JP2805448B2 - Insecticidal peptides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide effective for insecticide. More particularly, the present invention controls, inter alia, 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 pest. About the method.

In recent years, people have become aware of the environmental hazards and mammalian toxicity associated with the use of synthetic insecticides. 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.

[0003]

BACKGROUND OF THE INVENTION The most widely used microbial insecticides are derived from the bacterium Bacillus sulindiene Switzerland (hereinafter Bt). This bacterial agent is used to control various leafy caterpillars, Japanese beetles and mosquitoes. U.S. Pat.
No. 797,279 (issued January 10, 1989)
B. t. A gene encoding Klustachy delta-endotoxin; t. Hybrid bacterial cells containing the gene encoding Tenebrionis delta-endotoxin and methods for making them have been described.
B. t. The hybrid is B.A. t. Against pests sensitive to K. kurstakii strains; t. Active against pests sensitive to Tenebrionis strains. 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.

Bacteria B. t. Other derivatives from Gilloy and Wilcox (W
ilcox) EP-A-0 325 540.
No. 0A1. The present invention relates to a hybrid virulence gene that is toxic to lepidopteran insects. More specifically, the present invention relates to t. var. Part of the Klustakyi HD-73 virulence gene and t. va
r. The present invention relates to a hybrid delta-endotoxin gene comprising a portion of a virulence gene from Klustachy strain HD-1. A hybrid virulence gene encoding a protein active against lepidopteran insects has been disclosed.

[0005] Bacterium B. t. Is also known from Wilcox et al., European Patent Application Publication No. 03.
U.S. Pat. No. 40,948 discloses its insecticidal properties. The present invention relates to hybrid toxins, which are characterized in that t. The hybrid B. coli active against lepidopteran insects by fusing the insect intestinal epithelial cell recognition region of the gene to the diphtheria toxin B chain. t. Manufactured by producing toxins. Hybrid B. t. It has been suggested that the gene can be inserted into plants or cloned into a baculovirus to produce a recoverable toxin. Alternatively, hybrid B. t. Hosts containing the gene can also be used as pesticides by direct application to the target insect environment.

In the study of pesticidal compounds, scorpion venom has been identified as a possible source of compounds that confer insecticidal properties. Scorpion Ray Ursque ink Estriatas (Sco
rpion Leiurus quinquestri
atus), two insect-selective toxins isolated from the venom of Zlotkin et al., "An Excitatoria."
nd a Depressant Insert To
xin from Scorpion Venom b
of Affect Sodium Conduct
ance and Possess a Common
Binding Site "Arch Bioche
man and Biophysics, 240: 877-
87 (1985). 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.

Zlotkin et al., Canadian Patent No. 2,000
No. 5,658 (issued June 19, 1990) includes Scorpion (Scorpion) Laeurus quince Estriatas Hebraeus Butinae (Hebraeus).
Buthinae), a protein that is effective in killing insects derived from Buthidae.
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".

[0008] Griskin's "Toxic Comp"
onents from Buthus eupeus
and Lucosa singoriensis
venoms, "Shemyakin Institute
te of Bioorganic Chemistr
y, USSR Academy of Science
s, Moscow 117988, GSP-1, USS
R is Scorpion (Scorpion) Bathus
Four toxins that are toxic to insects isolated from the venom of Eupeus have been described. Also,
Tarantula okosa Swingolien Switzerland (tar
Antula Lucosa singoriensi
The isolation and properties of the toxic components of the venom of s) are also described.
Crude toxicity is non-toxic to insects.

In response to research and development on various pesticidal compositions, researchers have been engaged in developing methods for producing pesticidal genes and introducing them into target hosts. U.S. Pat. No. 4,879,236 issued to Smith and Summers et al., Issued Nov. 7, 1989, introduces a selected gene in combination with a baculovirus promoter into the baculovirus genome. And a method for constructing a recombinant baculovirus expression vector capable of expressing a selected gene in insect cells. The method involves cleavage of baculovirus DNA to generate a DNA fragment containing a polyhedrin gene or a protein thereof containing a polyhedrin promoter. To make a recombinant transfer vector,
The NA fragment is inserted into the cloning bihirk, and 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 Outgrapher California (b
aculovirus Autographa cal
ifornica) (AcMNPV) and its related polyhedrin promoter have been found to be useful in constructing viral expression vectors that can achieve very high levels of expression of selected genes in eukaryotic host cells. .

The inventor has selected a gene that produces a protein that is toxic to a particular insect or to the spectrum of insects, and
Cloning in an MNPV expression vector suggests that the expression vector can be used in a system for controlling insects. They suggest that this vector is applicable to plants or animals to be protected. The recombinant virus can be ingested by insects and invade cells of the later intestinal wall. 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.

[0011] A further method of creating insecticidal genes and introducing them into targets to be protected is Cutle.
r) “Electroporation: Being
Developed to Transform C
rops: Success With Model C
rop Confirmed, "AG Biotech
* News vol. 7 (5): 3 & 17 (1990)
It is described in. This reference teaches that DNA can be electroporated directly into germinating pollen, and that pollen returns to flowers to seed, 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 because the ultimate goal is to pollinate the flowers and "use the flowers" rather than regenerate the plants. This method collects pollen, germinates it in a germination medium for 30-60 minutes, after which the pollen tube begins to release pollen grains,
Adding the desired DNA to the pollen-containing liquid suspension, applying an electric shock to open the pollen tube pores, removing excess DNA, and placing the altered pollen on the stigma of the plant;
It consists of waiting for seeds to form. This is an easy way to transfer genes to cereal plants.

[0012] A further transmission system is Barnes.
s) and Edwards U.S. Pat. No. 4,861,595, issued Aug. 29, 1989. This invention relates to the use of treated, substantially intact microbial cells as a delivery system for protein compounds to animals and humans. 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 destroyed at the desired location in the animal or human digestive system, thus timing or targeting the product encapsulated by the present invention. Allows for a structured release. After appropriate treatment, the protein-producing microbial cells themselves are used as a feed system without requiring purification of the product compound. Can be produced by microbial means,
All proteins, polypeptides, amino acids or compounds, including pesticides, can be the starting materials for this invention.

