearance, that comprise beta-galactosidase

Carried out following test and produced reorganization PxNPV, wherein, lacked viral ecdysteroids Transglucosylase (egt) gene, and (β-gal) marker gene substitutes with colon bacillus (E.coli) beta-galactosidase enzymes.

The routine operation (" rhabdovirus expression vector: laboratory manual " that application O ' Reilly etc. describes, Oxford Univ.Press, New York, NY, 1994) plaque purification original seed production of the wild-type PxNPV that gone down to posterity by insect be used for engineered clone's original seed.Confirmed the integrity of described original seed (with " PxNPV-3 " expression) by parental generation original seed comparison restriction enzyme pattern with PxNPV.Biological assay to one group of insect kind has confirmed that also its virulence is comparable to not clone's original seed.

Fig. 1 has set forth the method that is used to make up transfer vector, and described transfer vector is used to by inserting the egt gene among the beta-galactosidase gene destruction PxNPV.The close similarity of the restriction enzyme pattern of AcNPV and PxNPV has shown the homology at dna level, and it makes the transfer vector can use v8 strain based on AcNPV (it is disclosed U.S. Patent No. 5,662,897 in).Made up the carrier (with " pmd 216.1 " expression) that comprises β-gal box by following method.Separated the BamH I～Xba I fragment [Zuidema etc., general virology magazine 71:2201 (1990)] that comprises the β-gal gene that is subjected to the control of fruit bat hsp70 promotor from pAcDz1.Then, this fragment subclone is gone into the BamH I site of polylinker and the pBluescript between the Xba I site ^TMSK ⁺(Stratagene, La Jolla, CA).Then, Pst " G " fragment [it comprises described egt gene and surrounding zone (referring to U.S. Patent No. 5,662, the 897)] subclone of AcNPV strain V8vEGTDEL is gone into the polylinker of pUC 9 on the Pst I site.The plasmid that generates is represented with " pmd 205.1 ".

Last transfer vector is to make up by carrying out following four fragments connection: (ⅰ) pBluescript that digests with BamH I and Pst I ^TMSK ⁺, used the calf intestinal phosphatase enzyme with its dephosphorylation; (ⅱ) 0.975 kb Bg1 II～Pvu II fragment of pmd 205.1; (ⅲ) 3.86 kb Sma I～Xba I β-gal fragment of pmd216.1; And (ⅳ) 1.6 kb Xba I～Pst I fragment of pmd 205.1.The hsp70 that the transfer vector that generates (with " pmd 220.2 " expression) comprises the site that is cloned into the egt disappearance drives β-gal marker gene.

PxNPV egt disappearance, β-gal mark is the pmd 220.2 of the Sf-9 cell going into to cultivate by cotransfection and the homologous recombination construction between the PxNPV-3 DNA.Be to mix among the TNM-FH (JRHBiosciences, Lenexa KS) of 200 μ l 1 μ g pmd220.2 DNA and 250 ng PxNPV-3 DNA and 25 μ g Lipofectin (GibcoBRL, Gaithersburg MD) at final volume.After at room temperature being incubated 15 minutes, (TNM-FH adds the final volume that 10% foetal calf serum and 1%pluronic F68 (GibcoBRL, Gaithersburg, MD)) are adjusted to 2.0ml with the TNM-FH perfect medium.This mixture is coated in 2.75 * 10 ⁵On the individual SF-9 cell, this cell has been fixed in the hole of standard 6 hole tissue culturing plates.After 24 hours,, crossed the virus that results sprout after 96 hours again with fresh culture culturing cell again.Recombinant virus is inclusion body male (occ+), and expresses described β-gal gene.Identified recombinant chou (occ+/blue plaque) by the three-wheel plaque purification in the presence of 100 μ g/ml 5-bromo-4-chloro-3-indyl-β-D-galactopyranosides (X-gal).The virus that generates is represented with " T96-19.1.1.1 ".The structure that directly connects the PxNPV virus vector of embodiment 2:Egt disappearance

Carry out following test and made up the direct connection carrier (referring to for example international patent application US 94/06079) that derives from PxNPV.This carrier comprises one and is inserted into the genomic polylinker of PxNPV on the egt site (with " Bsu-Sse joint " expression, seeing below).

Synthesized two kinds of oligonucleotide and formed described Bsu-Sse joint with following sequence: 5 '-CCTCAGGGCAGCTTAAGGCAGCGGACCGGCAGCCTGCAGG-3 ' (Oligo32) and 5 '-CCTGCAGGCTGCCGGTCCGCTGCCTTAAGCTGCCCTGAGG-3 ' (Oligo33)

In case after the annealing, these two kinds of oligomer are just encoded, and several are applicable to the restriction endonuclease site (comprising Bsu 36I site and Sse 8387I site) of construction of recombinant virus.In annealing buffer (comprising 10mM Tris-HCl (pH7.5), 100mM NaCl and 1mM EDTA), each oligomer is diluted to the concentration of 30 pmol/ μ l.Mixture heating up to 95 ℃ is reached 10 minutes, then slow cool to room temperature in a few hours.

The oligonucleotide of pressing after following method will be annealed inserts pmd 205.1 transfer vectors.With Spe I digestion pmd 205.1 carriers, described Spe I is in the single site cutting (Fig. 2) of the upstream that is in the egt disappearance just.The Klenow fragment of using the archaeal dna polymerase I is mended the end of flat digested plasmid in the presence of all four kinds of Nucleotide.Then, in the cumulative volume of 10 μ l, the 100ng linearization plasmid is connected on the double-stranded joint of 15pmol with the T4 dna ligase.After ligation was finished, hot deactivation T4 ligase enzyme was used the polynucleotide kinase treating mixture.By electrophoresis purifying 8.8 kb DNA band in 1% low melting point preparation scale sepharose (BioRad, Richmond VA).Melt the gel slice that comprises the linear DNA of 8.8 kb down at 65 ℃, the interior ligation of gel of using about 1/10 this gel slice is used to the described DNA of recirculation, then, it is used to transform colon bacillus DH5 α cell.

