JP2591705B2 - Insecticidal peptides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a peptide which is effective for insecticide. More particularly, the present invention relates, inter alia, to a bactericidal peptide which can be isolated from Diguetia spider venom, a DNA encoding such a pesticidally effective peptide, a process for preparing the peptide, and an invertebrate vermin. How to control.

In recent years, people have become aware of the environmental hazards and mammalian toxicity associated with the use of synthetic pesticides. As a result, the use of these pesticides declined rapidly. However, the need for effective insect control has not changed. This has prompted researchers to develop new methods of insect control.

The most widely used microbial insecticide is derived from Bacillus bacillus sulindiene Switzerland (hereinafter Bt). This fungicide is used to control various foliate caterpillars, Japanese beetles and mosquitoes. No. 4,797,279 to Nakamura et al. (Jan. 1, 1989)
(Published on Jan. 0) describes a hybrid bacterial cell containing a gene encoding Bt crustachy delta-endotoxin and a gene encoding Bt tenebrionis delta-endotoxin, and methods for producing them.
The Bt hybrid is active against pests sensitive to the Bt kulstachyi strain, as well as against pests sensitive to the Bt tenebrionis strain. In general, these hybrids have useful insecticidal properties, in terms of insecticidal activity, or in terms of the spectrum of activity, or both, as compared to those observed by physical mixtures of the parent strains. Is also excellent. Insecticidal compositions containing such microorganisms can be used to combat insects by adding an insecticidally effective amount of the hybrid to the insect or its environment.

Another induction from bacterium Bt is Gilrlo (Gilr
oy) and Wilcox in EP-A-0 325 400 A1. The present invention relates to a hybrid virulence gene that is toxic to lepidopteran insects. More particularly, the present invention relates to a hybrid Delta-Endotoxin gene comprising a portion of the Btvar. Kurstachy HD-73 virulence gene and a portion of the virulence gene from Btvar. A hybrid virulence gene encoding a protein active against lepidopteran insects has been disclosed.

Bacterium Bt is also known as Wilcox
U.S. Patent Application Publication No. 0340948 describes their insecticidal properties. The present invention relates to a hybrid toxin, which is produced by fusing the insect intestinal epithelial cell recognition region of the Bt gene to the diphtheria toxin B chain to produce a hybrid Bt toxin active against lepidopteran insects. It has been suggested that the hybrid Bt gene can be inserted into plants or cloned into a baculovirus to produce a recoverable toxin. Alternatively, a host containing the hybrid Bt gene can be used as an insecticide by direct application to the target insect environment.

In studies of pesticidal compounds, scorpion venom has been identified as a possible source of compounds that confer insecticidal properties. Scorpion Leiurus
quinquestriatus), two insect-selective toxins isolated from the venom of Zlotkin et al., “An Excitatory and a Depres.
ssant Insect To xin from Scorpion Venom both Affec
t Sodium Conductance and Possess a Common Binding
Site "Arch Biochem and Biophysics, 240: 877-87 (19
85). Studies on its chemical and pharmacological properties have shown that one toxin induces a rapid stimulatory paralysis of the fly larva and another induces a slow inhibitory flaccid paralysis. Both affected sodium conductance.

Zloktin et al. In Canadian Patent No. 2,005,658 (issued June 19, 1990) include Scorpion (Scorpion) Laeirus quince estriatus (Hebraeus Buthinae), which is effective against insects derived from Buthidae. Various proteins are described. In this invention, the venom is lyophilized and separated into several fractions. The fraction with the highest toxicity for larvae and the lowest for mice is subjected to further purification and the final product is what is called "Lgh P35".

Griskin's “Toxic components from Buthus eupeus a
nd Lucosa singoriensis venoms, “Shemyakin Instit
ute of Bioorganic Chemistry, USSR Academy of Scienc
es, Moscow 117988, GSP-1, USSR describe four toxins that are toxic to insects isolated from the venom of Scorpion (Scorpion) Butus (eupeus). It also describes the isolation and characterization of toxic components of the venom of tarantula Lucosa singoriensis. Crude toxicity is non-toxic to insects.

In response to research and development on a variety of pesticidal compositions, researchers have been engaged in developing methods for producing pesticidal genes and introducing them into target hosts. Smith
And Summers et al., US Patent No. 4,879,236.
(Issued Nov. 7, 1989) discloses a recombinant baculovirus expression system capable of introducing a selected gene in combination with a baculovirus promoter into a baculovirus genome to express the selected gene in insect cells. It relates to a method for constructing a vector. The method involves cleavage of baculovirus DNA to generate a DNA fragment containing the polyhedrin gene or protein thereof, including the polyhedrin promoter. To create a recombinant transfer vector, insert the DNA fragment into the cloning bihirk,
The selection gene is then inserted into this modified cloning vehicle so that it is under the control of the polyhedrin promoter. The recombination transfer vector is then contacted with wild-type baculovirus DNA to effect homologous recombination and incorporation of the selected gene into the baculovirus genome. Baculovirus Autographa californica (AcMNP
V) and its related polyhedrin promoter have been found to be useful in constructing viral expression vectors capable of achieving very high levels of expression of selected genes in eukaryotic host cells.

By selecting a gene that produces a protein that is toxic to a particular insect or to the spectrum of insects, and by cloning the gene in an AcMNPV expression vector, the expression vector Can be used in systems to control They suggest that this vector is applicable to plants or animals to be protected. The recombinant virus can invade cells of the gut wall after being consumed by insects. Another suggestion is to insert the genes into the baculovirus genome and fuse them to a polyhedrin structural sequence such that the polyhedron coating dissociates under the alkaline conditions of the insect gut to release toxic products.

A further method of creating insecticidal genes and introducing them into targets to be protected is Cutler's "Electropor
ation: Being Developed to Trawsform Crops: Success W
ith Model Crop Confirmed, “AG Biotech.News vol.7
(5): 3 & 17 (1990). This document,
It teaches that DNA can be electroporated directly into germinated pollen, and that pollen returns to flowers and seeds, which can then grow into transformed plants. This method is convenient for tobacco and also for corn and eel palm. This method can be easier than protoplast electroporation, since the ultimate goal is to pollinate the flowers and "use the flowers" rather than regenerate the plants. This method collects pollen and gives it 30 to
Germinate in germination medium for 60 minutes, after which the pollen tube begins to release pollen grains and the desired DNA is added to the liquid suspension containing the pollen.
And applying an electric shock to open the pollen tube pores, remove excess DNA, and place the altered pollen on the stigma of the plant and wait for seeds to form. This is an easy way to transfer genes to cereal plants.

Additional transmission systems are described in US Pat. No. 4,861,595 to Barnes and Edwards, issued Aug. 29, 1989. This invention has been processed as a system for supplying a tactic compound to animals and humans.
It relates to the use of substantially native microbial cells. The microbial cell first produces protein in the cell via a homologous gene. The protein producing microorganism is treated by chemical or physical means, but the cells remain substantially intact. The operation of this process results in non-growth treated microbial cells without a noticeable loss of activity of intracellular compounds. Since the cells are not replicating and have a stable cell wall, this cell wall can then be disrupted at the desired location in the animal or human digestive system, and thus the timed or encapsulated product encapsulated by this invention Allows targeted release.

After appropriate treatment, the protein-producing microbial cells themselves are used as a feed system without requiring purification of the product compound. Any protein, polypeptide, amino acid or compound, including insecticides, that can be produced by microbial means can be the starting material for this invention.

The possibility of using DNA technology to incorporate synthetic genes encoding neurotoxins found in scorpion venom is described by Carbonell et al., “Synthesis of a gene cod.
ing for an insect specific scorpion neurotoxin and
attempts to express it using baculovirus vectors,
"Gene 73: 409-18 (1988).
This document scorpions, Bathus eupeus
US) teach the potential of improving the baculovirus insecticide by incorporating a synthetic gene encoding a neurotoxin found in the venom of DNA into the baculovirus genome using DNA technology. AcMNPV based on polyhedron promoter
Three expression methods using the expression system to produce toxic production were studied. Expression of the 36 codon gene alone resulted in small production of the toxin. Some success has been found by attaching the signal peptide to the toxin. Significant levels of the protein were produced when the toxin gene was fused to the N-terminus of the polyhedron gene. However, production was 10-20 fold less than that observed for polyhedron itself. The limit of expression was not believed to be at the level of transcription, but was believed to be at one post-transcription level, including translational and protein stability. No paralytic activity of the toxin product was detected.

EP-A-0 433 829 describes transgenic plants which contain insect-specific toxins of insect predators in an amount sufficient to cause toxicity to selective insects feeding on plant tissue. It is effectively expressed in those cells.
The specific toxin described has been isolated from the venom of Scorpion (Scorpion) androctonas australis.

Researchers have also been able to isolate toxins extracted from spider venom. Geren's “Neuro
toxins and Necrotoxins of Spider Venoms, “J. Toxco
l.-Toxin Reviews 5 (2): 161-170 (1986) reviews neurotoxins and necrotic toxins and suggests that spider venom molecules may provide a model for certain pesticides.

Spider, US Pat. No. 4,925,664 to Jackson and Parkes, issued May 15, 1990.
A method for treating cardiac and neurological diseases by applying toxins derived from Agerenopswiss Alpeta and Holononacurta has been described. This toxin is also effective as a specific calcium channel or excitatory amino acid receptor blocker that can be used against insects and related pests.

EP-A-0 395 357 describes polyamines and polypeptides isolated from the venom of spider agelenopsis aperta. This polyamine antagonizes excitatory amino acid neurotransmitters. Polypeptides, and one of the polyamines, block calcium channels in living cells of various organisms. The use of such calcium channel blockers in controlling invertebrate pests is suggested.

EP-A-0 374 940 describes a toxicant isolated from the venom of the spider holorena cruta. Polypeptides are useful as insecticides and in medicine, for example, as calcium channels and glutamate antagonists.

Bower et al.'S “Identifacation and purification of
an irreversible presynaptic neurotoxin from the v
enom of the spider Hololena curta, “Proc.Natl.Aca
d. Sci. 84: 3506-3510 (1987) describes a protein neurotoxin isolated from the venom of the spider (spider) Holorena cruta and its inhibition of neuromuscular transmission in Drosophila larvae. The authors suggest that the toxin blocks presynaptic calcium channels in Drosophila motor neurons.

"Paralytic and Insecticidal Toxins"
from the Funnel Web Spider, Hololona Curta, “Toxion
29 (3): 329-336 (1991) describes the effect of one peptide purified from the venom of Hololena carta and 10 cartatoxins and when injected into lepidopterous larvae.

“Curtatoxins: Neurotoxic insecti” by Stapleton et al.
cidal polypeptides isolated from the funnel−wed s
pider Hololena curta, "J. Biol. Chem. 265 (4); 2054-
2059 (1990) describes three polypeptide neurotoxins isolated from the venom of the spider (spider) holorena carta and its effect on cricket aceta domestica.

"Extended Summari" from Quicke and Yussia Wood
es Pesticides Group and Physicochemical and Biophy
sical Panel Symposium Novel Approaches in Agrochem
Physic Research, "Pestic. Sci. 20: 315-317 (1987) states that the toxicants present in the venom of the parasitic wasp and of some spider venoms cause rapid paralysis when injected into insects. The authors suggest that this spider toxicant is a blocker of a receptor-gated cation-selective membrane channel that interacts with an open channel that gates through glutamine receptors in locust muscle. This publication refers to low molecular weight toxicants in the venoms of Argiop and Araneus black and venoms isolated from the venom glands of Juro spider (Nefira clavata).

Another study of the nature of toxicants from isolated spider venom has revealed the ability of low molecular weight factors isolated from spider (funnel-web spider) venom to reversibly bind to calcium channels. Chelskiy WO
89/07608 (published August 24, 1989) states that these active low molecular weight factors reversibly bind to calcium channels with sufficient specificity and affinity to eliminate calcium conductance in neurons, It is described that it allows the isolation and purification of calcium channel structures. These venoms have been found to be toxic to humans.

Another application of spider toxicants is Jackson and Perks' “Spider Toxins: Recent Applications in Neurobiolog
y, "Ann Rev Neurosci 12: 405-14 (1989). This reference teaches that there is a very heterogeneous toxicity of various toxicants. It has been suggested that the species specificity of the channel is suggested and that spider venom may provide calcium channel antagonists, and that the spider venom discussed is found to adversely affect vertebrates. The venom has been identified as a possible source of insect-specific toxicants for agricultural use.

Adams et al. “Isolation and Biological Activity
of Synaptic Toxins from the Venom of the Funnel We
b Spider, Agelenopsis Aperta, “in Insect Neuroche
mistry and Neurophysiology 1986, Bork ovec and
Edited by Gelman, Humana Press, New Jersey, USA In 1986, it was described that a multiple peptide toxicant that antagonizes insect synaptic transmission was isolated from the spider Agelenopsis aperta.

U.S. Pat. No. 4,855,405 issued to Yoshioka et al., Issued on Aug. 9, 1989, describes a receptor inhibitor obtained from the venom gland of a female spider and a method for producing the same. This compound has an insecticidal effect when an insect contacts the liquid or solid supported compound.

U.S. Pat. No. 4,918,107 issued to Nakajima et al. (Issued on Apr. 17, 1990) describes a compound having glutamate receptor inhibitory activity, a method for producing the compound, and an insecticide composition containing the compound. I have. The compound is carried in a liquid or solid carrier with a dispersing agent and applied directly to the plant or animal to be protected. Low dosages are effective as pesticides, have very low toxicity to mammals and fish, and have only minor adverse effects on the environment.