The possibility of using DNA technology to incorporate a synthetic gene encoding a neurotoxin found in scorpion venom is described by Carbonell et al.
synthesis of a gene coding
for an insect-specifics
corporation neurotoxin and att
empts to express it using
baculovirus vectors, "Gen
e 73: 409-18 (1988). This document is a scorpion, Bassus Eupeus (But)
hus eupeus), which teaches the possibility of incorporating a synthetic gene encoding a neurotoxin found in the venom of DNA into the baculovirus genome using DNA technology to improve baculovirus insecticides. Polyhedron
Three expression methods using the promoter-based AcMNPV expression system for toxic production were studied. Expression of the 36 codon gene alone resulted in low production of 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 times 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-0431829 describes transgenic plants, which are insect-specific for insect predators in amounts sufficient to cause toxicity to selective insects that ingest the plant tissue. The sex toxin 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 the venom of spiders. "Neurotoxins and" by Geren
Necrotoxins of Spider Ven
oms, "J. Toxicol.-Toxin Rev.
views 5 (2): 161-170 (1986) reviews neurotoxins and necrotic toxins and suggests that spider venom molecules may provide a model for certain pesticides.

In US Pat. No. 4,925,664 to Jackson and Parks (issued May 15, 1990), a heart is obtained by applying a toxin derived from a spider (spider) agerenop swiss apelta and holononakulta. And methods of treating neurological disorders. 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. One of polypeptides and polyamines,
Blocks 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 (spider) Holorena cruta. Polypeptides are useful as insecticides and in medicine, for example, as calcium channels and glutamate antagonists.

Bower et al., "Identificatiti."
on and purification of an
irreversible presynaptic
neurotoxin from the veno
m of the spider Hollena
curta, "Proc. Natl. Acad. Sc.
i. 84: 3506-3510 (1987) describes a protein neurotoxin isolated from the venom of the spider (spider) holorenacreta and its inhibition of neuromuscular transmission in Drosophila larvae. The authors suggest that the toxin blocks bresnapaptic calcium channels in Drosophila motor neurons.

"Paralytic an" by Kistad et al.
d Insectical Toxins Flo
m the Funnel Web Spider, H
olena Curta, "Toxion 29
(3): 329-336 (1991)
One peptide purified from the venom of the carta and the effects of 10 cartatoxins and injections into lepidopterous larvae are described.

"Curtatoxin" by Stapleton et al.
s: Neurotoxic insectical
polypeptides isolated fo
rmthe funnel-web spider H
olena curta, "J. Biol. Che
m. 265 (4); 2054-2059 (1990) describe three polypeptide neurotoxins isolated from the venom of the spider (spider) holorena carta and their effect on cricket aceta domesica.

"Ext" by Quicke and Yusiya Wood
ended SummeriesPesticides
Group and Physicochemicala
land Biophysical Panel Sy
mposium Novel Approaches
in Agrochemical Research
h, "Pestic. Sci. 20: 315-317.
(1987) describe that the toxins present in the venom of parasitic wasps and in some spider venoms cause rapid paralysis when injected into insects. The authors suggest that the spider venom is a blocker of a receptor-gated cation-selective membrane channel that interacts with an open channel that is gated by the locust muscle glutamate receptor. This publication refers to a toxic gland isolated from the venom glands of the naphthenic spider spider (Nefira Crabata), a low molecular weight toxicant in the venoms of Argiope and Araneus spiders.

Another study of the nature of toxicants from isolated spider venom reveals the ability of low molecular weight factors isolated from spider (funnel-web spider) venom to reversibly bind to calcium channels. did. WO89 / 07608 by Chelski et al. (August 24, 1989)
Published in Japan), 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 purification. These venoms have been found to be toxic to humans.

Another application of spider poisons is Jackson and Parkes, "Spider Toxins: Recen.
Applications in Neurobi
ology, "Ann Rev Neurosci 1
2: 405-14 (1989). This reference teaches that there is a very heterogeneous toxicity of the various poisons. It acknowledges that experiments have suggested the species specificity of calcium channels and that spider venom may provide calcium channel antagonists.
The spider venoms discussed are found to adversely affect vertebrates. This document also identifies spider venom as a possible source of insect-specific toxicants for agricultural use.

Adams et al., "Isolation an
d Biological Activity of
Synaptic Toxins from the
Venom of the Funnel Web S
pider, Agelenopsip Apert
a, "in Insect Neurochemist
ry and Neurophysiology 19
86, edited by Borkovecand Gelman, Humana Press, NJ, 1986, describes that a multiple peptide toxin that antagonizes insect synaptic transmission was isolated from the spider Agelenopsis aperta.

No. 4,855,40 to Yoshioka et al.
No. 5 (issued on Aug. 9, 1989) describes a receptor inhibitor obtained from Jiro spider venom gland and a method for producing the same. This compound has an insecticidal effect when an insect contacts the liquid or solid supported compound.

No. 4,918,10 to Nakajima et al.
No. 7 (issued on April 17, 1990) describes a compound having a glutamate receptor inhibitory activity, a method for producing the compound, and an insecticide composition containing the compound. 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 has also been 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.

[0029]

SUMMARY OF THE INVENTION A combination of problems associated with synthetic pesticides, including toxicity, environmental hazards, and loss of efficacy due to resistance, can render the host more disabling than the baculovirus infection itself. There is a continuing need to develop new means of invertebrate control, including the development of genetically engineered recombinant baculoviruses that express.
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 insecticidal 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. .

According to the present invention, a peptide having the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence of a portion thereof
A tide, peptide, characterized in that the peptide directing secretion and / or folding of the mature peptide as prepro sequences are provided.

According to the present invention, a peptide having the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence of a portion thereof
And the peptide is secreted and / or secreted by the mature peptide.
Or pepti as a prepro sequence to direct folding
A peptide is provided that is operatively linked to the peptide .

Furthermore, according to the present invention, there is provided a promoter sequence characterized by a sequence having substantial sequence homology to the sequence set forth in SEQ ID NO: 8 or a portion thereof, wherein the promoter sequence is a gene in a recombinant construct. A promoter sequence is provided which directs the expression of

Further according to the present invention, a recombinant construct having substantial sequence homology to the sequence set forth in SEQ ID NO: 8 or a portion thereof, and characterized by a sequence operatively linked to the gene to be expressed Is provided.

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

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

Further in accordance with 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 to express the peptide. New recombinant host cells are provided.

Further in accordance with the present invention, there is provided a novel recombinant baculovirus expression capable of expressing in a host or host insect cells a DNA sequence encoding a pesticidally effective peptide which can be substantially isolated from Diguetia spider venom. A vector is provided.

Further, according to the present invention, there is provided a novel method for producing a pesticidally effective peptide which can be substantially isolated from Diguetia spider venom, and a peptide produced by the method. The method comprises that the recombinant expression vector transformed or transfected in the host cell comprises a DNA sequence encoding a pesticidally effective peptide that can be substantially isolated from Dietia spider venom, and 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, there is provided a DNA sequence encoding a pesticidally effective peptide which can be substantially isolated from Diguetia spider venom, which is introduced into the germline of a plant or plant ancestor to produce the DNA. Novel alternation plants are provided wherein the trait of expression of the sequence is attracted to the next generation of the plant by sexual or asexual transmission.