Using polymerase chain reaction (PCR) screens the plasmid that generates and determines the orientation of Bsu-Sse joint with respect to the egt gene.With Oligo 32 and 33 respectively with oligomer EGT 1 (5 '-GCGGCCAATATATTGGCCGTGTTT-3 ') pairing, the egt gene 5 of 1 pair of disappearance of oligomer EGT ' the district be specific.Fig. 2 has indicated the orientation of Bsu-Sse joint among the LAB 50.2.

Use 1 μ g LAB50.2 and 250 ng T96-19.1.1.1 viral DNAs by the homologous recombination construction between LAB50.2 and the T96-19.1.1.1 PxNPV viral DNA (the Sf9 cell of having gone into to cultivate by cotransfection as described in the embodiment 1) the direct connection PxNPV carrier of egt-disappearance.In this case, recombinant virus is the inclusion body male and does not express β-gal gene.Identified reorganization (occ+/white) virus by the three-wheel plaque purification in the presence of 100 μ g/ml X-gal.The virus that generates is called as " T97-8.1.1.1 " (the ATCC preserving number is VR-2608).Embodiment 3: the structure that is applicable to the module expression vector of producing reorganization PxNPVs

A．pMEV?1-4

International patent application US 94/06079 discloses carrier pMEV1, pMEV2, pMEV3 and the pMEV4 design aspect the expression alien gene, structure and application under the control of bacilliform virus promoter.These carriers have the general structure shown in Fig. 3.Each carrier derives from pBluescript SK+ plasmid (Stratagene, La Jolla CA), promptly, by obtaining with the district between Sst I site and the Xho I site in the alternative pBluescript polylinker site of the expression cassette that comprises following elements: (ⅰ) lack synthetic linker DNA, it has the recognition site of restriction enzyme Sst I, Sse8387 I and Stu I; (ⅱ) promotor module, it comprises the complete 5 ' non-translational region (UTR) of promotor and the AcMNPV virogene of selecting; (ⅲ) polylinker module is in order to promote the insertion of target protein matter coding region; (ⅳ) 3 ' UTR module, it comprises the complete 3 ' non-translational region of AcMNPV6.9K (basic protein) gene; (ⅴ) lack synthetic linker DNA, it has the recognition site of restriction enzyme Stu I, Bsu 36 I and Xho I.

As shown in Figure 4, the promotor module among carrier pMEV1～pMEV4 gets the gene that the different times of comfortable virus in life cycle expressed.DA26 gene promoter module among the pMEV1 and the 35K gene promoter module among the pMEV4 derive from the gene that early stage (that is, the DNA synthetic beginning before) of virus in life cycle expressed.6.9K gene promoter module among the pMEV2 derives from " late period " structure gene, it is expressed in the synthetic beginning of DNA back, and the polyhedrin among the pMEV3 (polh) gene promoter module then derives from " phase very late " gene (the primary structure component of its coding viral occlusion).

Design polylinker module makes can be replaced the protein coding region of the 5 ' UTR that is close to described promotor module but not introduce external joint sequence.As shown in Figure 5, polylinker comprises Esp3 I site, and it is placed in cutting and the contact place cutting between promotor module and the polylinker module in the chain of bottom between the position 4 and 5 in the chain of the top of promotor module when applying Esp3 I digested vector of such position.With archaeal dna polymerase four kinds of standards 2 '-DNA that handles the digestion of Esp3 I in the presence of the deoxynucleoside triphosphate ester (dNTPs) produces linearized vector, this carrier is at the lucky 3 ' terminal flush end of being done into of promotor module.This sections can be connected to 5 ' blunt-ended fragment (its sequence starts from the ATG initiator codon of the protein coding region of expection).In order to promote the segmental directed cloning of protein coding, made up described segmental 3 ' end, so that it is included in the recognition site of one of splitted enzyme (having set forth with the BamH I) in the polylinker module in Fig. 5, and has digested described protein coding fragment and carrier with this kind of enzyme before ligation.

A key feature of these carriers is the recognition sites that have restriction enzyme Sse8387 I and Bsu36 I at arbitrary end of described expression cassette.This makes and can connect into direct connection PxNPVs carrier of the present invention (referring to for example the foregoing description 2) from the sequence of module expression vector excision insertion and with them.

B.pMEV5 and pMEV6

Make up two kinds of carriers (pMEV5 and pMEV6) and mixed fruit bat hsp70 (leading heat shock) gene promoter.PMEV5 comprises the sections of 724 bp fruit bat hsp70 promotor/5 ' UTR (in Fig. 4 with " hsp70 Bam " expression), and this sections-493 is stretched over position+231 (with respect to hsp70 gene transcription initiation site) from the position.PMEV6 comprises the sections of 475 bp identical promoters/5 ' UTR (in figure PD2 with " hsp70Xba " expression), and this sections-244 is stretched over position+231 from the position.

The promotor module that is used to make up pMEV5 and pMEV6 is to use plasmid phcHSP70PL[Morris etc., Journal of Virology 66:7397 (1992)] make template by the pcr amplification synthetic.The sequence that is used for the Oligonucleolide primers of each reaction and amplified production is as shown in Fig. 6 and 7 (respectively about pMEV5 and pMEV6).Each primer has bipartite texture.5 ' section and phcHSP70PL template do not have sequence homology, so be used to the specificity restriction site is mixed the end of last PCR product.These specificity restriction sites comprise: Esp3 I site and Xba I site (referring to Fig. 6 and 7) of being in Sst I site, Sse8387 I site and the Stu I site of PCR product 5 ' end and being in PCR product 3 ' end.3 ' section of every kind of primer all comprises with phcHSP70PL template homologous sequence and limits one of border of hsp70 specific sequence in each module.