The use of baculovirus as a biopesticide was also explored. The main disadvantage of wild-type baculoviruses as biopesticides is that they are slow-acting. Larvae consuming wild-type baculovirus generally die within 5-7 days. Infected larvae continue to consume food for a significant portion of this time, and substantial grain damage can occur.

A genetically engineered recombinant baculovirus that expresses a protein toxicant that can render a host more quickly than a baculovirus infection itself due to a combination of problems associated with synthetic insecticides, including toxicity, environmental hazards, and loss of efficacy due to antagonism There is a continuing need to develop new means of invertebrate control, including the development of invertebrates. According to the present invention there is provided a novel insecticidally effective peptide which is substantially purified and isolated from Diguetia spider venom and its agriculturally or horticulturally acceptable salts.

The insecticidal peptides of the present invention are believed to have a high degree of selectivity for invertebrates, especially insects. Moreover,
The insecticidally effective peptides of the present invention have been demonstrated to be highly effective insecticides against agriculturally important pests and are therefore believed to represent an important contribution in the field of invertebrate pest control.

Further in accordance with the present invention, there is provided a novel substantially isolated DNA sequence encoding a pesticidally effective peptide which can be substantially isolated from Diguetia spider venom.

Further according to the present invention, a DNA encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom
A novel recombinant expression vector comprising the sequence is provided. This vector is capable of expressing the coding sequence in transformed cells.

Further according to the present invention, a DNA encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom
Novel recombinant host cells are provided that have been transformed or transfected with the sequences such that the host cells express the peptide.

Further according to the present invention, a DNA encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom
Novel recombinant baculovirus expression vectors are provided that are capable of expressing the sequences in a host or mixed host cells.

Further in accordance with the present invention, there is provided a novel method of producing a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom, and a peptide produced by the method. The method comprises that a recombinant expression vector transformed or transfected in a host cell has a DNA sequence encoding a pesticidally effective peptide that can be substantially isolated from Dietia spider venom, and that the vector is transformed in the transformed cell. Culturing a recombinant host cell capable of expressing the coding sequence; and collecting a pesticidally effective peptide from the culture of the recombinant host cell.

Further in accordance with the present invention, comprising a DN sequence encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom, which is introduced into the germline of the plant or plant ancestor to express the DNA sequence A new generation of alternate plants is provided in which the trait of A. is attracted to the next generation of plants by sexual or asexual transmission.

Further in accordance with the present invention, there is provided a method for controlling an invertebrate pest comprising contacting the pest with an effective amount of a pesticidally effective peptide and an agriculturally or horticulturally acceptable salt substantially isolated from Diguetia spider venom. A new method is provided.

Further in accordance with the present invention, a pest is contacted with a recombinant baculovirus capable of expressing an effective amount of a pesticidally effective peptide and its agriculturally or horticulturally acceptable salt that can be substantially isolated from Diguetia spider venom. A novel method for controlling invertebrate pests comprising:

Further according to the present invention, there is provided a novel insecticidally effective peptide and its agriculturally or horticulturally acceptable salt, substantially isolated from Diguetia spider venom, comprising a novel agriculturally or horticulturally acceptable carrier. An insecticidal composition is provided.

Further according to the present invention there is provided a novel antibody which is substantially immunoreactive with an insecticidally effective peptide which can be substantially isolated from Diguetia spider venom and its agriculturally or horticulturally acceptable salts.

Further in accordance with the present invention, there is provided a novel method of using the antibodies of the present invention to detect the presence of an insecticidally effective peptide that can be substantially isolated from Diguetia spider venom, comprising the following steps: A novel method is provided comprising the steps of: obtaining a spider venom with an antibody bound to a detectable label; and detecting the labeled antibody bound to the peptide.

Further in accordance with the present invention is a novel method of using an antibody of the present invention to purify insecticidal peptides that can be substantially extracted from Diguetia spider venom, comprising the following steps: Binding; contacting a solution containing the peptide with an antibody bound to a solid support to attach the peptide present in the solution to the antibody; removing the peptide attached to the antibody bound to the solid support; And collecting the purified peptide.

Further, according to the present invention, there is provided a novel DNA probe derived from a DNA sequence encoding a pesticidally effective peptide which can be substantially isolated from Diggia spider venom.

Further in accordance with the present invention, a novel method for detecting the presence of a nucleic acid encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom, comprising the following steps: obtaining a spider nucleic acid; A novel method is provided that comprises contacting a nucleic acid with a DNA probe of the present invention; and detecting a probe bound to the nucleic acid.

FIG. 1 shows the RP HPLC of the whole Digutia canis venom, a RP HPLC using a gradient of 0.1% TFA: acetonitrile showing the three groups of components tested in mice. Fraction 2
Represents a TBW active ingredient.

Figure 2 shows Scha in slices of rat hypocampal.
FIG. 9 shows DK9-2 tested at 1 μM of synaptic transmission (excited population spike) at ffer colletal CA-1 pyramidal cell synapses. The data drawn is (a) DK9.2
(B) Recording of time averaged population spikes 5 minutes before addition and (b) 15-20 minutes after addition of DK9.2. These records are the same, indicating that at this concentration, DK9.2 has no detectable rat CNS activity in this assay.

FIG. 3 is a map of the baculovirus conversion vector pWR9 carrying the gene encoding preDK9.2.

FIG. 4 is a continuous virus feeding assay of Neonate tobacco caterpillars (1000,000 FIB / gm diet).

FIG. 5 shows the ability of TTX to block or reverse DK9.2-induced bursting.

A. Definitions As used herein, a "pesticidally effective peptide that can be substantially isolated from a Diguetia spider venom" is a peptide that is pesticidally effective as well as the peptide is substantially isolated from Diguetia by any of the techniques known in the art. And insecticidal fragments of peptides from all techniques as long as they can be isolated. Such resources include, for example, recombinantly produced insecticidally effective peptides.

The “expression vector” used here is the DN contained therein.
Includes vectors that can express the A sequence and that such sequence is operably linked to other sequences capable of effecting its expression. Although not always explicitly stated,
It is implicitly stated that these expression vectors must be reproducible in the host organism either as episomes or as an integral part of chromosomal DNA.
Obviously, the lack of replication capability makes them effectively inoperable. In summary, an `` expression vector '' is given a functional definition, in that all DNA sequences capable of effecting the expression of the specialized DNA code dealt with here are utilized for the specific sequence. Is included in this term. Generally, expression vectors utilized in recombinant DNA technology are often in the form of "plasmids", which
Called heavy helical DNA, it is not attached to the chromosome in the form of its vector. "Plasmid" and "vector" are used interchangeably. Plasmids are the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors that serve equivalent functions and become known in the art.

"Recombinant host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA technology.

The spider family Diguetidae currently consists of a single genus Diguetia. Diguetia species are commonly found in the southwestern United States (including California) and Mexico. These little spiders lay vast irregular nests on many kinds of plants and shrubs, including cacti. The various Diguetia species, like most spiders, are generally carnivores and readily prey on the many types of captive insects they encounter.

Although the invention is not meant to be limited by any theoretical reason, the unusual symptoms seen when injecting insects with the insecticidally effective peptides of the invention are ilkaloid Na ⁺ channel activators. Very similar to the symptoms seen when insects are injected with scorpion toxicants from the family Buthidae and the genus Buthus, known as veratridine or Na ⁺ channel activators. It is believed that this insecticidally effective peptide can cause partial depolarization of the muscle and / or nerve membrane, possibly affecting Na ⁺ or K ⁺ channels. More specifically, it is believed that this insecticidally effective peptide induces a repetitive burst discharge in nerves sensitive to tetrodotoxin blocking. Thus, perhaps this is a voltage-sensitive sodium channel on the neural membrane that is the site of action of the peptide.

B. Isolation of peptides from Diguetia venom Spider venom can be removed from Diguetia by known methods, such as spider venom gland extraction from Cephalotrax. However, to avoid spider venom and impurities inside the isolated venom, the spider venom is preferably obtained by electrically stimulating the spider to release the venom, and then collecting the released venom by suction.
Prevention of venom contamination by exhalation or hemolymphocytes is described in U.S. Pat. No. 4,925,664.

If the spider venom is obtained by an electrical milking technique, it is a high performance liquid chromatography ("HPLC")
Alternatively, the peptide (toxic) component can be fractionated using gel filtration chromatography or other useful separation techniques. It is also often desirable for the final fractionation of spider venom performed by HPLC.

Thus, the spider electro-milking technique is used in combination with gel filtration chromatography and / or high performance liquid chromatography and other related techniques such as hydrophobic interaction chromatography to obtain a substantially purified spider venom. be able to. However, it will be appreciated that other equivalent techniques can be used within the scope of the present invention to isolate spider venom. The toxicant thus isolated can be tested for amino acid sequence and insecticidal activity by methods well known to those skilled in the art.

C. Insecticidally Effective Peptides In one aspect, the invention relates to insecticidally effective peptides and insecticidally effective fragments thereof that can be substantially purified and isolated from Diguetia spider venom, and agriculturally or horticulturally acceptable. Provide their salts.

Once the pesticidally effective peptide-containing fraction has been isolated and purified, the amino acid sequence can be determined by methods well known to those skilled in the art, such as N-terminal amino acid sequencing and the use of an automated amino acid sequencer.

From this disclosure, it will be appreciated that additional insecticidally effective proteins are expected to be within the scope of the present invention. That is, it is believed that other pesticidally active peptides can be isolated from Diguetia in addition to the three species detailed herein. Members of this family of insecticidal peptides isolated from Diquetia are thought to share the following properties:

1) Size: all range from about 6200 to about 7200 daltons and from about 55 to about 65 amino acids in length; 2) Conserved amino termini: SEQ ID NOs: 1, 3 and 5 are the same for the first 5 amino acids 3) SEQ ID NOs: 1, 3 and 5 have greater than 40% sequence homology; 4) Sequences: 1, 3 and 5 have about 7 or 8 cysteine residues; 5) Genes Clustering: The genes for one or more of these toxicants appear to be co-localized portions of genomic DNA, possibly because all of these genes were dissociated from those of a single ancestor. This is a sign that

More specifically, three insecticidally effective peptides were isolated and confirmed. First, DK9.2 has been isolated and its amino acid sequence appears as defined in SEQ ID NO: 1, as translated from the isolated cDNA. Mass spectroscopy data suggests two isoenzymes containing a conservative substitution at position 26. One isoenzyme contains trenion at position 26 and the other contains glutamine at position 26. Glutamine isoenzyme is about 27 atomic units larger than threonine isoenzyme. All references to D9.2 hereinafter should be construed as referring to either and / or both isoenzymes. DK9.2 is characterized by a molecular weight of about 6371-6397 daltons as determined by mass spectrometry and by migration as a single peak on reverse phase HPLC. Second, when DK11 is isolated and determined by mass spectrometry,
It is characterized by a molecular weight of 6700 daltons or about 6740 daltons and by migration as a single peak on reverse phase HPLC. More specifically, this active HPLC fraction was identified as having the amino acid sequence when translated from the isolated cDNA, as shown in SEQ ID NO: 3. Finally, to date, DK, the third member of the protein family that is effective in killing insects
12 is about when determined by mass spectrometry
It was characterized by a molecular weight of 7100 daltons or about 7080 daltons and by migration as a single peak in reverse phase HPLC. More specifically, it was tentatively shown that this active ingredient fraction had an amino acid sequence as shown in SEQ ID NO: 5. The three peptides isolated correspond to acid SEQ ID NOs: 1, 3 and 5 and show substantially the same insecticidal activity. Acid sequence number 1, 3
Alternatively, other peptides having about 55-65 amino acids and greater than about 40% sequence homology and having about 7 or 8 cysteine residues are expected to exhibit substantially the same insecticidal activity. Such peptides are naturally occurring or can be produced by recombinant DNA technology methods known to those skilled in the art. Such peptides, whether naturally occurring or produced by recombinant technology,
It is intended to be within the scope of the present invention.

D. Identification of the Insecticidally Effective Peptide Coding Sequence of the Invention According to another aspect of the present invention, a substantially isolated pesticidally effective peptide encoding a substantially pesticidally effective peptide from spider venom is provided. A DNA sequence is provided.

Using the partial amino acid sequence data obtained as described above, genes that can be involved in the production of proteins that can be isolated from spiders can be isolated and identified. Numerous methods are available for obtaining genes that can be involved in peptide production. For example, Fuqua, S. et al., “A
Simple PCR method for detection and cloning low ab
undant transcript, “Biotechnique, vol. 9 No. 2 (199
March 2010); “Race: Rapi” by Frohman (MA)
d amplification of cDNA ends ", edited by PCR protocols, Inisis et al., published in Academic Press (1990) in San Diego, CA; and US Patent 4,70.
No. 3,008, “DNA Sequences Encoding Erythropoiet
in ". The above U.S. patent specification is incorporated herein by reference.