Further in accordance with the present invention, an invertebrate 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 novel method of controlling pests is provided.

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

Further in accordance with the present invention, an insecticidally effective peptide substantially isolated from Diguetia spider venom and its agriculturally or horticulturally acceptable salt are converted to an agriculturally or horticulturally acceptable carrier. A novel insecticidal composition is provided.

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

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 a pesticidally effective peptide which can be substantially isolated from Diguetia spider venom, comprising the following steps: Obtaining a spider venom; contacting the spider venom with an antibody conjugated to a detectable label;
And detecting a labeled antibody bound to the peptide.

Further in accordance with the present invention, there is provided a novel method of using an antibody of the present invention to purify an insecticidal-effective peptide substantially extractable from Diguetia spider venom, comprising the following steps: Binding to a support;
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 A novel method is provided that includes a collecting step.

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 Diguetia spider venom.

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

A. DEFINITIONS As used herein, "pesticidally effective peptide substantially isolable from Diguetia spider venom" refers to a pesticidally effective peptide as well as a peptide that 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 are obtained. Such resources include, for example, recombinantly produced insecticidally effective peptides.

As used herein, "expression vector" includes a vector capable of expressing the DNA sequence contained therein, and operably linked to another sequence 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 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 any DNA sequence capable of effecting the expression of a particular DNA code addressed herein is utilized in that particular sequence. Is included in this term. Generally, expression vectors utilized in recombinant DNA technology are often in the form of "plasmids", which are referred to as circular double helical DNAs, which in those vector forms are not capable of binding to the chromosome. Not in. "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.

[0051] "Recombinant host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA technology.

The spider family Diguetidae
ae) is currently a single genus Digueti
a). Diguetia species are commonly found in the southwestern United States (including California) and Mexico. These small spiders lay vast irregular nests on many types 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 present invention is not meant to be limited by any rationale, the abnormal symptoms observed when an insecticidally effective peptide of the present invention is injected into an insect include:
Alkaloid Na ⁺ channel activator veratridine or butidae (B) known as Na ⁺ channel activity
It is very similar to the symptoms seen when scorpion venoms from the family Uthidae and the genus Buthus are injected into insects. The insecticidally effective peptides are probably muscle and / or K ⁺ channels that affect Na ⁺ or K ⁺ channels.
Alternatively, it is believed that partial depolarization of the neural membrane can occur. 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. The prevention of venom contamination by exhalation or hemolymphocytes is described in U.S. Pat. No. 4,925,664.

Once the spider venom has been obtained by electro-milking, it can be fractionated into its peptide (toxic) components using high performance liquid chromatography ("HPLC") or gel filtration chromatography or other useful separation techniques. can do. Also, it is HPL
It is often desirable for the final fractionation of spider venom performed by C.

Thus, substantially purified spiders can be used using the electro-milking technique of spiders in combination with gel filtration chromatography and / or high performance liquid chromatography and other related techniques such as hydrophobic interaction chromatography. You can get venom. 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. In one aspect, the present invention relates to insecticidal peptides and fragments thereof that can be substantially purified and isolated from Diguetia spider venom, and agriculturally or horticulturally acceptable thereof. Provide salt.

Once the peptide-containing fraction effective for insecticide 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 automatic 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 Diguetia appear to share the following properties:

1) Size: All about 6200 to about 720
2) Conserved amino termini: SEQ ID NOs: 1, 3 and 5 are the same for the first 5 amino acids; 3) SEQ ID NO: 1, 3 and 5 have greater than 40% sequence homology; 4) SEQ ID NOs: 1, 3 and 5 have about 7 or 8 cysteine residues; 5) Gene clustering: one of these toxins The genes for the above appear to be co-localized portions of genomic DNA, indicating that perhaps all of these genes can be dissected from single ancestral genes.

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 threonine at position 26 and the other contains glutamine at position 26. Glutamine isoenzyme is about 27 atomic units larger than threonine isoenzyme. Subsequent D
All references to 9.2 should be construed as referring to either one 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, DK11 is isolated and characterized by a molecular weight of about 6700 daltons or about 6740 daltons as determined by mass spectrometry and by migration as a single peak on reverse phase HPLC. More specifically, this active HP
The LC fraction was isolated c as shown in SEQ ID NO: 3.
It was identified as having an amino acid sequence when translated from DNA. Finally, to date, the third member of this pesticidally active protein series, DK12, has been determined to be about 7100 daltons or about 70 daltons as determined by mass spectrometry.
It was characterized by a molecular weight of 80 daltons and by migration as a single peak in reverse phase HPLC. More specifically, it was tentatively shown that this active fraction had an amino acid sequence as shown in SEQ ID NO: 5. The three peptides isolated correspond to SEQ ID NOs: 1, 3 and 5 and show substantially the same insecticidal activity. It is expected that other peptides having about 55 to 65 amino acids and about 7 or 8 cysteine residues with greater than 40% sequence homology to SEQ ID NOs: 1, 3 or 5 will exhibit substantially the same insecticidal activity. Is done. 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, are intended to be within the scope of the present invention.

D. Identification of a Pesticidally Effective Peptide Coding Sequence of the Invention According to another aspect of the present invention, a substantially isolated DNA sequence encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom Is provided.

The partial amino acid sequence data obtained as described above can be used to isolate and identify genes involved in the production of proteins that can be isolated from spiders. Numerous methods are available for obtaining genes that can be involved in peptide production. As an example, Fuqua S
a, S. ) Et al., “A simple PCR meth
od for detection and clon
ing low abundant transcri
pt, "Biotechnique, vol.
2 (August 1990); Frohman,
M. A. ) Of “Race: Rapid amplifi
site of cDNA ends ”, PCR
Academic Press, San Diego, Calif., USA (1990)
And U.S. Pat. No. 4,703,008, entitled "DNA Sequences Encoding E."
rythropoietin ". The above US patent is incorporated herein by reference.

In summary, encoding the determined amino acid sequence, or representing a DNA strand complementary to such a DNA molecule encoding the determined amino acid sequence,
A DNA molecule is synthesized. 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 the genomic DNA of organisms such as spiders
DNA sequences derived from cDNA copies of mRNA derived from, or isolated from, cells or tissues of an organism such as a spider. Generally, DNA molecules of 15 or more nucleotides are required for identification of homologous DNA specificity. This number must be at least 5 in the sequence.
Requires the determination of the specificity of the individual amino acids. Since each amino acid can encode up to six unique trinucleotide DNA sequences or codons, a different DNA can encode the determined amino acid sequence.
It will be appreciated that the number of 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 only one DNA molecule in the probe mixture will have exact sequence homology to the gene in question, some synthetic DNA in the pool will be required since only a high degree of homology is required.
It will also be appreciated that the molecule is capable of specifically identifying the gene. 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. fact,
Single sequence DNA probes are made by including only the DNA codon most frequently utilized 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. Nos. 4,683,195 and 4,68
See 3,202 specification. Essentially, PCR is
When two material parts are known, the selected DNA
Allows the production of sequences. Primers corresponding to each end of the sequence in question, i.e. oligonucleotides
A probe is obtained. Using PCR, a central portion of the DNA sequence is then produced synthetically.