Before PCR reaction, by in 37 ℃ of following 10ml reactants with 200pmol primer insulation 30min its 5 ' terminal to (-) strand primer (HSP70Esp) phosphorylation, described reactant comprises 70mM Tris-HCl (pH7.5), 10mM MgCl ₂, 5mM DTT, 1mM ATP and 10 T4 of unit polynucleotide kinases (New England Biolabs, Beverly MA).Then, in independent MicroAmp pipe (Perkin Elmer, Norwalk CT), carried out the PCR reaction as follows.Each is incorporated in the 50 μ l reactants with 0.25～1.0ng phcHSP70PLDNA with every kind of primer of 50pmol, this reactant comprises 200 μ M dNTPs, 1X clone's Pfu polymerase buffer (Stratagene, La Jolla CA) and 5 units clones' Pfu archaeal dna polymerase (Stratagene, La Jolla CA).Sample is placed Perkin-Elmer 9600 type thermal cycler (Perkin Elmer, Norwalk CT) in, experience is at 94 ℃ of 1min (denaturing step), at 50 ℃ of 1.5min (annealing steps) with in 2 circulations of 72 ℃ of 3min (stretching, extension step), then at 94 ℃ of 1min, at 55 ℃ of 1.5min with in 28 circulations of 72 ℃ of 3min.Circulation is in the end stretched reaction and is carried out other 7min, and reactant is dropped to 4 ℃.Use phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracts amplified production once, is adjusted to the 0.3M sodium acetate, uses ethanol sedimentation again.

After in suitable reaction buffer, dissolving, with Pst I (it is in the cutting of Sse8387 I site) dna digestion, by (CA) the electrophoresis purifying on comprises the fragment of predetermined hsp70 promotor module for BioRad, Richmond at 1.2% low melting point preparation scale sepharose.The underlying carrier that is used to make up pMEV5 and pMEV6 is preparation like this: with EcoR I cutting pMEV1, mend flat end with the Klenow fragment of archaeal dna polymerase I in the presence of whole four kinds of dNTPs.After making terminal dephosphorylation by the described carrier of Sse8387 I secondary digestion and with calf intestinal alkaline phosphatase, purifying comprises big (3.1 kb) fragment of pBluescript skeleton and 3 ' UTR and polylinker module on the low melting-point agarose gel, is connected with hsp70 promotor module behind the purifying by oneself and forms pMEV5 and pMEV6.

C.pMEV5/ADK-AaIT and pMEV6/ADK-AaIT

Further artificial reconstructed above-mentioned pMEV5 and pMEV6 carrier and mix the gene of coding AaIT, AaIT is a kind of insect specificity toxin, it is [Zlotkin etc., biological chemistry (Biochimi) 53:1073 (1971)] that is expressed in the venom of North Africa scorpion " Andoctuonusaustralis (Hector) ".Be injected into the body cavity of insect larvae as AaIT after, it causes temporary transient shrinkability paralysis optionally in conjunction with the sodium channel of voltage-sensitive.The described toxin of long-term application (this can by implementing with the baculovirus infection insect larvae that produces AaIT) can cause prolonging palsy and final death [Stewart etc., natural 352:85 (1991); Maeda etc., virusology 184:777 (1991); McCutchen etc., biotechnology 9:848 (1991)].United States Patent (USP) 5,547,871 disclose a kind of codon optimizing ADK-AaIT gene order, and wherein, the secretion of AaIT toxin is to instruct by the signal peptide that derives from maduca sexta adipokinetic hormone (ADK) gene.International patent application US 94/06079 discloses the insertion of ADK-AaIT coding region in pMEV1～pMEV4 carrier and (has produced plasmid pMEV1/ADK-AaIT～pMEV4/ADK-AaIT).

In order to make up pMEV5 and the pMEV6 derivative of coding ADK-AaIT, using pMEV3/ADK-AaIT is that template has been synthesized the fragment that comprises the ADK-AaIT gene order by PCR.(+) strand primer (with " PD30 " expression) starts from the initial ATG codon of ADK signal peptide.DNA is caused in selected (-) strand primer (with " 69K3UT " expression) so that it in the position in polylinker module downstream (that is, in 3 ' UTR at pMEV3/ADK-AaIT) synthetic.The sequence of described primer and the dna fragmentation of amplification are given among Fig. 8.

Before amplification, by above-mentioned about HSP70Esp like that with the 5 ' terminal phosphateization of (+) strand primer (PD30).Also carried out the PCR reaction like that by above-mentioned, different is: carried out at 94 ℃ of 1min, at 52 ℃ of 1.5min with in 25 round-robin amplifications of 72 ℃ of 3min.After synthetic, with BamH I (it cuts described polylinker module) dna digestion, will comprise 274bp 5 '-flush end/3 of ADK-AaIT coding region '-BamH I fragment insertion pMEV5 and pMEV6.Determined to confirm the structure of the plasmid (pMEV5/ADK-AaIT and pMEV6/ADK-AaIT) of generation by restriction analysis and partial dna sequence.

D. the MEVS carrier that comprises ADK signal peptide module

Carried out following test and produced a series of module expression vectors, this carrier comprises the DNA of coding ADK signal peptide (seeing above), and the ADK signal peptide can be used to instruct the secretion of any insertion sequence.Made up a series of carriers (with " pMEV1/ADK～pMEV6/ADK " expression), wherein, the 57bp codon optimizing sequence (residue 1～57 among Fig. 8) of ADK signal peptide has been placed between promotor and the polylinker module.Expression cassette in these carriers has the general structure shown in Fig. 9.

In order to make up pMEVx/ADK carrier series, comprise the dna fragmentation of described promotor module and the ADK signal peptide that is connected from corresponding pMEVx/ADK-AaIT carrier recovery by PCR.(+) strand primer of each reaction is the specific oligonucleotide of promotor, and the DNA that it causes at 5 of described promotor module ' end is synthetic, and the restriction site of Sst I, Sse8387 I and Stu I is mixed the segmental 5 ' end of amplification.The template of each reaction and (+) strand primer are listed in following table:

Template	Primer	Sequence
			pMEV1/ADK- ????AaIT	?DA26FZ	?5′-AGCAGCGAGCTCCTGCAGGCCTACGCGT ?AATTCGATATAGAC-3′
pMEV2/ADK- ????AaIT	?69KFZ	?5′-AGCAGCGAGCTCCTGCAGGCCTATGCCG ?TGTCCAATTGCAAG-3′
			pMEV3/ADK- ????AaIT	?PHF	?5′-AGCAGCGAGCTCCTGCAGGCCTGACGCA ?CAAACTAATATCAC-3′
pMEV4/ADK- ????AaIT	?35KPRO1	?5′-AGCAGCGAGCTCCTGCAGGCCTCTTGAT ?GTCTCCGATTTC-3′
			pMEV5/ADK- ????AaIT	?HSP70Bam	?5′-AGCAGCGAGCTCCTGCAGGCCTGATCCT ?TAAATTGTATCCTA-3′
pMEV6/ADK- ????AaIT	?HSP70Xba	?5′-AGCAGCGAGCTCCTGCAGGCCTAGAATC ?CCAAAACAAACTGG-3′