In summary, encodes the determined amino acid sequence,
Or such encoding the determined amino acid sequence
A DNA molecule is synthesized that represents a complementary DNA strand to the DNA molecule. This synthetic DNA molecule can then be used to probe for DNA sequence homology in the cell clone containing the recombinant DNA molecule. Recombinant DNA molecules are cDNAs of mRNA derived from genomic DNA of an organism such as a spider, or isolated from cells or tissues of an organism such as a spider.
It partially contains the DNA sequence derived from the copy. Generally, DNA molecules of 15 or more nucleotides are required for identification of homologous DNA specificity. This number requires a determination of the specificity of at least 5 amino acids in the sequence. Each amino acid is a unique trinucleotide DNA up to 6
Can encode a sequence or codon, and thus can encode a different amino acid sequence.
It will be appreciated that the number of DNA molecules can be very large. Therefore, it is not practical to test every possible synthetic DNA probe individually, and a pool of some such DNA molecules is used concomitantly as a probe. The production of such pools, called "denatured" probes, is well known in the art. Although the only DNA molecule in the probe mixture will have exact sequence homology to the gene in question, only a high degree of homology is required, so some synthetic DNA molecules in the pool may It will also be appreciated that the gene can be specifically identified. Therefore, satisfactory isolation of the gene in question can be achieved using a pool of synthetic DNA probes that do not contain all possible DNA probe sequences.
In general, codons rarely used by an organism need not be represented in the probe pool. In fact, single-sequence DNA probes are made by including only the DNA codon most frequently used by the organism for each amino acid. However, it will be appreciated that this attempt is not always successful.

One technique for identifying gene sequences uses the replication chain reaction (PCR). For example, U.S. Pat.
See No. 95 and 4,683,202. In essence, PCR allows the production of a selected DNA sequence when the two terminal portions of the sequence are known. Primers corresponding to the respective ends of the sequence in question, oligonucleotide probes, are obtained. Using PCR, a central portion of the DNA sequence is then produced synthetically.

One way to use such PCR to obtain a gene encoding a unique spider venom gene is to use RNA
Is isolated from spiders and purified. The spider RNA is then reverse transcribed into cDNA using the deoxythymidylate terminal oligonucleotide as a primer. As in the denatured probes described above, the synthetic DNA molecule or mixture of synthetic DNA molecules is produced as being capable of encoding the amino terminal amino acid sequence of the venom protein determined above. This DNA
The mixture is used with a deoxythymidylate-terminated oligonucleotide to initiate a PCR reaction. Since the synthetic DNA mixture used to initiate the PCR reaction is specific for the desired mRNA sequence, only the desired cDNA is effectively amplified. The synthetic product represents an amplified cDNA that can be ligated into any of a number of well-known cloning vectors. Without opposing this, the "family" of peptides can be present in spider venoms with similar amino acid sequences, and in such cases, the use of mixed oligonucleotide primer sequences will reduce the number of these related peptides It will be appreciated that this results in the amplification of one or more of the cDNAs involved. Genes encoding related peptides are also within the scope of the invention. This is because related peptides also have useful insecticidal activity.

Finally, the cDNA sequence produced can be cloned into a suitable vector using conventional techniques, analyzed and the nucleotide sequence determined. DNAs encoding proteins effective for insecticide are, for example, acid SEQ ID NOs: 2 and 4
Is represented by Direct amino acid translation of these PCR products will reveal that they correspond to the complete coding sequence for the mature protein.

In addition to the cDNA encoding mature DK9.2, the upstream of the cDNA sequence of this DK9.2 coding sequence was cloned and acidified (acid sequence number 7). Translation of this complete mRNA revealed that DK9.2 was synthesized as a precursor protein containing the signal peptide, propeptide and its mature toxicant. The function of the signal peptide is considered to be important as a target for secreting the synthetic polypeptide. Signal sequences play an important role in ensuring the proper localization of newly synthesized proteins. Generally, they are "topogenic signals" (Proc. Nat. Acad. Sci., Blobel, G., USA 77, 1496-1500 (1980)).
Which target protein sequences attached for various purposes internal or external to the cell. This is particularly important for secreted proteins whose target sites are extracellular. When it can be easier for recombinant protein production, it is also useful to purify the expressed protein from the extracellular medium and from whole cell extracts rather than to degrade the cells . CDNA encoding the precursor protein of DK9.2
The signal peptide encoded by 17 of the following sequence
It is believed that it consists of amino acids.

In addition to the Met-Lvs-Val-Phe-Val-Val-Leu-Leu-Cys-Leu-Ser-Leu-Ala-Ala-Val-Tyr-Ala signal peptide, it was located upstream of the DK9.2 coding sequence. Translation of the mRNA represented the propeptide. The function of the propeptide is not known. The propeptide is a 21 amino acid peptide of the sequence

-Leu-Glu-Glu-Arg-Leu-Asp-Lys-Asp-Ala-Asp-Ile-Met-Leu-Asp-Ser-Pro-Ala-Asp-Met-Glu-Arg- These precursor peptides, namely prepro sequences Can greatly contribute to the stability, expression and folding of DK9.2 or other recombinant proteins when expressed in vivo and / or in vitro. It is also foreseen that these sequences, or portions thereof, may prove useful for the expression of other molecules, for example for the construction of genes.
As part of the present invention, we also use other signals and / or
Alternatively, data could be provided to confirm that the propeptide sequence can be used for expression of recombinant DK9.2.

E. Expression of Recombinant The present invention further provides a recombinant expression vector comprising DNA encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom. This vector allows expression of the coding sequence in transformed cells.
Also according to the present invention, a host cell is transformed or transfected with a DNA sequence encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom, such that it expresses the peptide. , A recombinant host cell is provided.

Provision of the appropriate DNA sequence encoding the desired protein allows the production of the protein to be performed using recombinant techniques now known in the art. The cryptographic sequence is
It can be obtained by recovering the cDNA or genomic sequence from the natural source of the protein, or it can be produced synthetically using the exact amino acid sequence determined from the nucleotide sequence of the gene. When this coding DNA is produced synthetically, it has the advantage that it is possible to adopt well-known codons of the intended host.

The necessary regulatory sequences, such as promoters, and, preferably, expression machinery, including enhancers and terminal controls, are readily available and are known in the art for a wide variety of hosts. For example, Molecular cloning a Laboratory by Sambrook et al.
See Manual, 2nd Edition, Cold Spring Harbor Press (1989).

Thus, the desired protein can be produced in both prokaryotic and eukaryotic systems, which in many cases result in a spectrum of the processing system.

The most commonly used prokaryotic system is E. coli, but other systems, such as B. subtilis and Pseudomonas, are also expected to be useful. Suitable regulatory sequences for prokaryotic systems include both constitutive and inducible promoters, including the Iac promoter, trp promoter, hybrid promoters such as the tac promoter, lambda phage PL promoter.
In general, heterologous proteins can be produced in these hosts as either fusion or mature proteins.
When the desired sequence is produced as a mature protein, the sequence produced may be preceded by methionine, which is not necessarily effectively removed. Thus, the peptides and proteins required here can be preceded by an N-terminal methionine when produced in bacteria. Moreover, the structure is created such that the coding sequence of the peptide precedes the actuation signal peptide that results in secretion of the protein. When so produced in a prokaryotic host, the signal sequence is removed upon secretion.

A wide variety of eukaryotic hosts are now available for the production of recombinant heterologous proteins. As in bacteria, eukaryotic hosts can be transformed with expression mechanisms that produce the desired protein directly, but more usually a signal sequence is provided to achieve secretion of the protein. Eukaryotic systems have the added advantage of being able to handle possible introns in genomic sequences encoding proteins of higher organisms. Eukaryotic systems also provide various processing mechanisms that result, for example, in glycosylation, oxidation or derivatization of some amino acid residues, acid site control, and the like.

Eukaryotic systems commonly used include yeast, insect cells, mammalian cells, bird cells, and higher plant cells. This list is not all. Suitable promoters that are compatible and operable for use in each of these host systems can be utilized, as well as termination sequences and enhancers, such as the baculovirus polyhedrin promoter. As mentioned above,
Promoters can be either constitutive or inducible. For example, in a mammalian system, the MTII promoter can be induced by the addition of heavy metal ions.

The structural features of the expression machinery suitable for the desired host are known to those skilled in the art. For recombinant production of a protein, the DNA encoding the protein is suitably ligated to the selected expression, the system is then transformed into a suitable host, and then cultured under conditions that permit expression of the foreign gene. Will be retained. The insecticidally active components of the invention thus produced are recovered from the culture by lysing the cells or by separation from a medium suitable and well known to those skilled in the art.

It is understood that slight variations in the main amino acid sequence can result in proteins having substantially equal or increased activity compared to the peptides exemplified herein. These mutations can be attributed to site-directed mutagenesis or to accidental mutations, such as by mutation of the host producing the peptide of the invention. All these mutations are included as long as insecticidal activity is retained. Mutations in a protein alter its primary structure based on changes in the nucleotide sequence of the DNA that encodes it (usually occurring or specifically described proteins). These mutations particularly include allelic variants. Mutational changes in the primary structure of the protein are attributable to deletions or substitutions. "Deletion" is defined as a polypeptide in which one or more internal amino acid residues are absent. "Addition" is defined as a peptide having one or more additional internal amino acid residues compared to wild type. "Substitution" results from the replacement of one or more amino acid residues by other residues. A protein "fragment" is a polypeptide consisting of a primary amino acid sequence that is equal to a portion of the primary amino acid sequence of the protein to which the polypeptide relates.

Preferred "substitutions" are those that are conservative, that is, one residue is replaced by another substituent of the same general type. As will be appreciated, natural amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and non-polar and / or aromatic.
It is generally preferred that encoded peptides that differ from the native form contain a substitution codon for an amino acid that is derived from the same group as the substituted amino acid.

Thus, in general, the basic amino acids Lys, Arg, and His
Are interchangeable; acidic amino acids Asp and Glu are interchangeable; neutral polar amino acids Ser, Thr, Cys, Gln, and Asn are interchangeable; non-polar aliphatic acids Gly, Ala, Va
l, Ile, and Leu are mutually conservative (but Gly and Ala are more closely related due to their dimensions, and
Val, Ile and Leu are more closely related); and the aromatic amino acids Phe, Trp, and Try are interchangeable.

Although Pro is a non-polar neutral amino acid, it presents a drawback because of its influence on the conformation. Pr except when the same or similar conformational results can be obtained
Substitution by o or in place of Pro is not preferred. Polar amino acids that represent conservative changes include Ser, Thr, Gln, Asn and, to a lesser extent, Met. In addition, although classified into different categories, Al
a, Gly and Ser appear to be interchangeable, and Cys
Can additionally fit into this group or be classified as polar neutral amino acids. Substitution of codons for amino acids from different classes may also be useful.

Because of the provision of recombinant materials for the proteins of the present invention, these proteins can be produced by recombinant techniques, as well as by automated amino acid synthesizers.
Due to the changes in post-translational properties conferred by various host cells, various modifications can be made to naturally occurring proteins. As a result of the glycosylation, amidation or lipidation pattern of a protein, or post-translational changes that alter the primary, secondary or tertiary structure of a protein, "modification (mo
dified) "proteins are different from unmodified proteins but are, of course, included within the scope of the invention as claimed.

It should further be noted that when the proteins described herein, for example SEQ ID NO: 1, are made synthetically, substitutions by amino acids not encoded by the gene may also be made. Examples of alternative residues include, for example, the formula H ₂ N (C
H ₂ ) _n COOH (n is 2 to 6) ω amino acid. These are neutral, non-polar amino acids such as sarcosine (Sar), t-butylalanine (t-BuAla), t-
Butylglycine (t-BuGly), N-methylisoleucine (N-MeIle), and norleucine (Nleu). For example, phenylglycine can be substituted for the aromatic neutral amino acids Trp, Tyr or Phe, citrulline (Cit) and methionine sulfoxide (MS
O) is polar but neutral, cyclohexylalanine (Cha) is neutral and non-polar, cysteic acid (Cya) is acidic, and ornithine (Orn) is basic. The conformational identity of proline is obtained when one or more of these is replaced by hydroxyproline (Hyp).

F. Transgenic plants Further according to the present invention, DNA encoding a pesticidally effective peptide which can be substantially isolated from Diguetia spider venom
Provided is a transformed plant comprising the sequence introduced into the germline of the plant such that expression of the DNA sequence is passed to the next generation of the plant through sexual or asexual growth.

According to the present invention, a gene encoding a peptide effective for insecticide can be introduced into plants by genetic engineering techniques. The production of peptides in plant cells by this technique is expected to be useful as a means for controlling insect pests. Therefore, it is possible to produce insect-resistant plants that are larger than natural species.

The coding region for a pesticidally effective peptide gene that can be used to transform plants can be full length or partially active length of the gene. However, it is necessary to express the gene sequence encoding the peptide and produce it as a functional peptide in the resulting plant cells. Genomic DNA and cDN encoding peptides that are effective in killing insects
Transformation can be performed using both A and synthetic DNA. In addition, it may be partially composed of gene cDNA clones, partially genomic clones, and partially composed of synthetic genes and various combinations thereof. Also, the DNA encoding the peptide gene can include portions from various species other than the isolated peptide source.

Moreover, the pesticidally effective peptide can be combined with another compound (s) and can produce unexpected pesticidal properties in transformed plants containing the chimeric gene and expressing this compound. I believe I can. These other compounds include, for example, protease inhibitors with oral toxicity to insects or Bacillus.
And a polypeptide derived from thuringiensis. Bacillus thuringiensis protein causes a change in the potassium permeability of the intestinal cell membranes of insects and generates pores in the membrane. Other pore-forming proteins can also be used in combination with insecticidal peptides. Examples of such small forming proteins include magainin, cecropin, atasin, mayitin, gramicidin S, sodium channel protein, and synthetic fragments, Staphylococcus aureus α-toxin, apolipoproteins and fragments thereof, alamethicin and various Is a synthetic amphipathic peptide. Lectins that bind to cell membranes and enhance endocytosis are another class of proteins that can be used in combination with the insecticidal peptides of the present invention to genetically alter plants to insect resistance.