In one method using such PCR to obtain a gene encoding a unique spider venom gene, RNA is isolated from the spider 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, synthetic DNA
A mixture of molecules or synthetic DNA molecules is prepared that can encode the amino-terminal amino acid sequence of the venom protein determined above. This DNA mixture is used with a deoxythymidylate-terminated oligonucleotide to perform PCR.
Initiate the reaction. Only the desired dDNA is effectively amplified since the synthetic DNA mixture used to initiate the PCR reaction is specific for the desired mRNA sequence. The synthetic product represents an amplified cDNA that can be ligated into any of a number of well-known cloning vectors. Without contradicting 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 relevant cDNAs that encode. 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 that are effective in killing insects are represented by, for example, SEQ ID NOs: 2 and 4. 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 sequenced (SEQ ID NO: 7). This complete mRNA
Translation 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 "topological signals"
nicsignals) "(Brobel, Gee's Pro
c. Nat. Acad. Sci. , U.S. S. A. 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 . It is also promising that it is easier to purify the protein. DK9.
The signal peptide encoded by the cDNA encoding the two precursor proteins is believed to consist of 17 amino acids of the following sequence: Met-Lys-Val-Phe-Val-Val-L
eu-Leu-Cys-Leu-Ser-Leu-Ala-Ala-Val-T
yr-Ala

In addition to the signal peptide, translation of the mRNA located upstream of the DK9.2 coding sequence represented a 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, or prepro sequences, are expressed in vivo and / or in vitro when expressed in D
It can greatly contribute to the stability, expression and folding of K9.2 or other recombinant proteins. 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 will also find that other signal and / or propeptide sequences
Data could also be provided to confirm that it could be used for expression of 9.2.

E. Expression of recombinants In accordance with the present invention there is provided on a dish a recombinant expression vector comprising DNA encoding a pesticidally effective peptide which can be substantially isolated from Diguetia spider venom. This vector allows expression of the coding sequence in transformed cells.
According to the present invention, there is also provided a DNA encoding a pesticidally effective peptide which can be substantially isolated from Diguetia spider venom.
A recombinant host cell is provided that has been transformed or transfected with a host cell with the sequence so that it expresses the peptide.

Appropriate DNA encoding desired protein
Provision of the sequence allows production of the protein to be performed using recombinant techniques now known in the art. A coding sequence can be obtained by recovering a cDNA or genomic sequence from the natural source of the protein, or can be produced synthetically, using the exact amino acid sequence determined from the nucleotide sequence of the gene. When the 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 expression mechanisms, preferably including enhancers and terminal controls, are readily available and are known in the art for a wide variety of hosts. For example, Molecula from Samblock et al.
r cloning a Laboratory Ma
See Nual, 2nd Edition, Cold Spring Harbor Press (1989).

Thus, the desired protein can be produced in both prokaryotic and eukaryotic systems, and in many cases will yield the spectrum of the processed system.

The most commonly used prokaryotic system is E. coli (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, the trp promoter, hybrid promoters such as the tac promoter, the 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 is preceded by an activation signal peptide that leads to 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 by expression mechanisms that produce the desired protein directly,
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, conformational control, and the like.

Eukaryotic systems commonly used include yeast, insect cells, milk 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 are available, as well as termination sequences and enhancers, such as the baculovirus polyhedrin promoter.
As mentioned above, a promoter can be either constitutive or inducible. For example, in a milk animal system, the MTI
The I promoter can be triggered 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.

The slight variation in the main amino acid sequence
It is understood that proteins can be obtained that have substantially equal or increased activity compared to the peptides exemplified herein. These mutations can be considered to be due to site control / mutagenesis, or to be accidental, such as due to mutations in the host producing the peptide of the invention. All these mutations are included as long as insecticidal activity is retained. Mutations in proteins alter their primary structure based on changes in the nucleotide sequence of the DNA they encode (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.

Therefore, in general, the basic amino acids Lys, A
rg and His are interchangeable; acidic amino acid A
sp and Glu are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln, and Asn are interchangeable; non-polar aliphatic acids Gly, Ala, Val,
Ile and Leu are mutually conservative (although Gly and Ala are more closely related because of size and Val, Ile and Leu are more closely related); and the aromatic amino acids Phe, Trp , And Tyr are interchangeable.

Although Pro is a non-polar neutral amino acid, it presents a drawback because of its effect on the conformation. Substitution by or in place of Pro is not preferred except when identical or similar conformational results can be obtained. Se is a polar amino acid that represents a change in conservation.
r, Thr, Gln, Asn and to a lesser extent Met. Furthermore, although classified into different categories, Ala, 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.

[0082] Because recombinant materials for the proteins of the invention are provided, 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 the protein, or post-translational changes that alter the primary, secondary or tertiary structure of the protein,
"Modified" proteins are different from unmodified proteins, but are, of course, included within the scope of the invention as claimed.

It should be further noted that when the proteins described herein, eg, SEQ ID NO: 1, are made synthetically, substitutions with amino acids not encoded by the gene may also be made. Examples of residues of alternative example formula _{H 2 N (CH 2) n} COOH (n is 2-6)
Amino acids of ω. These are neutral, non-polar amino acids such as sarcosine (Sar), t-butylalanine (t-BuAla), t-butylglycine (t-butylglycine).
BuGly), N-methylisoleucine (N-Me11)
e), and norleucine (Nleu).
For example, phenylglycine is an aromatic neutral amino acid Tr
can replace p, Tyr or Phe; citrulline (Cit) and methionine sulfoxide (MSO) are 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 that one or more of these are hydroxyproline (Hyp)
Obtained when replaced by

F. Transgenic plants Further according to the present invention, DNA encoding a pesticidally effective peptide that 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 via sexual or asexual growth.

A gene encoding a peptide effective for insecticide according to the present invention 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 plants that are more resistant to bizotes than natural species.