(-) strand primer of each reaction [with " ADKRev (5 '-CGGATCTAGACACGTCTCGGGCCTCAGCGATAATCACGAAGGC-3 ') " expression] caused from the synthesizing of 3 of ADK signal peptide ' end, and the site of Esp3 I and Xba I was incorporated into the segmental 3 ' end of amplification.Carried out the PCR reaction by above-mentioned like that about pMEV5～6, different is at 94 ℃ of 1min, has carried out 25 round-robin amplifications at 52 ℃ of 1.5min with at 72 ℃ of 3min.From the whole template synthetic DNA except that pMEV5/ADK-AaIT, the fragment that will comprise described promotor module and signal peptide is inserted between Pst I (Sse8387 I) site and Xba I site in one of existing pMEVs carrier (for example pMEV1) with Pst I (it cuts the Sse8387 I site of described promotor upstream) and Xba I (downstream of its cutting ADK signal peptide) digestion.

Because the hsp70Bam promotor module among the pMEV5 comprises site in the Xba I, so the hsp70Bam/ADK module must insert the pMEVx framework with two sections.Therefore, use the part of Pst I and Xho I digestion, with Xho I and Xba I digestion second section from pMEV5/ADK-AaIT template synthetic DNA.With representing the Pst I/Xho I fragment of 5 ' sections of hsp70Bam/ADK module to merge, connect into from the Pst I/Xba I carrier segments of pMEV 1 preparation with the Xho I of representing 3 ' sections of hsp70Bam/ADK module/Xba I fragment.

Each of the pMEVx/ADK carrier that generates is structurally identical with corresponding pMEVx carrier, just inserts 57 bp ADK signal peptides between promotor module and polylinker module.Embodiment 4: the structure of the module expression vector of coded insect-killing toxin

Carry out following test and produced the module expression vector of the coded insect-killing toxin that is fit to mix reorganization PxNPVs of the present invention.

A．Txp-Ⅰ

Straw itch mite toxin T xp-I is a kind of isolating paralytic neurotoxin of venom from predatism straw itch mite [lice shape Pu mite (Pyemotes tritici)] [Tomalski etc., toxin 27:1151 (1989)].291 amino acid whose precursor proteins of tox34 genes encoding [Tomalski etc., natural 352:82 (1991)].

In order to test the effectiveness of tox34 gene in reorganization PxNPV meaning, prepared three kinds of different Txp-1 constructions, they are instructing toxin from difference aspect the amino terminal signal sequence of emiocytosis.A kind of construction keeps natural tox34 amino terminal sequence; A kind of ADK signal peptide (replacing preceding 40 residues of proteinic aminoterminal of tox34) that comprises; Also has a kind of ADK of comprising signal peptide (replacing tox 34 preceding 26 residues of proteinic aminoterminal).

Synthesized three different segments corresponding to the tox34 gene of codon 1～291 (with " tox34 " expression), 27～291 (" tox34L ") and 40～291 (" tox34S ") by PCR, they are applicable to and are cloned into one or more said module expression vectors.The template that is used for amplified reaction is plasmid pHSP70tox34[Lu etc., biological control (Biological Control) 7:320 (1996)].The positive strand primer of each amplification is shown in following table:

Fragment	Primer	Sequence
			tox34	?TOX34ATG	?5′-ATGAAAATTTGTACATTTTTTATTCC-3′
tox34L	?TOX34NT1	?5′-GTTAAACCTTTTAGGTCTTTTAATAATATTTCC ?-3′
			tox34S	?TOX34NT2	?5′-GATAATGGCAATGTCGAATCTGTA-3′

(-) strand primer [is represented with " TOX34CT2 ", promptly, 5 '-GTACCCCCGGGATCCAATTTAACACAGTCTTGAATCACTT-3 '] cause from 3 of tox34 coding region ' the synthesizing of end, and the restriction site of BamH I and Xma I (Sma I) is mixed the segmental 3 ' end of amplification.Before amplification, use the method described about the primer HSP70Esp among the as above embodiment 35 ' terminal phosphateization with each (+) strand primer.Carried out the PCR reaction by as described in the embodiment 3, different is, described circulation comprises: at 94 ℃ of 1min, at 45 ℃ of 1.5min with in 2 circulations of 72 ℃ of 3min, then at 94 ℃ of 1min, at 55 ℃ of 1.5min with in 28 circulations of 72 ℃ of 3min.

After synthetic, with Xma I dna digestion, purifying 5 '-flush end/3 '-Xma I tox34 coding region fragment (representing) with tox34, tox34L or tox34S, be cloned into the MEVS carrier by preparation as described in the embodiment 3, different is to cut polylinker with Xma I (it is in the cutting of Sma I site) rather than with the BamH I.

Following table has been concluded and has been made up the MEVS carrier component utilized that each expresses the TxP-I:

Construction	Carrier	Txp-I encode fragment
			?pMEV1/Tox34	?pMEV1	?tox34
?pMEV5/Tox34	?pMEV5	?tox34
			?pMEV6/Tox34	?pMEV6	?tox34
?pMEV1/ADK-Tox34L	?pMEV1/ADK	?tox34L
			?pMEV1/ADK-Tox34S	?pMEV1/ADK	?tox34S
?pMEV2/ADK-Tox34L	?pMEV2/ADK	?tox34L
			?pMEV2/ADK-Tox34S	?pMEV2/ADK	?tox34S

B．LqHIT2

Except excitotoxin (for example AaIT), a lot of Buthinae scorpion venoms comprise second kind of insect specificity toxin, and this toxin causes slowly, progressive flaccid paralysis [Zlotkin etc., biological chemistry 30:4814 (1991)].To this group research is toxin LqhIT2 the most completely, and it is isolating from scorpion Leiurus quinquestriatus hebreaeus.Clone cDNA about LqhIT2 has disclosed 21 amino acid signal peptides and three C terminal amino acids (Gly-Lys-Lys), they are the toxin [Zlotkin etc., insect biochemistry and physiology document (Arch.Insect Biochem.Physiol.) 22:55 (1993)] that remove after the translation.