Peptide gene promoters are expected to be useful for expressing chimeric gene sequences, but other promoters are also expected to be useful. Effective plant promoters that are useful are hyperproducer promoters. The promoter, which is operably linked to the gene sequence of the peptide, should be capable of promoting expression of the peptide such that the transformed plant has increased resistance to the pest. Superproducing plant promoters that are expected to be useful in the present invention are well known. A chimeric gene sequence containing a pesticidally effective peptide gene operably linked to a promoter can be ligated into a suitable cloning vector to transform the desired plant. Generally, a plasmid or viral (bacteriophage) vector containing replication and regulatory sequences derived from a species compatible with the host cell is used. Cloning vectors will typically have a source of replication and at the same time have a phenotypic selectable marker in the transformed host cell, typically a specific gene that can provide resistance to the antibiotic. Transformation vectors can be selected by these phenotypic markers after transformation in the host cell.

Bacterial hosts such as E.coli, Salmonella reifimlium (ryphim
urium), and Serratia marcesce (marcesce)
s); and eukaryotic hosts such as yeast or filamentous fungi.

Cloning vectors and host cells transformed with the vectors are generally used to amplify the copy number of the vector. The amplified copy number can be used to isolate a vector containing the peptide gene and used, for example, to introduce the gene sequences described herein into plants or other host cells.

Methods for producing plants that express foreign genes are known.
For example, a plant tissue is called A. tumefa
by direct infection or co-culture of plants, plant tissues or cells: direct gene transfer of prosthetic DN to protoplasts; inoculation of PEG; microinjection and microprojectile bombardment. Can be transformed.

Transformation of tobacco by electroporation is described in Ag. Biotechnology News, Vol. 7, p.
17 (September / October 1990 issue). In this technique, plant protoplasts are electroporated in the presence of a plasmid containing an insecticidal peptide gene structure. The high field strength electrical impulse permeates the biological membrane to allow for plasmid transfer. Electroporated plant protoplasts reconstitute the cell wall and differentiate to produce plant callus.
Selection of transformed plant cells with the expressed insecticidally effective peptide is accomplished using the phenotypic markers described above.
Exogenous DNA can be in any form, such as naked linear, circular or supercoiled DNA, liposome-encapsulated DNA, DNA in spheroplasts, D in other plant protoplasts.
NA, salt-complexed DNA and other forms can be added to protoplasts.

All plant cells that can be transformed by Agrobacterium, and all plants regenerated from the transformed cells, are also transformed according to the present invention to produce transformed whole plants that contain the transformed insecticidally effective peptide gene. You can also. Transformation of rice is carried out by DM Rainin et al., "Agrobacterium-mediated tra
nsformation of rice (Oryza satival.) ", Biote chno
logy, Vol. 8, pp33-38 (January 1990).

Another method for introducing a peptide gene effective for sterilization into plant cells is to transform a plant cell with a peptide gene effective for insecticide. Infection with Tumefaciens. Under appropriate conditions known in the art, the transformed plant cells are grown to germinate and produce roots,
It is also to grow a transformed plant. Peptide sequences effective for insecticide can be provided in suitable plant cells, for example, A. It can be introduced by the T. faecalis Ti plasmid. Ti plasmid is A. It is transmitted to plant cells during infection with Tumefaciens and is stably integrated into the plant genome.

The Ti plasmid contains two regions that are believed to be essential for the production of transformed cells. One of these is called transfer DNA (T DNA) and induces tumor formation.
The other is called a toxic zone and is essential for tumor formation but not for its maintenance. T DNA transferred to plant genome
The region can be increased in size by insertion of the gene sequence of the enzyme without affecting its transposition ability. By removing the oncogenic genes so that they no longer interfere, the improved Ti plasmid can then be used as a vector for transferring the gene constructs of the present invention to appropriate plant cells.

Genetic material also includes polyethylene glycol (PEG)
Can be used to transfer to plant cells. PEG forms a precipitate complex with the genetic material taken up by cells.

Transfer of DNA into plant cells can also be accomplished by injection into isolated protoplasts, cultured cells and tissues and into meristems of young plants and plants. Transformed plants and their progeny can be obtained by conventional methods known in the art.

Another method of introducing foreign DNA sequences into plant cells consists of attaching DNA to the particles and forcing them into the plant cells with a shooting device called a "gene gun". All plant tissues or plant organs can be used as targets in this method, including embryos, apical and other meristems, buds, body tissues and reproductive tissues in vivo and in test subjects. However, the present invention is not limited to these. Transformed cells and calli are selected by the following established methods. The tissue of interest is induced to form somatic embryos or regenerating shoots, and transformed plants are provided by established methods known in the art. Suitable methods are selected according to the plant species used. Transformed corn plants have been produced by transferring genes to embryogenic cells using high speed microprojectiles. “Inheritance and expression of Chimeric g
enes in the progeny of transgenic maize plants ", B
See iotechnology, Vol. 8, pp833-838 (September 1990).

The regenerated plant can be chimeric to the incorporated foreign DNA. If cells containing foreign DNA develop into either microspores or macrospores, the synthesized foreign DNA is transmitted to the reproductive organs. If the cell containing the foreign DNA is a somatic cell of the plant, the non-chimeric transformed plant can be harvested in vivo or in vitro from cutting or stem cutting by the following established methods known in the art. Produced by normal methods of (asexual) propagation. Such a method is selected depending on the plant species used.

After transformation of the plant cells or plants, those plant cells or plants that have been transformed to express the peptide are selected by a suitable phenotypic marker. These expression markers include, but are not limited to, antibiotic resistance. Other phenotypic markers are known in the art and can be used in the present invention.

A variety of different transformation systems can be used to transform all plant types, in principle, so that they express the insecticidally effective peptides of the invention.

There is increasing evidence that virtually all plants can be regenerated from cultured cells or tissues. Examples of these include, but are not limited to, all major cereals, sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Knowledge of whether all of these plants can be transformed by Agrobacterium is currently limited. Species that are natural plant hosts for Agrobacterium can be transformed in vitro. Monocotyledonous plants, especially cereals and grasses, are not natural hosts for Agrobacterium. Attempts to transform them using Agrobacterium have been unsuccessful to date. There is now increasing evidence that certain monocotyledonous plants can be transformed by Agrobacterium. Cereal and grass seeds can also be transformed using the new experimental methods now available.

Additional plant species that can be transformed by Agrobacterium include Ipomoea, Passiflora, Cyclamen, Malus, Prurus, Rosa, Rubus, Populus, Populas), Santalion, Allium, Liliu
m), Nacissus, Ananas, Arachis, Phaseolus and Pisum.

Regeneration varies from plant species to species, but generally a suspension of transformed protoplasts containing multiple copies of a pesticidally effective peptide gene is first provided. Embryogenesis is then induced to the stage of maturation and germination like a natural embryo from a protoplast suspension. The medium generally contains various amino acids and hormones. Shoots and roots usually occur simultaneously. Efficient regeneration depends on the medium, the genotype and the origin of the culture. If these three variables are controlled, regeneration is fully reproducible and repeatable.

Mature plants grown from transformed plant cells can self-propagate to give rise to inbred plants. This inbred plant produces seeds containing the gene for the insecticidally effective peptide. These seeds can be grown to produce plants that express the insecticidally effective peptide. Insect breeding can be used to develop insect resistant hybrids. In this way, an insect resistant inbred line crosses the inbred line to produce a hybrid.

In a diploid plant, typically one parent can be transformed with a pesticidally effective peptide (toxin) gene sequence and the other parent is a wild species. After mating of the parents, the first generation hybrid (F ₁ ) shows a distribution of 1/2 toxin / wild type: 1/2 toxin / wild type. These first generation hybrids (F ₁ ) self-propagate to produce second generation hybrids (F ₂ ). Gene distribution of F ₂ hybrid 1/4 toxin / toxin: 1/2 toxin wild species: 1/4 wild type / wild species. F ₂ hybrids with the genetic properties of the toxin / toxin are chosen as the insect tolerant plants.

Variants as used herein are stable and heritable, and describe phenotypic changes that include inherited mutations transmitted sexually to plant progeny. However, the mutant still expresses the insecticidally effective peptide of the present invention. Also, as used herein, a mutant is a result of environmental conditions, for example, as a result of a genetic variation in which the trait is meiotically transmitted by radiation or by well-known rules of inheritance. Describe changes. However, the mutant plant must still express the peptide of the invention.

In general, the ideal pesticidally effective peptide selected to be expressed in the transformed plant is one that is characterized by its safety against non-target insects and vertebrates. The expression system is selected such that the level of expression confers insecticidal efficacy. Therefore, this technical ease of obtaining such agriculturally important transformed plants is an additional benefit of using complete pest tubing to reduce insect damage to crops in an environmentally responsible manner. It is expected to give weapons to farmers.

G. Application of Peptides as Insecticides The insecticidally effective peptides of the present invention can be obtained by contacting a pest with an effective amount of a peptide of the present invention to produce Lepidoptera
pideptera) is effective in controlling invertebrate pests. Conveniently, insects are the preferred pests to control.

Methods of controlling an invertebrate pest by contacting the pest with a peptide are known. Examples include proteins for oral inoculation of insect pests that are synthetically encapsulated. A recombinant host expressing the protein of the present invention, for example, Pseudomonas
Fluorescens, killed by heat, can be suitable for pests for subsequent ingestion and control.

Of course, the method of controlling invertebrate pests using the proteins of the present invention can also be used in combination with other pest control methods. For example, the above-described transformed plants and E. coli can be treated to express other invertebrate toxins, depending on the type of pest to control and other key variables present.

Also provided is an insecticide composition comprising a pesticidally effective amount of a peptide according to the invention and an agriculturally or horticulturally acceptable salt thereof in an agriculturally or horticulturally acceptable carrier.

H. Antibodies against insecticidally effective peptides According to another aspect of the present invention, there are provided antibodies against the insecticidally effective peptides of the present invention. In the following description, reference is made to various methodologies well known to those skilled in the art of immunology for detecting and purifying peptides reactive with the antibodies described herein.

An antibody is said to be "bindable" to a molecule if the antibody specifically reacts with the molecule to bind the molecule to the antibody. The term "epitope" refers to that portion of a peptide antigen that can be recognized and bound by an antibody. An antigen can have one or more epitopes.
An "antigen" can elicit an animal to produce antibodies capable of binding an epitope of the antigen. The above specific reaction is meant to indicate that the antigen immunoreacts with its corresponding antibody in a highly selective manner and does not react with many other antibodies that can be induced by other antigens.

As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) refers to an intact molecule capable of binding to an antigen and fragments thereof (eg, Fab and F (ab ')).
₂ fragments). Fab and F (ab ') ₂ fragments, more readily apparent from circular, lack the F _c fragments of intact antibody, may have fewer non-specific tissue binding of an insect antibody.

The antibodies of the present invention can be produced by any of a variety of methods. Methods for producing such antibodies are well known and are fully described in the literature. For example, "Molecular Cloning a laborato" from Sambrook
ry manual ", 2nd edition, Cold Spring Harbor Press, Vol. 3, Ch. 18 (1989). For example, cells expressing a pesticidally effective peptide or fragment thereof may be administered to animals. To induce the production of serum containing a polyclonal antibody capable of binding to the pesticidally effective peptide.In general, pesticidally effective peptide fragments are produced and purified to substantially eliminate natural contaminants. Peptide fragments, either free or insecticidally effective, are synthesized by methods known in the art, either purified fragments or synthesized fragments or purified natural fragments and / or synthetic. The combined fragments are administered to animals to produce polyclonal antisera with high specific activity.

Monoclonal antibodies can be produced using well-known hybridoma technology. In general, such methods involve immunizing an animal with a peptide antigen that is effective in killing insects. The spleen cells of such animals are extracted and fused with a suitable myeloma cell line. All suitable myeloma cell lines can be used according to the present invention. After fusion, the resulting hybridoma cells are selectively kept in a suitable medium and then cloned by limiting dilution. The hybridoma cells obtained by such a selection are then analyzed to identify clones secreting antibodies capable of binding to the insecticidal peptide antigen.

If the peptide source is impure, only a few of the hybridoma cells will produce antibodies that can bind to the peptide (other hybridoma cells will produce antibodies that can bind to peptide contaminants). Therefore, it is necessary to search (screen) hybridoma cells for those capable of secreting antibodies capable of binding to the peptide. Such a screen may culture a sample of the peptide (or venom) in the presence of a monoclonal antibody secreted from each of a particular group of hybridoma cells and neutralize or attenuate the ability of the venom to paralyze insects. It is preferably achieved by identifying a hybridoma capable of secreting the antibody. Once such a hybridoma has been identified, it can be propagated as a clone by methods known in the art to produce a peptide-specific monoclonal antibody.

In order to purify insect-selective toxins, natural or recombinant, using antibody affinity chromatography, insecticidal requires the use of antibodies that can bind to effective peptides. Generally, such antibodies are monoclonal antibodies. Once a peptide-specific monoclonal antibody is obtained, it can be immobilized by conjugation to a solid support and the peptide from natural toxins or other sources can be purified using immunoaffinity chromatography by methods well known in the art. And can be used for purification. Such methods can arbitrate for a high degree of purification and produce peptides that are substantially free of natural contaminants. As used herein, if a peptide is present in a form that lacks naturally and normally associated compounds (ie, other proteins, lipids, hydrocarbons, etc.), the peptide will "substantiate natural contaminants" Not included in
It is said to be a thing.