The coding region for a pesticidally effective peptide gene that can be used to transform plants can be the 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. Transformation can be achieved using both genomic and cDNA and synthetic DNA encoding the insecticidally effective peptide. In addition, genes are partially derived from cDNA clones, partially from genomic clones,
And synthetic genes and various combinations thereof. Also, the DNA encoding the peptide gene can include portions from various species other than the isolated peptide resources. Moreover, the pesticidally effective peptide can be combined with another compound (s), which can produce unexpected pesticidal properties in transformed plants containing the chimeric gene and expressing this compound. I can be trusted. These other compounds include, for example, protease inhibitors having oral toxicity to insects or polypeptides derived from Bacillus 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 pore-forming proteins are 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 type of protein that can be used in combination with the insecticidally effective peptides of the present invention to genetically alter plants to insect resistance.

Although the peptide gene promoter is expected to be useful for expressing the chimeric gene sequence,
Other promoters are 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.

Host cells that are expected to be useful include bacterial hosts, such as E. coli, Salalmonella ryphimurium, and Serratia marcescens; and eukaryotic hosts, such as yeast or filamentous fungi. .

The cloning vector and host cells transformed with the vector 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 expressing foreign genes are known. For example, plant tissues can be directly infected or co-cultured with plants, plant tissues or cells by A. tumefaciens; direct gene transfer of exogenous DNA into protoplasts;
It can be transformed by inoculation with EG; microinjection and microprojectile bombardment.

Transformation of tobacco by electroporation was carried out using Ag, Biotechnolo.
gy NeWs, Vol. 7, p, 3 and 17 (19
(September / October 1990 issue). In this technique, plant protoplasts are electroporated in the presence of a plasmid containing a peptidically effective 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 may be in any form, for example, naked linear, circular or supercoiled DNA, liposome-encased DN
A, DNA in spheroplasts, DNA in other plant protoplasts, DNA complexed with salts 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, and all transformed cells that contain the transformed insecticidal peptide gene (s) are effective. Plants can also be produced. Transformation of rice is carried out by DM Raineri et al., "Agr
obacterium-mediated trans
formationof rice (Oryza sa
tiva 1. ) ", Biotechnology, V
ol. 8, pp33-38 (January 1990).

Another method of introducing a bactericidal peptide gene into plant cells is to transform a plant cell with an insecticidal peptide gene. Infection with Tumefaciens. Under suitable conditions known in the art, growing transformed plant cells to germinate, produce roots, and further grow transformed plants. Peptide sequences effective for insecticide can be provided in suitable plant cells, for example, A. It can be introduced by the Ti plasmid of Tumefaciens. The Ti plasmid is A. Transmitted to plant cells during infection by Tumefaciens,
It 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. The TDNA region that transfers to the plant genome can be increased in size by insertion of the oxygen gene sequence without affecting its transfer ability. Except for tumorigenic genes,
By ensuring 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 can also be transferred to plant cells by using polyethylene glycol (PEG). PEG forms a precipitate complex with the genetic material taken up by cells. DNA for plant cells
Can also be achieved 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 for introducing a foreign DNA sequence into plant cells is to attach DNA to the particles and use this as a "gene gun".
It consists of pushing into plant cells by a shooting device called. All plant tissues or plant organs can be used as targets in this method, including embryos, in vivo and in vitro, apical and other meristems, bud bodies and reproductive tissues, including Not limited. 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 a
nd expression of Chimeric
genes in the progeny of
transgenic maizeplants ", B
iotechnology, Vol. 8, pp833-
838 (September 1990).

The regenerated plants 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 foreign DNA
Transformed plants that are not chimeric as the cells containing somatic cells of the plant are derived from sprouts or stem cuttings in vivo or in vitro by the following established methods known in the art, Produced by normal methods of 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 which have been transformed to express the peptide are selected by means of a suitable phenotypic marker. These phenotypic 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, a natural plant host for Agrobacterium, are monocotyledonous (monocotyledonas) that can be transformed in vitro
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 Ipomoe (Ipomoe).
a), Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubas, R
ubus), Populus, Santalion, Allium
m), Lilium, Nazis (Naci)
ssus), Ananas, Arachis (A)
rachis), 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.

[0101] Mature plants grown from transformed plant cells can self-propagate to produce 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 another 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 wild.
After mating of the parents, the first generation hybrid (F ₁ )
/ 1/2 toxin / wild type: distribution of 1/2 toxin / wild type is shown.
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.

As used herein, variants are stable and hereditary, and describe phenotypic changes that include inherited mutations transmitted sexually to the progeny of the plant. 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 mutation 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 insecticidal peptides selected to be expressed in transformed plants are those that are characterized by their 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 management methods to reduce insect damage to crops in an environmentally responsible manner. It is expected to give weapons to farmers.

G. Application of the Peptide as an Insecticide The insecticidally effective peptide of the present invention can be obtained by contacting a pest with an effective amount of the peptide of the present invention to produce Lepidoptera (Le
It is effective in controlling invertebrate pests such as P. doptera. Conveniently, insects are the preferred pests to control.

Methods for controlling invertebrate pests by contacting the pests with a peptide are known. Examples include proteins for oral ingestion of pests that are synthetically encapsulated. Recombinant hosts expressing the proteins of the invention, such as Pseudomonas fluorescens, can be killed by heat and suitable for pests for subsequent oral uptake and control.

Needless to say, the method for controlling invertebrate pests using the protein of the present invention can be used in combination with other pest control methods. For example, the transformed plants described above and E. coli. E. coli can be processed to express other invertebrate toxins, depending on the type of pest to control and the other key variables present.

There is also provided 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 to 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 or a fragment thereof (eg, Fab).
And F (ab ') ₂ fragment). Fab
And F (ab ') ₂ fragments lack the Fc fragment of the intact antibody and may have less nonspecific tissue binding of insect antibodies, as more readily apparent from the circles.

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, Sambrook et al.'S "Molecu1"
ar Cloning laboratory ma
Nual ", 2nd edition, Cold Spring Harbor Press, Vol. 3, Ch. 18 (1989). For example, cells expressing a pesticidally effective peptide or fragment thereof are administered to animals. To induce the production of serum containing a polyclonal antibody capable of binding to the pesticidally effective peptide.In general, a pesticidally effective peptide fragment is produced, purified and substantially free of natural contaminants. Peptide fragments that are effective or are pesticidally effective are synthesized by methods known in the art, either purified fragments or synthesized fragments or purified natural fragments and / or synthesized fragments. The combination of fragments obtained is administered to animals to produce polyclonal antisera with large specific activity.

[0113] 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 resulting from such a selection are then analyzed to identify clones that secrete antibodies capable of binding to the pesticidally effective peptide antigen.

If the peptide source is impure, only a few of the hybridoma cells will produce antibodies capable of binding to the peptide (other hybridoma cells will produce antibodies capable of binding 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 peptide (or venom) material in the presence of monoclonal antibodies 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.