In order to study the ability that the LqhIT2 toxin improves the insecticidal activity of PxNPV, assembled the dna sequence dna of encoding mature LqhIT2 toxin and be cloned into four kinds of different pMEVx/ADK expression vectors: pMEV1/ADK, pMEV2/ADK, pgEV5/ADK and pMEV6/ADK.The sequence of the toxin coding region shown in Figure 10 has 27 different with natural cDNA sequence in 61 codons; Introduced these change so that synthetic LqhIT2 coding region in the codon purposes reflected in the AcMNPV genome all purposes [Ayres etc. (1994)] of codons.

Mark step has carried out comprising the assembling of the dna fragmentation of LqhIT2 coding region.At first, synthesized four kinds of oligonucleotide, they represent two chains and a small amount of 3 ' flank joining region DNA (Figure 10) of LqhIT2 coding region jointly.Before application, it comprises the 3 ' part of (+) chain with oligonucleotide LqhIT2F2[to adopt the method for describing among the embodiment 3] and LqhIT2R3[it comprise the 3 ' part of (-) chain] 5 ' terminal phosphateization.By be heated to 95 ℃, cooling mixture to 60 ℃ and each oligonucleotide of 40pmol is annealed lentamente then momently in 10 μ l 50mM NaCl.Regulate the cumulative volume of mixture to 40 μ l, connect the two halves (LqhIT2F1:LqhIT2R3 and LqhIT2F2:LqhIT2R4) of LqhIT2 coding region.Owing in each of oligonucleotide, have incomplete synthetic product, so initial ligation produces the segmental heterology mixture of length in 200～1000 bp scopes.Use 0.5 μ l ligation thing as the template source and use oligonucleotide LqhIT2 PCRF (in its 5 ' terminal phosphate change) and LqhIT2 PCRR as primer (Figure 10), by PGR from described mixture separation required product.As described in embodiment 3, increase, that is, and at 94 ℃ of 1min, carried out 25 circulations at 55 ℃ of 1.5min with at 72 ℃ of 3min.After synthetic, with BamH I (it cuts in the joint sections of contiguous terminator codon) dna digestion, 190 required bp 5 '-flush end/3 '-BamH I fragment is cloned into MEVS carrier: pMEV1/ADK, pMEV2/ADK, pMEV5/ADK and pMEV6/ADK (as summarizing among embodiment 3 and Fig. 5) after by the gel electrophoresis purifying.Confirmed the sequence of LqhIT2 coding region in each carrier by dna sequencing.

B．ω-ACTX-HV1

ω-atracotoxins is that a class is from the isolating insecticidal peptide toxin of Australian Atrax robustus.Research the most completely this group one of toxin be ω-atracotoxin-HV1 (ω-ACTX-HV1), that is, from isolating 37 the residue peptide toxin of venom (international patent application AU93/00039) of Blue Mountains Atrax robustus Hadronyche versuta.ω-ACTX-HV1 suppresses the voltage-controlled calcium channel of insect, and the Helicoverpa armigera young is caused death, but harmless to newborn mice.

Based on the aminoacid sequence of ω-ACTX-HV1, designed two kinds of oligonucleotide, they represent two chains of ω-ACTX-HV1 coding region and a small amount of 3 ' flank joining region DNA.Design the codon purposes in ω-ACTX-HV1 coding region and reflected in the AcMNPV genome all purposes [Ayres etc. (1994)] of codons.The sequence of these oligonucleotide as shown in Figure 11.Because the length of oligonucleotide ACTXHV1F and ACTXHV1R, this intensive possibility is arranged: each significantly is subjected to the pollution of incomplete synthetic product.Therefore, use with above-mentioned about the next synthetic ω-ACTX-HV1 coding region fragment that is fit to be cloned into the MEVS carrier of the similar PCR strategy of LqhIT2.In this case, merge each oligonucleotide of 0.1pmol and be used for the pcr amplification of required total length ω-ACTX-HV1 product as template.The primer that is used for this reaction is ACTXPCRF (as described above in its 5 ' terminal phosphate change) and ACTXPCRR.Carried out the PCR reaction like that by above-mentioned, different is, has carried out at 94 ℃ of 1min, at 55 ℃ of 1.5min with in 15 round-robin amplifications of 72 ℃ of 3min.After the amplification, with BamH I (it cuts in the joint sections of contiguous terminator codon) cutting DNA, required 118 bp, 5 ' flush end/3 '-BamH I fragment is cloned into MEVS carrier: pMEV1/ADK, pMEV2/ADK, pMEV5/ADK and pMEV6/ADK after by the gel electrophoresis purifying.Confirmed the sequence of ω in each carrier-ACTX-HV1 coding region by dna sequencing.Embodiment 5: the structure of reorganization PxNPVs

Carry out following test and made up the reorganization PxNPVs of coded insect-killing toxin.

Prepared viral DNA (O ' Reilly etc., 1994) from inclusion body.DNA (using about 5 units enzyme/μ g DNA) with Bsu 36 I and Sse8387 I digestion purifying.Application size exclusion chromatography or saccharose gradient method of purification are from the fragment isolated viral genomic dna of excision.The linearizing viral DNA that generates is resuspended to 10mMTris-HCl (pH 8.0)/1mM EDTA with the concentration of 0.2～1 μ g/ μ l after with ethanol sedimentation.

Then, under 15 ℃ 0.5 μ g linearizing DNA and 15ng are being connected a night from the Bsu 36 I/Sse 8387 I digestion fragments of suitable MEVs carrier purifying 5 μ l reactant volumes.This mixture is used for by transfection Sf-9 cell as described in the embodiment 1 again.After the transfection 24 hours, cultivate culture again with fresh culture.Results culture supernatant after having spent 72 hours again, dilution is used for infecting Sf-9 cell (for using of plaque separation) again.Select plaque at random, be used to infect 48 orifice plates and (fill 6 * 10 ⁴Individual Sf-9 cells/well is in the 0.5mlsTNM-FH perfect medium) the hole.Application is identified recombinant chou for the primer of the gene specific that inserts.Be used for purifying by the other plaque purification of two-wheeled and derive from the virus of the plaque that provides positive hole.