Once the peptide has been purified, it can be used to immunize an animal (eg, a mouse or rabbit) to effect the production of a peptide-specific polyclonal antibody.

A suitably sized, generally 10-50 nucleotide, DNA probe can be derived from a DNA sequence encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom. Such a probe may be obtained by contacting the DNA probe with the venom and the DNA probe, and contacting the DNA probe with a DNA encoding a pesticidally effective peptide, which can be substantially isolated from Diguetia spider venom. It can be detected by detecting in a method.

I. Genetically Engineered Pesticidal Microorganisms Pesticidally effective peptides alone or in combination with other insect toxins are expected to be useful in activating or increasing the virulence of microorganisms such as baculoviruses and hybrid bacteria. You.

Heliothis virescens
(Cotton bollworm), Orgia pseudodagger (Or
gyia pseudotsugata) (Douglas fir tussock mot
h), Limantia dispar (g)
ypsy moth), Autographer California (Auto
grapha Californica) (alfalfa looper), Neodiprion sertifer
(European pine fly) and Lanspcyresia pomonella (codling moth)
A variety of baculoviruses, including those that infect E. coli, are registered in several countries and used as pesticides. The introduction of at least one insect-selective toxin into the genome is expected to significantly increase the performance of such pesticides.

Recombinant expression vectors that are expected to be particularly suitable for use in the present invention include baculovirus expression vectors such as the type described in US Pat. No. 4,879,236 (ATCC catalog “Cell Line and Hybridoma” 6t).
h ed., (1988) pp. 166-167). The US patent is incorporated herein by reference. Also Carbonel
l) "Synthesis of a gene coding for an insect
−specific scor pion neurotoxin and attempts to ex
press it using baculovirus vectors, “Gene, 73: 409−
See also 418 (1988). Such a vector is
DNA sequence encoding a pesticidally effective peptide substantially isolated from Diguetia spider venom
It is expected to be useful in systems that can be cloned into a baculovirus, such as the California (AcMNPV) expression vector. See U.S. Patent No. 4,879,236 and the expression vector described in Miller et al., Science, 219, 715-721 (1983). The recombinant expression vector virus can then be applied to a plant or animal, wherein the insect is a pest and when the virus is ingested by the pest,
The recombinant virus enters cells of the intestinal wall and begins replicating.
Genes of proteins that are effective in replicating insects and killing insects are expressed,
Insects become disabled or die in less time than if the insects consumed wild-type AcMNPV virus.

Hybrid virus is also available in European Patent Application 034094
No. 8 teaches that it is expected to be useful. Hybrid viruses expressing the DNA of the present invention are expected to produce viruses with altered insect host range. For example, the fusion protein is a DNA of the present invention,
The expressed insecticidally effective peptide could be expressed as a single polypeptide product of a hybrid gene consisting of a specific insect intestinal cell recognition protein to target host insect targets.

Various prokaryotic and eukaryotic microorganisms can be transformed to express a hybrid toxin gene encoding a pesticidally effective protein by the methods taught in European Patent Application No. 0325400.

Hybrid bacterial cells comprising a plasmid having a gene encoding the protein of the present invention are expected to be useful in the method of the present invention. Insects are controlled by applying this hybrid to insects. See, for example, U.S. Pat. No. 4,797,279. This patent is hereby incorporated by reference as a good indication of the foregoing.

Another example using a baculovirus that is suitable for use in the present invention is Tomalski et al.
ct paralysis by baculovirus mediated expression of
mite neurotoxin gene ", Nature, 352: 82-85 (1991) and" Construction of an "by Stewart et al.
improved baculovirus insecticide containing an ins
et specific toxin gene ", Nature, 352: 85-88 (199
1) “Development” by McCutchen et al.
of a recombinant Baculovirus expressing an insect
seective Neurotoxin: Potential for Pes Cotrol, "Biot
echnology, 9: 848-851 (1991).

J. Cross-Hybridization: DNA Sequences as Probes of Related Compounds DNA probes of appropriate size can be derived from the DNA sequences of the present invention. Such probes can be used to detect the presence of a nucleic acid encoding a pesticidally effective peptide of the present invention by hybridization with nucleic acids from other sources. Screening of signal sequences, fragments of cDNA, or even whole cDNAs, under low control conditions, using oligonucleotide probes that encode, can lead to other active peptides with functional homology to the family of toxin molecules described herein. Allows access. Sources of nucleic acids that are good candidates for cross-hybridization using nucleotide probes derived from the DNA sequences of the present invention are not limited to spiders of the same genus but different species, spiders of related genera, and Spiders of the genus but in different places.

K. Identification of Promoter Sequences Another aspect of the invention provides promoter sequences that control the synthesis of spider DK9.2. The gene organization of DK9.2 was tested by screening a genomic library using the mature toxin cDNA as a probe. Analysis of genomic DNA upstream of transcription initiation (putted to be the 5 'end of the cDNA) indicates the presence of a putative promoter.
The DNA sequence of 421 base pairs of the promoter region is shown in SEQ ID NO: 8. This putative promoter contains many of the essential control signals normally present in the region immediately upstream of the transcription start site (for a description of promoter control signals, see McKnight et al., 1982, Transcirptio).
nal Control Signals of an Eucaryotic Protein
-See Coding Gene, Science 217: 316).
The canonical TATA box appears -30 from the proposed transcription start site, suggesting that it is important in controlling the start of RNA synthesis. Further upstream, there are two peripheral palindrome sequences, one of which contains a consensus CAAT box that is thought to be important for RNA polymerase II enzyme binding. It is foreseen that this promoter, or a region of this promoter, will have utility in the transcription and expression of various genes in plant, animal or bacterial cells, for example in recombinant structures.

EXAMPLES The following examples are intended to illustrate certain compositions and methods within the scope of the present invention, but they are not intended to limit the scope of the present invention.

Materials and Methods Spiders were obtained from Spider Farm, Inc., Black Canyon City, Arizona, USA, and species identification was provided by Spider Farm, Inc. Diguetia Cunnies spiders were electrically milked to collect venom. The use of a safety guard prevented the venom from reflux or contamination by blood lymphocytes.

Toxic purification: Crude venom (stored at -80 ° C) was thawed, mixed well, dissolved in 0.1% trifluoroacetic acid (TFA) and chromatographed. Crude venom was fractionated by reverse phase liquid chromatography (RPLC) incorporating a Beckman System Gold 126 solvent and a 160 photodiode array detector. Acetonitrile or isopropanol was used as an ion-pairing reagent in combination with TFA. The next column with a guard column of the same matrix was used for purification. Gynamax 300A RP C ₁₈ column (25 cm x 4.6 mm ID, 5 μm particle size), and Vydac
300A C ₁₈ semi-preparative column (25cm x 1
(0 mm inner diameter, 5 μm particle size). Peak detection was performed by monitoring at 220 nm and collecting fractions using a GILSON 208 differential collector. All fractions were lyophilized after fractionation and stored at -80 ° C.

First Atom Bombardment Mass Spectrometer (FAB-MS) Mass spectra were measured using standard AZB-SE mass spectrometers at the University of Illinois VG instrument under standard FAB conditions (xenon gas, resolution 1
000, acceleration voltage 8KV, raw material temperature 40 ° C).
Samples (about 1 μg) were analyzed in 4 μl of thioglycerol containing 25% aqueous TFA (1.0%).

Example 1 Initial Insecticidation and Identification of Peptide-Containing Fractions from the Total Toxin of Diguetia Cannies 25 μl of the frozen total venom obtained from the above Diguetia canis was dissolved in 0.1% trifluoroacetic acid (TFA),
Yami-prepared Vyda eluting at a flow rate of 3.5 ml / min
c Separation by reverse phase liquid chromatography on a RP _C18 column. A linear gradient eluent was used, starting with an 85:15 mixture of 0.1% TFA: 50% acetonitrile in water and ending after 180 minutes with a 50: 50% mixture. Fraction retention time (approximately minutes) 1 3.2 4 29.6 6 48.2 8 57.1 9 62.8 10 66.4 11 68.7 12 71.4 13 77.1 16 126.1 17 131.4 Each fraction was concentrated by lyophilization from the eluent and then by lyophilization from water. . The residue was stored at -80C. Each fraction was dissolved in 25 μl of buffered saline solution to evaluate insecticidal activity. The insecticidal activity of some fractions was confirmed by testing against tobacco caterpillar (Heliosis virescens) "TBW". (Table 1) Test solution 3 in TBW larvae, 5 in each fraction
μl was injected using a 50 μl Hamilton syringe equipped with a 33 gauge needle and a PB600 repeat microdispenser. Injections were made by injecting the needle at a shallow angle into the abdomen (near one of the front legs) at the lateral center to avoid damage to internal organs. After injection, each insect was placed in a separate container containing the inlet feed. Observations were made periodically and paralysis was measured 24 hours after injection. 0.3g about tobacco caterpillar
The average body weight of was used for dosage weighing. One set of control insects was injected with buffered saline solution only.

The paralysis gradually progressed, preceded by a muscular Keren. In severe cases, these keirens can be used for 15-30
It began within minutes, gradually increased in strength, and eventually became completely incapable of larvae. Occasionally, flue lasted more than 48 hours after injection. Occasionally this occurred even when a sublethal dose was administered, and the larva eventually recovered. Flue severity was the most reliable indicator of toxicity, and larvae affected up to the point of total paralysis and / or body contraction did not recover.

Example 2 Purification of Fraction 9 from Diguetia canyons The major component of TBW active fraction 9 was prepared using a linear (1.0% / min) solvent gradient of isopropanol / 0.1% TFA using Vydac RP C ₁₈ (25 cm × 10 mm id). One additional chromatography monitors the homogeneity (at a molecular weight range of approximately 6,500 daltons, SDS-PA
(One visible each by GE electrophoresis).

Peter detection and fraction collection were performed as described in Example 1. Two fractions were collected. First
Eluted at 21.81 minutes (fraction 9.1), the second at 22.39 minutes
Min (eluted at fraction 9.2).

Fractions 9.1 and 9.2 were concentrated by lyophilization from the eluent and then by lyophilization from water,
Residues of 9.1 and 9.2 respectively remained. The purity of the lyophilized fraction was estimated to be at least 99%. Residue 9.2 from 25 μl of total venom is almost 6 μl
g of pure protein. Residue 9.2 was tested for insecticidal activity against tobacco caterpillars. Inject each of the five insects with 6.3 μg of the residue in buffered saline and another five insects with buffered saline alone as a control as described in Example 1. The test was performed by The insects were tested after 24 hours and 48 hours. At the 24-hour reading, all of the insects treated with the 9.2 residue solution were paralyzed and then died, while the control insects appeared normal. No change was observed after 48 hours.

63 fast atom bombardment mass spectrometers
It showed a molecular weight of 71 ± 2. N-terminal amino acid sequence analysis yielded a partial amino acid sequence of residue 9.2, amino acids 1-33, substantially as shown in SEQ ID NO: 1.

Diguetia toxin in etch virescens (TBW)
The LD ₅₀ of 9.2 was about 1.0 nmol / g. Symptoms were similar to those associated with the administration of all venom as described in Example 1.

Example 3 Purification and fraction 11 of Diguetia canises In the same manner as in Example 2, fraction 11 isolated from the whole venom of Diguetia canises in the example 1 was further purified by reversed-phase liquid chromatography to obtain one fraction. . This fraction was concentrated by lyophilization from the eluent and subsequent lyophilization from water to leave a residue, labeled residue 11. This residue was tested for insecticidal activity against tobacco caterpillars at 5.0 WVE / g as described in Example 2. After 24 hours, 80% of the insects treated with residue 11 showed paralysis and after 48 hours 60% of the insects treated with this peptide showed paralysis.

A Fast Atom Bombardment mass spectrometer of this polypeptide showed a molecular weight of 6740 ± 2. N-terminal amino acid sequence analysis yielded a partial amino acid sequence substantially as set forth in SEQ ID NO: 3, amino acids 1-29.

Example 4 Purification and fractionation of Diguetia canises 12 In the same manner as in Example 2, fraction 12 isolated from the whole venom of Diguetia canises as in Example 1
Was further purified by reverse phase liquid chromatography to give 1
One fraction was obtained. This fraction was concentrated by lyophilization from the eluent and then lyophilization from water to leave a residue, labeled residue 12.
This residue was tested for insecticidal activity against tobacco caterpillars as described in Example 2. After 24 hours, 100% of the insects treated with residue 12 show paralysis, and 48 hours
80% of the insects treated in 2 showed paralysis. The control insects looked normal.

Analysis of this polypeptide by Fast Atom Bombardment Mass Spectrometer showed a molecular weight of 7080 ± 2. N-terminal amino acid sequence analysis resulted in a tentative partial amino acid sequence of residue 12, as shown in SEQ ID NO: 5.

This limited supply of material does not allow an accurate scale of toxicity, so that residue 9.2 was tested at a higher dose (nmol / g) than residues 11 and 12 by 39% and 67%, respectively. The dose chosen was intended to cover a range from minimal lethal to ineffective levels. Nevertheless, the results showed that these peptides had exactly the same toxicity. Residue 9.2 has a slightly greater capacity than the others, but the difference is probably less than a factor of three. The minimum 100% lethal dose for these peptides (residues 9.2, 11 and 12) appears to be 3.0-5.0 nmol / g. This compares favorably with commercial pesticides. For example, teflubenzuron has a typical LD ₅₀ (SAW) of 1.0 nmol / g.