An insect-selective toxin, natural or recombinant,
In order to purify using antibody affinity chromatography, it is necessary to use an antibody that can bind to a pesticidally effective peptide. 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.

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. DNA probes of suitable size, generally from 10 to 50 nucleotides, can be derived from DNA sequences encoding pesticidally effective peptides that can be substantially isolated from Diguetia spider venom. Such probes can be contacted with the venom and the DNA probe to determine the presence of a DNA encoding a pesticidally effective peptide, which can be substantially isolated from Diguetia spider venom, and the probe bound to the DNA is known to those skilled in the art. Can be detected.

I. Genetically Engineered Insecticidal 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. Heliothis virescens (cot
ton bollworm), orgiapsudotsugata (Doi)
uglas first tussock moth, Lymantia dispe
r) (gypsy moth), Autographa California (Autographacaliforni)
ca) (d1falfa leoper), Neodeprion 30
rtifer) (European pine fl)
y), and Lanpsilatesia pononnera (Lansp.
cyresia Pomenella) (Codlin
g mos) are registered in several countries and used as insecticides, including those that infect g. 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 which are expected to be particularly suitable for use in the present invention are described in US Pat.
Baculovirus expression vectors such as the type described in US Pat. No. 9,236 (ATCC catalog "Cel"
l Line and Hybridoma "6th
ed. , (1988) pp 166-167). The US patent is incorporated herein by reference. "Synthesis" by Carbonell et al.
of a gene coding for an i
nsect-specific scorpion n
eurotoxin and attempts to
express it using baculov
irus vectors, "Gene, 73: 409.
See also -418 (1988). Such vectors are useful in systems in which a DNA sequence encoding a pesticidally effective peptide that can be substantially isolated from Diguetia spider venom can be cloned into a baculovirus, such as an Autographa California (AcMNPV) expression vector. Is expected. US Patent No. 4,87
9,236 and Sci of Miller et al.
ence, 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 invades cells of the intestinal wall and starts replicating.
During replication, genes for protein that are effective in killing insects are expressed,
Insects become disabled or die in less time than if the insects consumed wild-type AcMNPV virus.

It is taught that the hybrid virus is also expected to be useful in European Patent Application 0340948. Hybrid viruses expressing the DNA of the present invention are expected to produce viruses with altered insect host range. For example, the fusion protein may be expressed as a single polypeptide product of a hybrid gene consisting of the DNA of the present invention and a specific insect intestinal cell recognition protein to direct the expressed insecticidal peptide to host insect targets. Was completed.

A variety of 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. .

A hybrid bacterial cell comprising a plasmid having the gene encoding the protein of the present invention is 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 baculoviruses suitable for use in the present invention is Tomalsky (Tomalsky).
ski) et al., “Insect paralysis b
ybaculovirus-mediated exp
response of amite neurotox
in gene ", Nature, 352: 82-85.
(1991) and Stewart et al., "Construction of an impro.
Ved baculovirus insectici
de containing an inspect-s
specific toxin gene ", Natur
e, 352: 85-88 (1991); McCutchen et al., "Development."
of a recombinant Baculov
irus expressing an insect
selective Neurotoxin: Pote
neutral for Pest Control, "Bi
technology, 9: 848-851 (199)
1).

J. Cross-Hybridization: DNA Sequences as Probes of Related Compounds DNA probes of appropriate dimensions 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. Signal sequence, c
Screening of DNA fragments, or even whole cDNAs, under low control conditions using oligonucleotide probes that encode, allows access to other active peptides with functional homology to the family of toxin molecules described herein. to enable. 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, and spiders of related genera and Spiders of the genus but in different places.

K. Identification of Promoter Sequences Another aspect of the present invention provides promoter sequences that control the synthesis of spider DK9.2. The gene composition of DK9.2 was determined using the mature toxin cDNA as a probe.
The library was tested by screening. Analysis of genomic DNA upstream of the transcription start (putted to be the 5 'end of the cDNA) indicates the presence of the putative promoter. 421 base pairs of D in the promoter region
The NA sequence 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 (McKni
ght) et al., 1982 Transcriptiona.
1 Control Signals of an E
ucaryotic Protein-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 palindromic sequences, one of which is
One contains a consensus CAAT box that is considered important for the binding of the RNA polymerase II enzyme. This promoter, or a region of this promoter, can be plant,
It is foreseen to have use in the transcription and expression of various genes in animal or bacterial cells, for example, in recombinant structures.

[0125]

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.

Purification of toxicant: Thaw crude venom (stored at -80 ° C), mix well, and add 0.1% trifluoroacetic acid (T
FA) and chromatographed. Beckman System Gold 126 solvent and 160
Crude venom was separated by reverse phase liquid chromatography (RPLC) incorporating a photodiode array detector.
Acetonitrile or isopropanol was used as ion-pairing reagent in combination with TFA. The next column with a guard column of the same matrix was used for purification. D
ynmax 300A RPC ₁₈ column (25c
m × 4.6 mm inner diameter, 5 μm particle size), and Vy
dac 300A _C18 semi-preparative column (25 cm × 10 mm inner diameter, 5 μm particle size).
Peak detection was performed by monitoring at 220 nm and collecting fractions using a differential collector. All fractions were lyophilized after fractionation and stored at -80 ° C.

First Atom Bombardment Mass Spectrometer (FAB-MS) Mass spectrum was converted to a VG instrument ZAB-S of the University of Illinois.
Standard FAB conditions using an E mass spectrometer (xenon gas, resolution 1000, acceleration voltage 8KV, raw material temperature 40 ° C)
Recorded under Sample (about 1 μg) is 25% aqueous TF
Analysis was performed in 4 μl of thioglycerol containing A (1.0%).

Example 1 Initial fractionation and identification of insecticidal peptide-containing fractions from total venom of Diguetia canis 25% of frozen total venom from the above-mentioned Diguetia canis was dissolved in 0.1% trifluoroacetic acid (TFA). Separation by reverse phase liquid chromatography on a semi-prepared Vydac RPC ₁₈ column eluted at a flow rate of 5 ml / min. 0.1% TF
A: A linear gradient eluent was used, starting with an 85:15 mixture of 50% aqueous acetonitrile in water and ending after 180 minutes with a 50: 50% mixture.

[0130] 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 a buffered saline solution to evaluate insecticidal activity. The insecticidal activity of some fractions was confirmed by testing against tobacco caterpillar (Heliosis virescens) "TBW" (Table I).

TBW larvae, 5 per fraction,
Was injected with a 50 μl Hamilton syringe equipped with a 33 gauge needle and a PB600 repetitive 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 artificial feed. Observations were made periodically and paralysis was measured 24 hours after injection.
An average body weight of 0.3 g for tobacco caterpillars was used for dose calculation. One set of control insects was injected with buffered saline solution only.