Following table provides the information about the various reorganization PxNPVs of preparation.

Virus	The MEVs that uses
		?PxEGTDEL/35K?ADK-AaIT	pMEV4/ADK-AaIT
?PxEGTDEL/hsp70Bam?ADK-AaIT	pMEV5/ADK-AaIT
		?PxEGTDEL/DA26?tox34	pMEV1/tox34
?PxEGTDEL/hsp70Bam?tox34	pMEV5/tox34
		?PxEGTDEL/hsp70Xba?tox34	pMEV6/tox34
?PxEGTDEL/DA26?ADK-tox34S	pMEV1/ADK-tox34S
		?PxEGTDEL/DA26?ADK-tox34L	pMEV1/ADK-tox34L
?PxEGTDEL/6.9K?ADK-tox34S	pMEV2/ADK-tox34S
		?PxEGTDEL/6.9K?ADK-tox34L	pMEV2/ADK-tox34L
?Ac(V8)EGTDEL/DA26?ADK-AaIT	pMEV4/ADK-AaIT
		?PxEGTDEL/DA26?ADK-LqhIT2	pMEV1/ADK-LqhIT2
?PxEGTDEL/6.9K?ADK-LqhIT2	pMEV2/ADK-LqhIT2
		?PxEGTDEL/hsp70?Bam?ADK-LqhIT2	pMEV5/ADK-LqhIT2
?PxEGTDEL/hsp70?Xba?ADK-LqhIT2	pMEV6/ADK-LqhIT2
		?PxEGTDEL/DA26?ADK-ω-ACTX-HV1	pMEV1/ADK-ω-ACTX-HV1
?PxEGTDEL/6.9K?ADK-ω-ACTX-HV1	pMEV2/ADK-ω-ACTX-HV1
		?PxEGTDEL/hsp70?Bam?ADK-ω-ACTX-HV1	pMEV5/ADK-ω-ACTX-HV1
?PxEGTDEL/hsp70?Xba?ADK-ω-ACTX-HV1	pMEV6/ADK-ω-ACTX-HV1

Embodiment 6: the effectiveness of expressing the reorganization PxNPVs of insect-killing protein

Carry out following test and tested the desinsection effectiveness of reorganization PxNPVs of the present invention.

(2～3 days sizes, 0.15～0.3mg) food that has carried out standard cover calibrating and reach the required dosage of 50% mortality ratio in the insect population of determination test (is LC to slough off the phase diamondback moth larvae to second ₅₀).With 0.01% sodium lauryl sulphate virus dilution original seed one by one, get 50 μ l viral suspension equal portions liquid and be used for surface contamination 15mm circle, Stoneyille food (the Southland Products Incorporated that has increased Toenol 1140 is contained in this place, LakeVillage, AK).One larva is placed every place, allow it at the duration of test contaminated food of ingesting.The larva that every day, twice counting die three day of test, then, every day, counting was till beginning nymphosis in six to seven days at least.The data of compiling revision test are analyzed by probit analysis (Finney, 1952).Determine to take place the half time (LT of lethality by the probable value analysis of following death time of single dose ₅₀).

The results are shown in following table.In order to compare, all inclusion body (OB) concentration are all with " OBs/16cm ²Food " expression.

rPxNPV	?LC 50(OBs/16cm 2)	LT 50(day) @4.5 * 10 4
			PxEGTDEL/35K?ADK-AaIT	?9.34×10 2	2.69
?wt?PxNPV	?5.97×10 4	4.39

Probable value produces from three revision tests (about 32 larvas/dosage is handled).

rPxNPV	?LC 50(OBs/16cm 2)	LT 50(day) @4.5 * 10 3	LT 50(my god) @ 4.5 * 10 4
				PxEGTDEL/35K?ADK?AaIT	?7×10 2	?3.7	?3.0
PxEGTDEL/h?sp70Bam?ADK-AaIT	?6×10 2	?3.7	?2.7
				Ac(V8)EGTDEL/DA26?ADK-AaIT	?6.3×10 5	?ND *	?4.0

^*The ND undetermined goes out probable value and produces from four revision tests (about 32 larvas/dosage is handled).

These results show: (ⅰ) add the speed of killing and wounding that toxin gene has not only increased virus in the PxNPV genome, and sharply reduced the effective LC to diamondback moth larvae ₅₀And (ⅱ) compare with closely-related reorganization AcNPV, reorganization PxNPVs has the LC of rapid reduction ₅₀With the much rapid speed of killing and wounding.Because small cabbage moth is a kind of important vegetables pest, so, just select recombinant baculovirus as biotic pesticide in the vegetables market, this discovery has decisive significance.

Following table provides the data that derive from the analysis of the reorganization PxNPVs that expresses second kind of insect selectivity toxin (tox34).

?rPxNPV	?LT 50(day) @4.5 * 10 3	LT 50(day) @4.5 * 10 4
			?PxEGTDEL/DA26?tox34	?4.5	?3.4
?PxEGTDEL/hsp70Bam?tox34	?2.6	?1.9
			?PxEGTDEL/hsp70Xba?tox34	?2.0	?1.8
?PxEGTDEL/DA26?ADK-tox34S	?3.7	?2.7
			?PxEGTDEL/DA26?ADK-tox34L	?3.1	?2.4
?PxEGTDEL/6.9K?ADK-tox34S	?2.9	?1.6
			?PxEGTDEL/6.9K?ADK-tox34L	?3.2	?1.8
?PxEGTDEL/hsp70Bam?ADK-AaIT	?3.0	?2.2

Probable value produces from four revision tests (about 32 larvas/dosage is handled).

?rPxNPV	Average LC 50(OBs/16cm 2)	LT 50(day) @4.5 * 10 3
			?PxEGTDEL/hsp70Xba?tox34	?0.8×10 2	?2.4
?PxEGTDEL/hsp70Bam?ADK-AaIT	?2.9×10 2	?3.3
			?wt?PxNPV	?165×10 2	?4.6@4.5×10 4

These data show that the effectiveness of the increase of reorganization PxNPVs is not limited to express the construction of AaIT.The PxNPVs that expresses tox34 shows and expresses the same low LT of recombinant chou of AaIT ₅₀And LC ₅₀(being PxEGTDEL/hsp70Xba tox34) in some cases expressed tox34 by reorganization PxNPV and caused LT ₅₀Significantly be lower than the recombinant chou of expressing AaIT.