Example 5 Residue isolated from Diguetia canis venom
Isolation of the 9.2 and 11 coding genes Step # 1: RNA isolation Spiders were taken from an external source and identified as Diguetia canines. Live spiders were frozen and the head and thorax removed under liquid nitrogen. Analytical Biochemistry, 162,1
RNs were extracted from head and chest using the protocol of Chomczynki and Sacchi described in 56 (1987). Oligo d (T)
Polyadenylation messenger using cellulose (Pharmacia LKB, Sweden) chromatography
RNA (mRNA) was purified.

Step # 2: cDNA Synthesis Mouse Leukemia Virus Reverse Transcriptase (Bethesda Research Laboratories, M.
In D), the messenger RNA was reverse-transcribed into cDNA. 20 μl of the reaction mixture was prepared with enzyme buffer, 50 ng mRNA, 2 units RNase H, 30 ng d (T) Notl primer (Promega, Madison, Wl) supplied by the cDNA synthesis kit (Burlinger-Maiheim, IN). ), 1 mM of each deoxynucleotide triphosphate, and 100 μg of reverse transcriptase. The reaction mixture was incubated at 37 ° C. for 1 hour and maintained at 42 ° C. for 10 minutes. The reaction mixture was precipitated with ethanol and resuspended in 20 μl of water.

Step # 3: Synthesis of primers Synonymous primer DNA sequence mixtures that were able to encode amino acid residues 1 to 8 according to SEQ ID NO: 1 were designed to reduce synonymity by using some Drosophila codon choices . This primer was synthesized by contract equipment at the Howard Hea's Medical Institute of Utah Stereology.

Step # 4: Amplification Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase was first described by Siki et al., Science, 239: 487 (1988). For our application, 5 μl of Aegutian head and chest c DNA was used as a template in a polymerase chain reaction containing the reagents included in the GeneAmp ^™ DNA amplification kit (Perkin Elmer Cetus, CA). The amplification reaction contained 2 μM concentrations of sense and antisense primers, 100 μM of each deoxynucleotide triphosphate and 4 units of thermostable recombinant Tag I polymerase. The reaction was performed in a DNA thermal cycler manufactured by Perkin Elmer Cetus. From a family of related toxins with NH ₂ -terminal amino acid sequences similar, residue 9.2
Was achieved using a stringent annealing temperature (58 ° C.). These conditions amplified a single DNA with a length of about 275 base pairs as measured by agarose gel electrophoresis. Using less stringent annealing conditions, more than 5 amplification products can be detected by agarose gel electrophoresis, ranging in size from about 200 to 360 base pairs. These various PCR products, obtained with low stringency, probably encode polypertides related to residue 9.2 in amino acid sequence, at roughly the size and in insecticidal activity. Such related polypeptides are identified as residue 11
And 12 are expected. The reason is that the N-terminal sequence of these polypeptides
Because they are very similar to one another and to residue 9.2.

Step # 5: Cloning of PCR products PCR products from both high and low stringency reactions
The unincorporated primers were purified using a Centricon-100 (Amicon) molecular size separator. The retained product was then converted to the restriction enzyme Not I (MBR,
(Milwaukee, WI). This restriction enzyme cleaves in the downstream (3 'end) primer leaving an adhesive tail. Vector nKS (Stratazan, La Jolla, CA) is Ec
Double digestion with oRV (US Biochemical) and Not I created a specific site for direct cloning. Vector DH5αF '
Was transformed. ^The colony lift was screened using a ³² P-labeled internal probe, and candidate colonies were screened for miniprep DN using an internal probe as a primer.
A sequence (US Biochemical Sequenase
Version 2.0) confirmed this further.

The total DNA sequence of the cDNA inserts of the two clones is SEQ ID NO: 2
And 4. Only the former was obtained in clones derived from stringent PCR reactions, whereas both types of cDNA insertion were found in clones obtained from the products of low stringent PCR reactions. The amino acid sequence of the polypeptide encoded by SEQ ID NO: 2 is shown in SEQ ID NO: 1. This polypeptide has the same N-terminal sequence as determined for residue 9.2. The calculated molecular weight of this polypeptide is 6377.9 daltons. Assuming that all cysteines in the native polypeptide exist in the form of intramolecular disulfide bonds (cystine), the calculated molecular weight will be 6369.7 daltons. Therefore, this polypeptide appears to be the same as the peptide isolated in residue 9.2 which showed a molecular weight of 6371 ± 2 by mass spectrometry. The amino acid sequence of the peptide encoded by SEQ ID NO: 4 is shown in SEQ ID NO: 3. This polypeptide has the same N-terminal sequence as determined for residue 11. Therefore, this peptide appears to be the same polypeptide isolated in residue 11.

Example 6 Toxicity to mammals Total venom 5 obtained from D. canies described herein
μl was lethal to mice by intraperitoneal injection (IP) in three separate tests. Treated mice were initially indistinguishable from saline controls. About 10-15 after injection
At minutes, the treated mice became slightly hyperactive and exhibited a characteristic jumping gait. This was followed by brief periods of poor coordination, tired breathing and Keren. Death occurred within 2 minutes of the first onset of symptoms.

Residues 9.2, 11 and 12 at 4.2, 0.9 and 1.2 respectively
Mice were injected intraperitoneally at a dose of mg / kg with no effect within 24 hours for all three peptides.

Residue 9.2 was injected into the cerebral ventricle of 4 mice (about 28 g).
Injections were made at a dose of 30 μg / mouse (about 1 mg / kg). After injection
No effect was observed at all times up to 48 hours. About 125 μg (4.2 mg / kg) for another mouse
Was administered (IP) with no effect.

To characterize the toxicity of this venom to vertebrates, a pooled reversed-phase fraction of the total venom was tested on mice (FIG. 1). Fraction 2 contained components with tobacco caterpillar (TBW) activity. At a dose equivalent to 25 μl whole venom (WVE), intraperitoneal injection of fractions 1 and 2 had no effect. Fraction 3 produced the same effect as observed for injection of total venom. These results suggest a clear division between insecticidal and vertebrate activity (toxicity) present in the components of the Diguetia canis venom.

Example 7 Electrophysiological Data Shafar Collaterial of Rat Hippocampal Slices
CA 1 pyramidal cell synapse (Schaffer collateral)
DK9.2 was tested at 1 μM for synaptic transmission in -CA lpyramidal cell synapse). The data shown in FIG. 2 show the time-average population spikes (ti) between (a) 5 minutes before DK.2 addition and (b) 10-20 minutes after DK9.2 addition.
me-averaged population spike) record. These records can be superimposed and at this density,
DK9.2 shows no activity in the rat CNS detectable by this assay. This assay can detect various effects on ion channels and neurotransmitter receptors in various mammals (Tay V. Dunwiedy, The Use of In Virto Brain Slices in Neuropharm.
acology, in Electrophysiological Techniques in Phar
macology. M. Edited by Geller, Alan R. Lis, Inc., New York, USA (19
1986). In particular, molecules that block sodium channel inactivation, such as certain scorpion venoms, cause a significant expansion of the last phase of the population spike response (Kaneda M, Miyama Y, Ikemoto Y and Akaique N. Scorpion toxin Prolongs an inactivation ph
ase of the voltage-dependent sodium current in ra
t isolated single hippocampal neurons, Brain Res.4
87: 192-195,1989: Alan El Mueller's Natural
Product Sciences, Inc., unpublished observation
s). In contrast, DK9.2 is active in insect preparations (house flies) at concentrations as low as 100-fold and in a manner suggesting that it prevents inactivation of insect sodium channels.

Example 8 Upstream cDNA Sequence Encoding the Precursor of DK9.2 To obtain the upstream sequence of the cDNA encoding DK9.2, nucleic acid residues # 159 to 159 on the antisense strand of the DNA sequence present in SEQ ID NO: 2 An internal oligonucleotide corresponding to 178 was synthesized. An Eco RI restriction site is located at the 5 'end of this primer. 10 μl of the single-chain venom gland cDNA was tailed at its 3 ′ end with a deoxyguanosine residue using an enzyme, terminal deoxynucleotide transferase enzyme (Bethesda Research Laboratories). 20 μl containing 14 μl enzyme and 500 μM dGTP
Was incubated at 37 ° C. for 15 minutes. Samples were precipitated with ethanol and resuspended in 20 μl H ₂ O.

The DNA sequence upstream of the gene-specific primer was amplified using an anchor PCR technique similar to that used for the downstream / mature toxicant cDNA sequence. The amplification reaction has its sense (a
The d (c) tail primer) and the antisense primer at a concentration of 2 μM contained 100 μM of each deoxynucleotide triphosphate, and 4 units of a thermostable recombinant Tag polymerase. The temperature profile was as follows: 2 minutes at 94 ° C, 2 minutes at 37 ° C
Min, 37 ° C for 1 min. This cycle was repeated twice, then the program was switched in a second step to the same profile, which included a 54 ° C. elevated annealing temperature. This cycle was repeated 32 times.

Anchor PCR was generated on a 380 bp fragment as shown on a 4% agarose gel in the presence of ethidium bromide. The reaction product was filled into the ends using a large (Klenow) fragment of E. coli DNA polymerase I (Molecular Biology Resources, Madison, WI) and precipitated by the addition of ethanol. This product was resuspended and digested with the restriction enzyme EcoRI. The digested fragment was digested with the enzyme T4 kinase at 1 mM.
Quinated in the presence of ATP, then EcoRI and EcoRV
Digested pB Ruescrips KS vector (Eco RV digested
pBluescripts KS vector). Subclones were analyzed by double-stranded DNA sequencing (Sequencing) using Sequenase 2.0 (USB).

Translation of the cDNA sequence contained in the upstream region of the mature peptide toxin revealed DK9.2 to be synthesized as a precursor protein. The upstream cDNA sequence is shown in SEQ ID NO: 6. The encoded precursor protein contains a signal sequence and a propeptide region (SEQ ID NO: 7). These are deleted to give the mature peptide toxin which can be isolated from spider venom. The signal and propeptide sequences may be required for production and secretion of DK9.2 in spiders.

Example 9 Construction of Recombinant Baculovirus Lepidopteran Signal Sequence (Jones et al., Molecular Cloning
Regulation and Complete Sequence of a Hemocyanin
Related Juvenile Hormone Supressible Proteim from
Insect Hemolymphs, J. Biol. Chem. 265: 8596 (1990))
According to the method of Rossi et al. (J. Biol. Chem. 257: 9226 (1982)).
Was used to construct from two synthetic oligonucleotides. Two 48 mers were purified by ion exchange chromatography. These two oligonucleotides share, at their 3 'end, 11 base pairs of the complementary sequence. This sequence is composed of four deoxyribonucleoside triphosphates and DNA-polymerase I
When annealed in the presence of the Klenow fragment, a double stranded product was synthesized. The reaction product was purified using hydroxylapatite chromatography and the double-stranded DNA molecule was converted to Aat II, cloned DK9.2.
The upstream of this sequence of the cDNA was extinguished with a restriction enzyme suitable for insertion into pKS-DK9c. Subclones were screened for signal sequence insertion and evaluated by DNA sequencing.

DNA sequencing is an in-sequence between two cDNA sequences.
Frame fusion was confirmed. All alloy gene structure is pBlueBa
c, performed and adapted for cloning into the Nhe I site of the baculovirus transfer vector (Vialard.
J. Virology 64: 3-50 (1990)]. Subclones were sequenced to confirm correct insertion of the construct. Plasmid WR9 DNA sequencing is a baculovirus conduction vector
The synthesized "caterspide in pBlueBac
r) Insertion of the "gene (preDK9.2)" was confirmed (FIG. 3).
The use of the BlueBac vector is a screening method, since insertion of our recombinant gene into the baculovirus genome involves co-expression of β-galactosidase and is detectable by a color change when grown on the indicated medium. Facilitate.

The recombinant baculovirus encoding preDK9.2 is 1
Using a mixture of μg of AcMNPV viral DNA and 2 μg of plasmid DNA, the protocol of Summers and Smith (“A Manual of Methods for Baculovirus Vectors an
d Insect Cell Culture Procedures "Texas Agricultur
al Experiment Bulletin No. 1555, 1988), Spodoptera frugiperda (Spodoptera
frugiperda) strain Sf9 (ATCC # CRL1711) produced by transcription. Four days after transfection, a dilution of the cell supernatant was inoculated with 5 × 10 ⁶ Sf9 cells
Spotted on a 100 mm plate and covered with agarose containing Bluo-gal (Gibso: BRL, Geiselberg, Md.) As substrate. Within 5-6 days, the recombinant was detectable by its pale blue color. Plaques were picked using a Pasteur pipette and eluted in 1 ml of medium. This eluate was used to re-infect Sf9 cells inoculated in T-25 flasks. Three days after infection, 6
Small amounts of supernatant were collected from different isolates of the species and used for the production of viral DNA. PCR using virus-specific primers from the region surrounding the polyhedrin gene
Amplification confirmed that five of the six virus isolates contained an appropriately sized insert and were free of wild-type contamination. Titration stocks of the recombinant virus were then prepared for in vivo and in vitro testing.