The paralysis gradually progressed and was preceded by muscular karen. In severe cases, these calendars began within 15-30 minutes after injection, gradually increased in strength, and eventually the larva became completely incapacitated. 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.

[0133]

[Table 1]

Example 2 Purification of Fraction 9 of Deguetia canyons The major component of TBW active fraction 9 was prepared using Vydac RP C using a linear (1.0% / min) solvent gradient of isopropanol / 0.1% TFA. ₁₈ (25cm x 1
One additional chromatograph (0 mm id) was monitored at 220 nm at 3.5 ml / min and homogenized (over the molecular weight range of approximately 6,500 daltons, SDS-P
AGE electrophoresis to one visible band each). Peak detection and fraction collection were performed as described in Example 1. Two fractions were collected. The first eluted at 21.81 minutes (fraction 9.1) and the second eluted at 22.39 minutes (fraction 9.2).

Fractions 9.1 and 9.2 were concentrated by lyophilization from the eluent and then by lyophilization from water, leaving residues of 9.1 and 9.2, respectively. The purity of the lyophilized fraction was estimated to be at least 99%. The residue 9.2 from 25 μl of the total venom was estimated to contain approximately 6 μg of pure protein. The residue 9.2 was tested for insecticidal activity against tobacco caterpillars. As described in Example 1, each of five insects is injected with 6.3 μg of the residue in buffered saline, and another five insects are injected with buffered saline alone as a control The test was performed by: 2
The insects were tested after 4 hours and 48 hours. At the 24 hour reading, all of the insects treated with the 9.2 solution of residue were paralyzed and then died, while the control insects appeared normal. No change was observed after 48 hours.

The Fast Atom Bombardment mass spectrometer showed a molecular weight of 6371 ± 2. N-terminal amino acid sequence analysis yielded a partial amino acid sequence of residue 9.2, amino acids 1-33, substantially as set forth in SEQ ID NO: 1.

The LD ₅₀ of Deguethia toxin 9.2 in Etch Viressense (TBW) is about 1.0 nmol / g
Met. Symptoms were similar to those associated with the administration of total venom as described in Example 1.

Example 3 Purification of Diguetia canities, fraction 1
1 In the same manner as in Example 2, fraction 11 isolated from the whole venom of Deguetia canis venom of Example 1 was further purified by reverse-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 show 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 of Diguetia canities, fraction 1
2 In the same manner as in Example 2, fraction 12 isolated from the whole venom of Diguetia canis 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 by 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 showed paralysis, and at 48 hours, 80% of the insects treated with residue 12 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 residue does not allow an accurate scale of toxicity, so that residue 9.2 has a higher dose (nmole / g) of 39% and 67% than residues 11 and 12, respectively. Tested. 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 can be said to have a slightly greater capacity than the others, but the difference is probably less than a factor of three. The minimum 100% lethal dose of 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 Isolation of Code Genes for Residues 9.2 and 11 Isolated from Diguetia canis Venom Step # 1: RNA Isolation Spiders were taken from an external source and identified as Diguetia canis. Live spiders were frozen and the head and thorax removed under liquid nitrogen. Analytical Bioch
RNA was extracted from the head and thorax using the Chomczynski and Sacchi protocol described in chemistry, 162, 156 (1987). Oligo d
(T) Cellulose (Pharmacia LKB of Sweden)
Polyadenylated messenger RNA (mRNA) was purified using chromatography.

Step # 2: Synthesis of cDNA Murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, M.V.) using the manufacturer's protocol.
In D), the messenger RNA was reverse transcribed into cDNA.
20 μl of the reaction mixture was supplied with enzyme buffer supplied with cDNA synthesis kit Mannheimerlinger Mannheim, Ind., 50 ng mRNA, 2 units of RNase H, 3
0 ng of d (T) Not I primer (Promega, Madison, WI) contained 1 mM of each deoxynucleoside triphosphate, and 100 μg of reverse transcriptase. The reaction mixture was incubated at 37 ° C. for 1 hour,
Sustained 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 could encode amino acid residues 1 to 8 according to SEQ ID NO: 1 were designed by using some Drosophila codon choices to Reduced. This primer was synthesized by a contract facility at the Howard Heath's Medical Institute at Utah State University.

Step # 4: Amplification Primer-directed enzymatic amplification of DNA with thermostable DNA polymerase is described by Siki et al., Science, 239:
487 (1988). For our application, 5 μl of Diguetia head-thoracic cDNA
Using the GeneAmp ™ DNA amplification kit (Perkin
Used as template in a polymerase chain reaction containing reagents contained in Elmer Cetus, CA. The amplification reaction was performed with 2 μM concentrations of sense and antisense primers, 100 μM of each deoxynucleotide triphosphate and 4 units of thermostable recombinant Tag I.
Polymerase was included. The reaction was performed in a DNA thermal cycler manufactured by Perkin Elmer Cetus. Selective amplification of the gene encoding residue 9.2 from a family of related toxins with similar NH ₂ -terminal amino acid sequences was achieved using stringent annealing temperatures (58 ° C). These conditions are:
About 275 as determined by agarose gel electrophoresis
A single DNA having a base pair length was amplified. Using less stringent annealing conditions, more than 5 amplification products can be detected by agarose gel electrophoresis, the size of which is in the range of about 200-360 base pairs. These various PCs obtained with low stringency
The R product encodes a polypeptide probably related to residue 9.2 in amino acid sequence, at roughly the size and in pesticidal activity. Such related polypeptides are expected to include residues 11 and 12. The reason is that the N-terminal sequence of these polypeptides
This is because they are very similar to each other and to residue 9.2, as shown in SEQ ID NOs: 1, 3 and 5.

Step # 5: Cloning of PCR products PCR products from both the high and low stringency reactions were:
Unincorporated primers were purified using a Centricon-100 (Amicon) molecular size separator. The retained product is then purified by the restriction enzyme Not I
(MBR, Milwaukee, WI). This restriction enzyme cleaves in the downstream (3'-end) primer, leaving no adhesion. The vector pKS (Stratagene, La Jolla, CA) was double digested with EcoR V (U.S. Biochemical) and Not I to create specific sites for direct cloning. The vector and the insect were ligated and transformed into the complex DN5αF ′. ³² P
Colony lift is screened using a labeled internal probe, and candidate colonies are sequenced with miniprep DNA using an internal probe as a primer (US
This was further confirmed by Biochemical Sequenase version 2.0).