Aforementioned all patents, patent application, paper, publication and test method all integral body to incorporate this paper into for referencial use.

Those skilled in the art understand by top detailed description a lot of changes that the present invention did.Obvious change like this belongs to scope of the present invention.

Claims (57)

1. An isolated recombinant diamondback moth baculovirus (PxNPV) having a genetic alteration relative to wild-type PxNPV, wherein the alteration is selected from: (a) introduction or deletion of a restriction site; (b) a viral encoding Modification, deletion or duplication of a gene; (c) introduction of a gene encoding a heterologous protein; and (d) any combination of any of the foregoing. 2. The recombinant baculovirus of claim 1, which has incorporated in its genome a nucleic acid sequence encoding an insect-modifying substance, said nucleic acid sequence being operably linked to a promoter capable of activating transcription in insect cells. 3. The recombinant baculovirus of claim 2, wherein said insect-modifying substance is an insecticidal toxin. 4. The recombinant baculovirus of claim 3, wherein the toxin is selected from the group consisting of: AaIT, AaHIT1, AaHIT2, LqhIT2, LqqIT1, LqqIT2, BjIT1, BjIT2, LqhP35, LqhαIT, SmpIT2, SmpCT2, SmpCT3, SmpMT, DK9.2, DK11 , DK12, μ-agatoxin, King Kong toxin, Pt6, NPS-326, NPS-331, NPS-373, Tx4(6-1), TxP-1, ω-atracotoxins, α-conotoxin, μ-conotoxin toxin, chlorotoxin and omega-conotoxin. 5. The recombinant baculovirus of claim 4, wherein the toxin is selected from the group consisting of: AaIT, TxP-1, LqhIT2 and ω-atracotoxin. 6. The recombinant baculovirus of claim 4, further comprising a sequence encoding a heterologous signal peptide, wherein said signal peptide encoding sequence is fused in-frame with the sequence encoding said toxin. 7. The recombinant baculovirus according to claim 6, wherein the signal peptide is selected from the group consisting of: the pBMHPC-12 signal sequence obtained from Bombyx mori; the adipokinin signal sequence obtained from Manduca sexta; the apolipophorin signal sequence obtained from Manduca sexta; The eggshell signal sequence from Bombyx mori; the cuticle signal sequence from Drosophila; the esterase-6 signal sequence from Drosophila; and the sex-specific signal sequence from Bombyx mori. 8. The recombinant baculovirus of claim 2, wherein the promoter is the hsp70 promoter. 9. The recombinant baculovirus of claim 8, wherein the promoter is selected from hsp70 Bam and hsp70 Xba. 10. The recombinant baculovirus of claim 3, wherein the promoter is selected from the group consisting of DA26, 35K, 6.9K, polyhedrin, hsp70 and actin promoters. 11. The recombinant baculovirus of claim 3, wherein said genome has been further modified to inactivate the viral egt gene. 12. The recombinant baculovirus of claim 3, further comprising a nucleic acid sequence encoding juvenile hormone esterase (JHE), said nucleic acid sequence being operably linked to a promoter capable of activating transcription in insect cells. 13. The recombinant baculovirus of claim 5, wherein the promoter is the hsp70 promoter. 14. A recombinant diamondback moth baculovirus incorporating into its genome a sequence encoding TxP-1 operably linked to the Drosophila hsp70 promoter. 15. A recombinant diamondback moth baculovirus incorporating into its genome a sequence encoding AaIT operably linked to the Drosophila hsp70 promoter. 16. The recombinant baculovirus of claim 1, wherein said genetic alteration creates a cloning site that is absent in wild-type PxNPV. 17. 16. The recombinant baculovirus of claim 16, wherein a nucleic acid sequence encoding an insect-modifying substance operatively linked to a promoter capable of activating transcription in insect cells is incorporated at said cloning site. 18. 17. The recombinant baculovirus of claim 17, wherein said insect-modifying substance is an insecticidal toxin. 19. The recombinant baculovirus of claim 18, wherein the toxin is selected from the group consisting of: AaIT, AaHIT1, AaHIT2, LqhIT2, LqqIT1, LqqIT2, BjIT1, BjIT2, LqhP35, LqhαIT, SmpIT2, SmpCT2, SmpCT3, SmpMT, DK9.2, DK11 , DK12, μ-agatoxin, King Kong toxin, Pt6, NPS-326, NPS-331, NPS-373, Tx4(6-1), TxP-1, ω-atracotoxins, α-conotoxin, μ-conotoxin toxin, chlorotoxin and omega-conotoxin. 20. The recombinant baculovirus of claim 19, wherein said promoter is selected from the group consisting of: DA26, 35K, 6.9K, polyhedrin, hsp70 and actin promoters. twenty one. The recombinant baculovirus of claim 20, wherein said genome has been further modified to inactivate the viral egt gene. twenty two. The recombinant baculovirus of claim 21, further comprising a nucleic acid sequence encoding juvenile hormone esterase (JHE), said nucleic acid sequence being operably linked to a promoter capable of activating transcription in insect cells. twenty three. A recombinant diamondback moth baculovirus selected from ATCC VR-2607, ATCC VR-2608 and ATCC VR2609. twenty four. A direct connection vector comprising genomic DNA isolated from the recombinant baculovirus of claim 1. 25． 24. The direct ligation vector of claim 24, wherein said DNA further comprises DNA encoding a modified insect material operably linked to a promoter capable of activating transcription in insect cells. 26． The direct link vector of claim 25, wherein said insect-modifying substance comprises a toxin selected from the group consisting of: AaIT, AaHIT1, AaHIT2, LqhIT2, LqqIT1, LqqIT2, BjIT1, BjIT2, LqhP35, LqhαIT, SmpIT2, SmpCT2, SmpCT3, SmpMT, DK9.2, DK11, DK12, μ-agatoxin, King Kong toxin, Pt6, NPS-326, NPS-331, NPS-373, Tx4(6-1), TxP-1, ω-atracotoxins, α-taro Spirotoxins, μ-conotoxins, chlorotoxins and omega-conotoxins. 27． The direct link vector of claim 26, further comprising a sequence encoding a heterologous signal peptide, wherein said signal peptide encoding sequence is fused in-frame to a sequence encoding said toxin. 