Example 10 Biological activity of recombinant DK9.2 Recombinant residue 9.2 purified from serum-containing tissue culture medium
(DK 9.2) was tested on the biologically active intermediate form of TBW larvae. The dose was calculated based on _A280 of the test solution. 16
At a dose of μg / g, the recombinant material caused significant muscle spasms within 2 hours after injection. Half of these larvae (3
(Or 6) paralyzed and contracted severely 48 hours later, while the other 3 larvae continued to show low to moderate muscular calendula. At the dose of 8 μg / g, 3 to 6 larvae were paralyzed, but at the dose of 5.2 μg / g, 2 to 6 larvae were paralyzed. These results confirm that recombinant DK9.2 is biologically active.

Example 11 Recombinant Baculovirus-In Vivo Test A. DK9.2 Recombinant nuclear polyhydrosis virus (vACDK9.
2) Biological activity 1) Titration of virus preparation Virus stocks were plaque assayed (Luri
a) et al., "General Virology", 1978, pp 21-32;
(Willie and Sons, New York). Titration is based on plaque forming units per unit volume (PF
U) expressed in units. In contrast, the dose was expressed as PFU / larva. One PFU is the functional equivalent of one mature virion (virus) in the preparation, a preparation in which all virions can infect one host cell (Lullia et al., Supra). For example, 103 host cells are 1
Each microliter of virus preparation containing 06 PFU / ml (ie 103 PFU / μl) could be infected.

2) Bioanalysis DK9.2 Biological activity of recombinant nuclear polyhydrosis virus (rNPV) vAcDK9.2 was tested in a series of dose response assays. In these analyses, TBW (larvae) (average mass about 250 mg) were injected with various doses of vAcDK9.2 in tissue culture medium or with tissue culture medium alone (n =
Ten). As in the toxin injection experiments described in Example 1, the treated larvae were kept in individual containers with food supply and observed periodically. The combined results of two such injection assays are shown in Table II. At 5000 PFU / larva and higher doses, typical symptoms of DK9.2 toxicity (muscle tremors and paralysis) begin to appear approximately 24 hours post-infection and at least half of the test insects are incapable within 48 hours (Ie, paralyzed or partially paralyzed). The lowest dose tested, ie, 50 PFU / larva, induced tremors and calendar within 48 hours, and at least 70% of the larva became incompetent within 96-120 hours.

B. Biological activity of vAcDK9.2 compared to wild-type NPV Further studies were performed to determine the efficacy of vAcDK9.2 in comparison to its parent wild-type virus, Autographa California NPV (wt-AcMNPV) did. The purpose of these analyses was to ensure that larvae infected with vAcDK9.2 had equal doses of wt AcMN
The goal was to determine if the larvae infected with PV became incompetent in less time.

1) Injection assay The biological activities of vAcDK9.2 and wt-AcMNPV were compared in a series of injection assays. 5 × 10 ⁵ PFU / larvae vAcDK 9.2 or wt- AcMNPV was injected. Control larvae were injected with tissue culture medium. The results, summarized in Table III, show that vAcDK9.2 showed wt-AcMN in all three
Demonstrates that it is substantially more effective than PV.

To test 2) breeding analysis activity of vAcDK9.2 by ingestion, the polyhedrin Negateibu (pol ^-) was necessary to produce the recombinant in the form of oral infection. This is an oral infection
Pol—Methods reported by other investigators for recombinant NPVs (eg, Kuroda et al., 1989, J. Virology).
63 (4): 1677-1685, and Price et al.
roc Natl. Acad. Sci. 86: 1453-1456) using vAcD
Achieved by co-occlusion of K9.2 with wt-AcMNPV. SF−
Nine cells were simultaneously infected with vAcDK9.2 and AcMNPV at a multiplicity of infection (MOI) of 10 and 10, respectively. At the same time, SF
The cells of the second group of −9 were isolated from wt-AcMNPV alone (MOI =
Infected in 2). Five days after infection, the cell inclusion bodies were disrupted and fractionated by centrifugation (eg, Wood, 1980, Virology).
104: 392-399). Cell inclusions were counted with a hemocytometer. Cells co-infected with vAcDK9.2 and wt-AcMNPV produced fewer and smaller cell inclusions than cells infected with wt-AcMNPV alone. The yield of polyhedral cell inclusions (PIB) from the mixed infection was 4.6 PIB / cell, while the yield from pure wt-AcMNPV infection was 33.3 PIB / cell.

A food uptake assay was used to assess the biological activity of co-occluded vAcDK9.2 in comparison to that of wt-AcMNPV. Mixed or wild-type PIB was incorporated into the non-agar-based insect diet at a concentration of 10 ⁵ PIB / g diet. The diet was dispensed into small containers and then fed to neonatal TBW larvae (one per container, n = 20). Control larvae received the same amount of untreated diet. Caterpillars on ad libitum
Was fed and the development of symptoms was observed periodically. Table of results
Shown in II. No effect was seen in the first 48 hours, but after 72 hours more than 50% of the larvae fed the mixed PIB were paralyzed. The wild-type virus had no effect at this point. After 96 hours, more than 90% of the recombinant-treated larvae were paralyzed, whereas only 10% of the wild-type treated larvae died or died. After 100 hours, 100% of the recombinantly treated larvae had 7% of the larvae treated with wt-AcMNPV.
Died or died compared to 5%. Therefore, in the breeding analysis, as in the injection analysis, the larvae treated with vAcDK9.2 became incompetent in a significantly shorter time than the larvae treated with wt-AcMNPV.

Example 12 Promoter of Gene Encoding DK9.2 A genomic library was prepared in order to evaluate regulators of the gene encoding DK9.2. Herman
mann) and Frischauf ("Methods in Enzymology", Vol. 152, Academic Press, Inc., pp. 180).
-183) was used to prepare DNA from spiders. This DNA
Was partially extinguished by Sau 3A and 25,0 using a TLS-55 rotor (Beckman Company, Limited).
Fractionated by centrifugation on a 10% -38% sucrose density gradient at 00 rpm for 16 hours. Pooled DNA fractions of 25-45 kb were prepared and inserted into the Xho I fire extinguishing cosmid vector using the partial filling method. 1/4 of binding mixture
Gigapak gold (registered trademark) (Stratage, LaJolla C
A) was used to load phage particles, and after transformation an equivalent of greater than 3 × 10 ⁹ bp of spider DNA was obtained. This library was plated on 25 150 mm Petri dishes and radiolabeled D
Mounted on nylon filter to probe with NA.

Colony lifts of the Diguetia genomic library were screened in a system using radiolabeled cDNA encoding DK9.2 and end-labeled oligonucleotides encoding the amino, carboxy and internal probes. One cosmid cDK2 hybridized with all of the probes. Southern blot of Eco R1 digested cosmid DNA showed that the 3.0 kb fragment hybridized to both internal and C-terminal probes. The double-stranded sequence of cosmid cDK2 using the previously identified three primers confirmed the isolation of the gene for DK9.2.
And this sequence is triggered by an amino-terminal oligonucleotide (with a high degree of homology to all peptides that are effective in killing insects in Dequetia) at a complex site on cosmid DNA,
Another (or other) gene from this family
This suggests that it may be on the same adjacent piece of DNA.

To gain access to the promoter region of the DK9.2 gene, we synthesized oligonucleotides corresponding to the pre / signal sequences on the sense strand. PCR amplification between this primer and our gene-specific primers mapped the signal sequence to be more than 3,000 bp upstream of the amino terminal exon. Then antisense
Strand primers were then used to sequence genomic DNA in the region immediately upstream of the 5 'end of the cDNA encoding the signal peptide.

DNA sequenced upstream of the signal sequence on the genomic DNA suggested another intron at bp-11 from the starting methionine codon. Synthesize oligonucleotide primers corresponding to the 5 'region of the precursor DK9.2 cDNA,
Used to start the PCR reaction, it was determined how large an intervening sequence was between the transcription and translation initiation sites. The 1,000 base pair amplification product confirmed the presence of the intron and gave an estimate of its size. DNA sequences upstream of the genomic DNA up to the start of transcription (assumed to be equivalent to the 5 'end of the cDNA) do not indicate the presence of a promoter. The DNA sequence of the promoter region is present in SEQ ID NO: 8. This imaginary promoter contains many essential control signals that are commonly present in other eukaryotic promoters in the region immediately upstream of the translation start site. This promoter or a segment of the promoter can be used for transcription / translation of other eukaryotic genes in bacteria, viruses, plants or animals.

Example 13 Neurophysiological studies related to the mode of action of DK9.2 A neurophysiological study was performed to determine the mode of action of DK9.2. Records from the maggot's neuromuscular junction and peripheral nerves indicate that DK9.2 elicits repeated burst discharges in nerves sensitive to tetrodotoxin blocking. That is, the site of action of this poison is probably the voltage-sensitive sodium channel of the neural membrane. Further studies have shown that DK9.2 constantly excites peripheral nerves at a threshold concentration of 10 nM. Also, it is the scorpion Leiurus (Quinq Estriatas)
uestriatus) is at least 50 times more potent than mammalian toxin 4.

The experimental animals used in this study were house flies (Musca domes
tica). Fix the larva with an insect pin,
The back was opened by a medical incision. The body was opened flat with a pin, excluding the internal organs, exposing the body wall musculature. Peripheral nerves coming out of the brain were cut off and the brain was removed. The preparation was poured in a large amount insects sodium chloride aqueous solution comprising from: (mM): Nacl (140) , KCl (5), CaCl 2 (0.75), MgCl
₂ (4), NaHCO ₃ (5) and HEPES (5) PH = 7.2.

Stimulation suction electrodes were attached to any convenient nerve trunk and neuromuscular transmission was recorded. The threshold of stimulation is muscle contraction fiber 6 ~
It gradually rose until seven were observed. Next, a recording intracellular microelectrode was pierced into the fiber, which was connected to an intracellular preamplifier, and the Excitatory Post Synapt as a response to nerve stimulation.
ic Potentials (EPSP) were monitored.

To record the increasing electrical activity of peripheral nerve fibers,
The suction electrode was attached to an AC preamplifier. 100 signals
Amplified by a factor of 2 and the output was filtered at 0.3 and 1 KHz.
All records were digitized and displayed (displayed) on a MacLab computerized instrument system and analyzed.

Initial experiments on the effects of DK9.2 used neuromuscular junction preparations. A single stimulus applied to the peripheral nerve resulted in a single EPSP in the body wall muscle. In the presence of DK9.2, a single stimulus resulted in a high frequency discharge of EPSP. Irritation
In addition to the induced discharges, spontaneous burst discharges also appeared, which lasted for several minutes. The duration of the burst was typically 1 second or longer, but the frequency of the EPSP during the burst was> 30 Hz. As the electrodes were released from the muscle during the contraction due to the onset of the burst, the presence of the burst discharge caused severe contractions in the body wall neuromuscular, which made it difficult to maintain intracellular readings. Therefore, most experiments suppress contraction,
High concentrations of sucrose that do not affect larval electrical activity (30
0 mM) in aqueous sodium chloride solution. Similar results were obtained using either normal or high concentration aqueous sodium sucrose chloride solutions.

Burst discharge observed after DK9.2 treatment is scorpion venom α
Similar to that observed in the presence of the toxin. The protein is known to interact with voltage-sensitive sodium channels in the neural membrane. Therefore, burst discharges should be blocked by the specific sodium channel blocker tetrodotoxin (TTX). The ability of TTX to block or reverse DK9.2-induced bursts is shown in FIG. In FIG. 5, EPSP is 1 μM.
It was easily blocked by TTX and rendered the preparation inactive for further treatment with 100 nM DK9.2. A similar experiment is shown in FIG. 5B, where the burst is triggered by DK9.2 and reversed by subsequent application of TTX. In this experiment, the onset of the burst removes the electrode from the muscle and produces a piercing artifact when attempting to redetermine the recording. Once a suitable recording site was found, the burst was monitored for about 2 minutes and the prepared sample showed a cessation of activity within seconds after TTX treatment.

To overcome the problems associated with intracellular recordings from contracting muscles, the above experiment was repeated using extracellular recordings from peripheral testing of maggots. These recordings are composed of the sensory nerve activity as well as the naturally occurring reverse activity of motor nerve fibers. At 70 nM, DK9.2 results in increased neuronal discharge unaffected by subsequent treatment with aqueous sodium chloride, but is easily blocked by 0.4 μTTX. Also, the application of TTX before DK 9.2 prevents the appearance of bursts.

Further studies were performed to determine the sensitivity of insect nerves to DK9.2 and compare its ability to that of a standard scorpion venom. DK9.2 was tested on peripheral nerve preparations starting at 1 nM, increasing the concentration every 5 minutes or doing so until an increase in activity was observed. In this preparation, 1 nM and 5 nM DK9.2 were inactive, but 10 nM.
M induced the activity of the prepared sample after a short delay. The results from the five nerve preparations were used to determine the effective threshold concentration of DK9.2 in insect cells. These results are summarized in Table IV.

Table IV Concentrations Number of treatments Number of responses% response 1 nM 200 nM 100 5 nM 3 133 10 nM 3 3 100 The last group of experiments was similarly performed with scorpions.
on Leiurus quinquestriatus (LgTX4) to determine the sensitivity of insect nerves to the toxicants. Preparation sample (n = 4)
Was exposed to concentrations of LgTX4 up to 500 nM with no effect. This finding is consistent with previous studies that found that LgTX4 was specific for mammalian sodium channels. The next challenge of these prepared samples with DK9.2 required 10-30 nM to get a response. The slight increase in the effective threshold concentration of DK9.2 is probably due to weak blocking by inactive LgTX4. These experiments demonstrate that DK9.2 is at least 50 times more active on insect nerves than LgTX4.