28． The direct connection vector of claim 27, wherein the signal peptide is selected from the group consisting of: the pBMHPC-12 signal sequence obtained from Bombyx mori; the alipokine signal sequence obtained from Manduca sexta; the apolipophorin signal sequence obtained from Manduca sexta; The eggshell signal sequence from Bombyx mori; the cuticle signal sequence from Drosophila; the esterase-6 signal sequence from Drosophila; and the sex-specific signal sequence from Bombyx mori. 29． The direct ligation vector of claim 25, wherein said genome has been further modified to inactivate the viral egt gene. 30． 25. The direct link vector of claim 25, further comprising a nucleic acid sequence encoding juvenile hormone esterase (JHE) operably linked to a promoter capable of activating transcription in insect cells. 31． A direct ligation vector comprising genomic DNA isolated from the recombinant baculovirus of claim 16. 32． A direct ligation vector comprising genomic DNA isolated from the recombinant baculovirus of claim 19. 33． A direct ligation vector comprising genomic DNA isolated from the recombinant baculovirus of claim 23. 34． A pesticidal composition comprising a recombinant diamondback moth baculovirus (PxNPV) genetically altered relative to wild-type PxNPV and an agriculturally acceptable vector, wherein the alteration is selected from: (a) a restriction site (b) modification, deletion or duplication of a virally encoded gene; (c) introduction of a gene encoding a heterologous protein; and (d) any combination of any of the foregoing. 35． The insecticidal composition of claim 34, wherein said recombinant baculovirus incorporates in its genome a nucleic acid sequence encoding a substance modifying insects, said nucleic acid sequence being operably linked to the initiation of transcription in insect cells. child connection. 36． The pesticidal composition of claim 35, wherein said insect-modifying substance is an insecticidal toxin selected from the group consisting of AaIT, AaHIT1, AaHIT2, LqhIT2, LqqIT1, LqqIT2, BjIT1, BjIT2, LqhP35, LqhαIT, SmpIT2, SmpCT2 , SmpCT3, SmpMT, DK9.2, DK11, DK12, μ-agatoxin, King Kong toxin, Pt6, NPS-326, NPS-331, NPS-373, Tx4(6-1), TxP-1, ω-atracotoxins, α-conotoxin, μ-conotoxin, chlorotoxin and omega-conotoxin. 37． The pesticidal composition of claim 36, wherein said recombinant baculovirus further comprises a sequence encoding a heterologous signal peptide, wherein said signal peptide encoding sequence is fused in-frame with the sequence encoding said toxin. 38． The pesticidal composition according to claim 37, wherein the signal peptide is selected from the group consisting of: the pBMHPC-12 signal sequence obtained from Bombyx mori; the alipokine signal sequence obtained from Manduca sexta; the apolipophorin signal sequence obtained from Manduca sexta; The eggshell signal sequence from Bombyx mori; the cuticle signal sequence from Drosophila; the esterase-6 signal sequence from Drosophila; and the sex-specific signal sequence from Bombyx mori. 39． The pesticidal composition of claim 36, wherein said genome has been further modified to inactivate the viral egt gene. 40． 36. The pesticidal composition of claim 36, wherein said genetic alteration creates a cloning site not present in wild-type PxNPV. 41． The insecticidal composition according to claim 40, wherein said baculovirus further comprises a nucleic acid sequence incorporated at said cloning site, said nucleic acid sequence encoding a substance modifying insects, operable and able to reproduce in insect cells Promoter linkage to activate transcription. 42． 41. The pesticidal composition of claim 41, wherein said insect-modifying substance is an insecticidal toxin. 43． The pesticidal composition of claim 42, wherein the toxin is selected from the group consisting of: AaIT, AaHIT1, AaHIT2, LqhIT2, LqqIT1, LqqIT2, BjIT1, BjIT2, LqhP35, LqhαIT, SmpIT2, SmpCT2, SmpCT3, SmpMT, DK9.2, DK11 , DK12, μ-agatoxin, King Kong toxin, Pt6, NPS-326, NPS-331, NPS-373, Tx4(6-1), TxP-1, ω-atracotoxins, α-conotoxin, μ-conotoxin toxin, chlorotoxin and omega-conotoxin. 44． An insecticidal product comprising the recombinant diamondback moth baculovirus according to claim 2 and an agriculturally acceptable carrier. 45． An insecticidal product comprising the recombinant diamondback moth baculovirus according to claim 4 and an agriculturally acceptable carrier. 46． An insecticidal product comprising the recombinant diamondback moth baculovirus according to claim 7 and an agriculturally acceptable carrier. 47． An insecticidal product comprising the recombinant diamondback moth baculovirus according to claim 14 and an agriculturally acceptable carrier. 48． An insecticidal product comprising the recombinant diamondback moth baculovirus according to claim 15 and an agriculturally acceptable carrier. 49． An insecticidal preparation comprising the recombinant diamondback moth baculovirus according to claim 20 and an agriculturally acceptable carrier. 50． An insecticidal product comprising the recombinant diamondback moth baculovirus according to claim 23 and an agriculturally acceptable carrier. 51． A method of killing insects, which comprises: contacting said pests with an insecticidally effective amount of the insecticidal preparation of claim 44. 52． A method of killing insects, which comprises: contacting said pests with an insecticidally effective amount of the insecticidal preparation of claim 45. 53． A method of killing insects, comprising: applying an insecticidally effective amount of the insecticidal preparation of claim 46. 54． A method of killing insects, comprising: applying an insecticidally effective amount of the insecticidal preparation of claim 47. 55． A method of killing insects, comprising: applying an insecticidally effective amount of the insecticidal preparation of claim 48. 56． A method of killing insects, comprising: applying an insecticidally effective amount of the insecticidal preparation of claim 49. 57． A method of killing insects, comprising: applying an insecticidally effective amount of the insecticidal preparation of claim 50.

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