List of Sequences 1) General Information (i) Applicant: Karen J. Crabco; Bradford Cal Bangwagenen; J. Hunter Jackson (ii) Title of Invention: Insecticidally Effective Peptide (iii) Sequence No .: 8 (iv) Address of communication (A) Addressee: Woodcock, Washbaum, Kurtz, Makiebitz and Norris (B) Street: One Library Place 46
Floor (C) City: Philadelphia (D) State: Pennsylvania (E) Country: USA (F) Zip code: 19103 (v) Computer readout form (A) MEDIUM TYPE: DISKETTE, 3.5INCH, 1.44Mb STORAGE (B) COMPUTER: IBM PS / 2 (C) OPERATING SYSTEM: PC-DOS (D) SOFTWARE: WORDPERFECT 5.0 (vi) Current application data: (A) Application number: (B) Application date: (C) Classification: (vii) Prior application data: (A) Application number: 662,373 (B) Filing date: March 1, 1991 (viii) Agent / agent information: (A) Name: John W. Caldwell (B) Registration number: 28,937 (C) Reference / docket number: FMC-0054 (ix) Telephone communication information: (A) Telephone: (215) 568-3100 (B) Telefax: (215) 568-3439 (2) Information of sequence number 1 ( i) Sequence characteristics: (A) Length: 56 amino acids (B) Type: amino acid (C) Topolo Over: Unknown (2) Information of SEQ ID NO: 2 (i) Sequence characteristics: (A) Length: 275 base pairs (B) Type: nucleic acid (C) Strand property: double (D) Topology: unknown (2) Information of SEQ ID NO: 3 (i) Sequence characteristics: (A) Length: 58 amino acids (B) Type: amino acid (C) Topology: unknown (2) Information of SEQ ID NO: 4 (i) Sequence characteristics: (A) Length: 204 base pairs (B) Type: nucleic acid (C) Strand property: double (D) Topology: unknown (2) Information of SEQ ID NO: 5: (i) Sequence characteristics: (A) Length: 62 amino acids (B) Type: amino acid (C) Topology: unknown (2) Information of SEQ ID NO: 6: (i) Sequence characteristics: (A) Length: 359 base pairs (B) Type: nucleic acid (C) Strand property: double (D) Topology: unknown (2) Information of SEQ ID NO: 7 (i) Sequence characteristics: (A) Length: 38 amino acids (B) Type: amino acid (C) Strand property: (D) Topology; unknown (2) Information of SEQ ID NO: 8: (i) Sequence characteristics: (A) Length: 421 base pairs (B) Type: nucleic acid (C) Strand property: double (D) Topology: unknown

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12P 21/08 G01N 33/53 N C12Q 1/68 9162-4B C12N 15/00 ZNAA G01N 33/53 (72) Inventor Clapcho, Karen, Joan 84109 Salt Lake City, San Rafael Avenue, Utah, USA 3840 (72) Inventor Van Wagenen, Bradford, Calif. 84102 Salt Lake City, Utah, United States 84102 Salt Lake City 1070 East 300 South Apartment 507 (72) ) Inventor Jackson, John, Randolph, Hunter Utah, USA 84109 Salt Lake City Gilroy Road 3661

Claims (55)

(57) [Claims] A substantially pesticidally effective peptide isolated from Diguetia spider venom and having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 Or agriculturally or horticulturally acceptable salts thereof. 2. The insecticidally effective peptide according to claim 1, wherein the spider venom is derived from Diguetia canities. 3. The pesticidally effective peptide or agricultural product according to claim 1, characterized by a molecular weight of 6371 to 6397 daltons as determined by mass spectrometry and migration as a single peak on reversed phase high performance liquid chromatography. Or horticulturally acceptable salts thereof. 4. The method of claim 1, characterized by a molecular weight of about 6740 Daltons determined by mass spectrometry and migration as a single peak on reversed phase high performance liquid chromatography.
An insecticidally effective peptide as described or an agriculturally or horticulturally acceptable salt thereof. 5. An insecticidal peptide or an agriculturally or pesticidally effective peptide according to claim 1, characterized by a molecular weight of 7080 daltons determined by mass spectrometry and migration as a single peak on reversed phase high performance liquid chromatography. Horticulturally acceptable salts thereof. 6. A DNA encoding a peptide effective for insecticide represented by SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.
SEQ ID NO: 2 encoding SEQ ID NO: 1, SEQ ID NO: 4 encoding SEQ ID NO: 3, and SEQ ID NO: 6 encoding SEQ ID NO: 1 or SEQ ID NO: 5
A substantially purified DNA characterized by a DNA sequence selected from the group consisting of the DNA sequences described in 1. 7. A recombinant expression vector characterized by a DNA sequence encoding a pesticidally effective peptide set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, which can be isolated from Diguetia spider venom. A recombinant expression vector, wherein the vector is capable of expressing the coding sequence in a transformed cell. 8. The recombinant expression vector according to claim 7, wherein the spider venom is derived from Diguetia canities. 9. The DNA sequence determined by mass spectrometry, 6371 to 6397.
8. The recombinant expression vector according to claim 7, which encodes a pesticidally effective peptide characterized by the molecular weight of daltons and migration as a single peak on reversed phase high performance liquid chromatography. 10. The recombinant expression vector according to claim 7, wherein the DNA sequence encodes the peptide shown in SEQ ID NO: 1. 11. The DNA sequence encodes a pesticidally effective peptide characterized by a molecular weight of about 6740 Daltons determined by mass spectrometry and migration as a single peak on reversed phase high performance liquid chromatography. The recombinant expression vector according to claim 7, 12. The recombinant expression vector according to claim 7, wherein the DNA sequence encodes the peptide shown in SEQ ID NO: 3. 13. A DNA sequence encoding a pesticidally effective peptide characterized by a molecular weight of about 7080 daltons as determined by mass spectrometry and migration as a single peak on reversed phase high performance liquid chromatography. The recombinant expression vector according to claim 7, 14. The recombinant expression vector according to claim 7, wherein the DNA sequence encodes the peptide shown in SEQ ID NO: 5. 15. The recombinant expression vector according to claim 7, wherein the DNA sequence is the DNA set forth in SEQ ID NO: 2. 16. The recombinant expression vector according to claim 7, wherein the DNA sequence is the DNA set forth in SEQ ID NO: 4. 17. The recombinant expression vector according to claim 7, wherein the DNA sequence is the DNA shown in SEQ ID NO: 6. 18. The method according to claim 7, wherein the transformed cells are derived from a plant susceptible to insect infestation.
The recombinant expression vector according to any one of the preceding claims. 19. A DNA sequence encoding a pesticidally effective peptide set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, which can be isolated from Diguetia spider venom, can be expressed in a host cell or in a host insect cell. A recombinant baculovirus expression vector. 20. The recombinant baculovirus expression vector according to claim 19, wherein the expression vector is a baculovirus genome characterized by a polyhedrin inserted with a DNA sequence and is under transcriptional control of a baculovirus polyhedrin promoter. 21. A DNA sequence encoding a peptide effective for insecticidal activity as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 which can be isolated from Diguetia spider venom in a manner capable of expressing the peptide in a host cell. A transformed or transduced recombinant host cell. 22. The recombinant host cell according to claim 21, wherein the spider venom is derived from Diguetia canities. 23. The DNA sequence determined by mass spectrometry, 6371-639.
22. Encoding a pesticidally effective peptide characterized by a molecular weight of 7 Daltons and migration as a single peak on reversed phase high performance liquid chromatography.
A recombinant host cell as described. 24. The recombinant host cell according to claim 21, wherein the DNA sequence encodes the peptide shown in SEQ ID NO: 1. 25. A DNA sequence encoding a pesticidally effective peptide characterized by a molecular weight of about 6740 Daltons determined by mass spectrometry and migration as a single peak on reversed phase high performance liquid chromatography. 22. The recombinant host cell according to claim 21, wherein 26. The recombinant host cell according to claim 21, wherein the DNA sequence encodes the peptide shown in SEQ ID NO: 3. 27. The DNA sequence encodes an insecticidal peptide characterized by a molecular weight of about 7080 daltons determined by mass spectrometry and migration as a single peak on reversed phase high performance liquid chromatography. 22. The recombinant host cell according to claim 21, wherein 28. The recombinant host cell of claim 21, wherein the DNA sequence encodes the peptide set forth in SEQ ID NO: 5. 29. The recombinant host cell according to claim 21, wherein the DNA sequence is the DNA set forth in SEQ ID NO: 2. 30. The recombinant host cell according to claim 21, wherein the DNA sequence is the DNA set forth in SEQ ID NO: 4. 31. The recombinant host cell according to claim 21, wherein the DNA sequence is the DNA set forth in SEQ ID NO: 6. 32. (a) The insecticide represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 wherein the recombinant expression vector transformed or transduced in the recombinant host cell can be isolated from Diguetia spider venom. Culturing a recombinant host cell having a DNA sequence encoding an effective peptide, wherein said vector is capable of expressing said coding sequence in transformed cells; and (b) transforming said insecticidally effective peptide into a recombinant host cell. A method for producing an insecticidally effective peptide, which is collected from a culture. (33) The insecticide described in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 wherein the recombinant expression vector transformed or transduced in the recombinant host cell can be isolated from Diguetia spider venom. Culturing a recombinant host cell having a DNA sequence encoding an effective peptide, wherein said vector is capable of expressing said coding sequence in a transformed cell; and (b) transforming said insecticidally effective peptide into a recombinant host cell. A peptide produced by a method of harvesting from a culture. 34. The DNA sequence determined by mass spectrometry 6371-639.
33. Encoding a pesticidally effective peptide characterized by a molecular weight of 7 daltons and migration as a single peak on reversed phase high performance liquid chromatography.
The described method. 35. The method according to claim 32, wherein the DNA sequence encodes the peptide set forth in SEQ ID NO: 1. 36. The DNA sequence encodes a pesticidally effective peptide characterized by a molecular weight of about 6740 Daltons determined by mass spectrometry and migration as a single peak on reversed phase high performance liquid chromatography. 33. The method of claim 32, wherein 37. The method according to claim 32, wherein the DNA sequence encodes the peptide set forth in SEQ ID NO: 3. 38. The DNA sequence encodes a pesticidally effective peptide characterized by a molecular weight of about 7080 daltons as determined by mass spectrometry and migration as a single peak on reversed phase high performance liquid chromatography. 33. The method of claim 32, wherein 39. The method according to claim 32, wherein the DNA sequence encodes the peptide set forth in SEQ ID NO: 5. 40. The method according to claim 32, wherein the DNA sequence is the DNA set forth in SEQ ID NO: 2. 41. The method according to claim 32, wherein the DNA sequence is the DNA set forth in SEQ ID NO: 4. 42. The method according to claim 32, wherein the DNA sequence is the DNA set forth in SEQ ID NO: 6. 43. An insecticidally effective peptide or an agriculturally or horticulturally acceptable salt thereof as set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 which can be isolated from Diguetia spider venom. A method for controlling an invertebrate pest, which comprises contacting with an invertebrate pest. 44. The method according to claim 43, wherein the spider venom is from Diguetia Cunnies. 45. The method according to claim 43, wherein the pest is an insect. 46. The method according to claim 43, wherein the pest is a lepidoptera. 47. A method comprising contacting a recombinant baculovirus capable of expressing an effective amount of the peptide of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 which can be isolated from Diguetia spider venom with a pest. How to control spine pests. 48. A recombinant baculovirus peptide or an agriculturally or horticulturally acceptable peptide capable of expressing an effective amount of the peptide of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 which can be isolated from Diguetia spider venom. A pesticidal composition comprising a pesticidally effective amount of a salt thereof, and an agriculturally or horticulturally acceptable carrier. 49. An antibody that is substantially immunoreactive with the peptide of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 that can be isolated from Diguetia spider venom. 50. The antibody according to claim 49, wherein the spider venom is derived from Diguetia canities. 51. is substantially immunoreactive with the peptide to detect the presence of a pesticidally effective peptide set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 which can be isolated from Diguetia spider venom A method using an antibody, comprising: (a) collecting a spider venom, (b) contacting the spider venom with an antibody conjugated to a detectable label, and (c) removing the labeled antibody conjugated to the peptide. A method for detecting the presence of a peptide, which is characterized by detecting. 52. The method according to claim 51, wherein the spider venom is from Diguetia Cunnies. 53. A DNA probe derived from a DNA sequence encoding a pesticidally effective peptide set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 which can be isolated from Diguetia spider venom. 54. A method for detecting the presence of a nucleic acid encoding an insecticidally effective peptide which can be isolated from a Digueti spider venom, comprising: (a) collecting the nucleic acid from the spider; SEQ ID NO: 1 and SEQ ID NO: 3 which can be isolated from
Or contacting with a DNA probe derived from a DNA sequence encoding a pesticidally effective peptide set forth in SEQ ID NO: 5, and (c) detecting a probe bound to the nucleic acid. A method for detecting the presence of a nucleic acid encoding a pesticidally effective peptide that can be isolated. 55. Substantially immunizing with a peptide according to SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. (A) immobilizing the antibody on a solid support, and (b) contacting a solution containing the peptide to be purified with the antibody immobilized on the solid support, A method comprising: binding a peptide present in a solution to the antibody; (c) removing the peptide bound to the antibody immobilized on a solid support; and (d) collecting the removed peptide.