Total DNA of cDNA Insertion of Two Clones
The sequences are shown in SEQ ID NOs: 2 and 4. Only the former is obtained in clones derived from a rigorous PCR reaction, whereas a strict PC with both types of cDNA insertion being low.
Found in clones obtained from the products of the R reaction.
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 63
77.9 daltons. Assuming that all cysteines in the native polypeptide exist in the form of intramolecular disulfide bonds (cystine), the calculated molecular weight is 636
9.7 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 All venoms 5 obtained from canies
μl was lethal to mice by intraperitoneal injection (IP) in three separate tests. Treated mice were initially indistinguishable from saline controls. About 10 after injection
At １５15 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.

Mice were injected intraperitoneally with the residues 9.2, 11 and 12 at doses of 4.2, 0.9 and 1.2 mg / kg, respectively, but no more than 24 hours for all three peptides. No effect was seen.

The residue 9.2 was treated with 4 mice (about 28 g).
Approximately 30 μg / mouse (approximately 1 mg / kg)
Injection. No effect was observed at any time up to 48 hours after injection. Approximately 125 μg (4.2 mg / kg) residue in another mouse 9.
2 was administered (IP), but no effect was observed.

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. 25μ
At a dose equal to 1 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-C of Rat Hippocampal Slices
A1 Pyramidal cell synapse (Schaffer c
ollateral-CA 1 pyramidal
DK9.2 was tested at 1 μM for synaptic transmission in cell synapse. The data shown in FIG. 2 are (a) 5 minutes before DK9.2 addition and (b) DK
9.2 Time-averaged population spikes between 10 and 20 minutes after addition
T. spike) record. These records can be superimposed, indicating that at this concentration, DK9.2 has no detectable activity in the rat CNS in this assay. This assay can detect various effects on various mammalian ion channels and neurotransmitter receptors (Tay V. Dunwiddy,
The Use of In Vitro Brain
Slices in Neuropharmacol
ogy, in Electrophysiological
al Techniques in Pharmaco
logy, etch. M. Edited by Geller, Alan R. Lis, Inc., New York, USA (1
986) published). In particular, molecules that prevent sodium channel inactivation, such as certain scorpion venoms,
Causes a significant expansion of the last phase of the population spike response (Kaneda M, Miyama Y, Ikemoto Y and Akaique N. Scorpion toxin pr
olongs an inactivationpha
se of the voltage-depende
nt sodium current in rat
isolated single hippocamp
al neurons, Brain Res. 487:
192-195, 1989; Alan El Mueller's Natural Product Science
s, Inc. , Unpublished observ
ations). In contrast, DK9.2 is 10
It is active in insect preparations (house flies) at concentrations as low as 0-fold and in a manner that suggests blocking the 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, a nucleic acid residue on the antisense strand of the DNA sequence present in SEQ ID NO: 2 was obtained. Internal oligonucleotides corresponding to # 159-178 were synthesized. An Eco RI restriction site is located at the 5 'end of this primer. 10 μl of single-chain venom gland cDNA contains enzyme, terminal deoxynucleotide transferase enzyme (Bethesda Research.
Laboratories) at its 3 'end with a deoxyguanosine residue. A 20 μl reaction containing 14 μl enzyme and 500 μM dGTP was added to 37 ° C.
For 15 minutes. The sample is precipitated with ethanol, 2
Resuspended in 0 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 contained 100 μl of its sense (ad (c) tail primer) and antisense primer at 2 μM concentration.
M each of the deoxynucleotide triphosphates and 4 units of thermostable recombinant Tag polymerase. The temperature profile was as follows:
2 minutes at 94 ° C, 2 minutes at 37 ° C, 1 minute at 37 ° C. This cycle is repeated twice, then the program is run in the second step for 5
The same profile was switched to incorporate a 4 ° C. elevated annealing temperature. This cycle was repeated 32 times.

The 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 is converted to the large (K) of E. coli DNA polymerase I (Molecular Biology Resources, Madison, WI).
The ends were filled in using the (now) fragment and precipitated by the addition of ethanol. This product was resuspended and digested with the restriction enzyme EcoRI. This digested fragment is quinated by the enzyme T4 kinase in the presence of 1 mM ATP and then digested with EcoRI and EcoRV.
B Ruskrips KS vector (EcoRV dige
sted pBluescripts Vecto
r). Subclones were analyzed by double-stranded DNA sequencing (sequencing) using Sequenase 2.0 (USB).

C contained in the upstream region of the mature peptide toxin
Translation of the DNA sequence 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 DK9.2 production and secretion in spiders.

Example 9 Construction of recombinant baculovirus Lepidopteran signal sequence (Molecule from Jones et al.)
ar CloningRegulation and
Complete Sequence of Hem
ocyanin-Related Juvenile
Hormone-Suppressible Prote
im from Insect Hemlymph
s, J.S. Biol. Chem. 265: 8596 (19
90)) by the method of Rossi et al. (J. Biol. Ch.
em. 257: 9226 (1982)).
Was constructed from two synthetic oligonucleotides. Two 48
Mars was purified by ion exchange chromatography. These two oligonucleotides share, at their 3 'end, 11 base pairs of the complementary sequence. When this sequence is annealed in the presence of the four deoxyribonucleoside triphosphates and the Klenow fragment of DNA-polymerase 1, 2
The main chain product was synthesized. The reaction product is purified using hydroxylapatite chromatography and
Aatll, cloned DK
The upstream of this sequence of 9.2 cDNA was digested with a restriction enzyme suitable for insertion into pKS-DK9c. Subclones were screened for signal sequence insertion and evaluated by DNA sequencing.

DNA sequencing involves two cDNAs.
An in-frame fusion between the sequences was confirmed. The total synthetic gene structure was implemented and adapted for cloning into the NheI site of pBlueBac, a baculovirus transfer vector [Vialard, J. et al. J. et al. Vi
rology 64; 3-50 (1990)]. Subclones were sequenced to confirm correct insertion of the construct. Plasmid WR9 DNA sequencing was performed using the synthetic "caterspider" gene (pr) in the baculovirus transfer vector pBlueBac.
eDK9.2) was confirmed (FIG. 3). pBlue
The use of the Bac vector is a screening method, since the insertion of our recombinant gene into the baculovirus genome involves co-expression of β-galactosidase and is detectable by a change in color as it grows on the indicated medium. Facilitate.

The recombinant baculovirus encoding PreDK9.2 contained 1 μg of AcMNPV viral DNA.
With a mixture of 2 μg of plasmid DNA and Su
mmers and Smith's protocol ("AM
annual of Methods for Bacu
lowvirus Vectors and Insec
t Cell Culture Procedure
s ", Texas Agricultural Exp
element Bulletin No. 1555,
1988) by using Spodoptera frugiperd.
a) Produced by transcription of strain Sf9 (ATCC # CRL1711) cells. After transfection 4
After a day, a dilution of the cell supernatant was spotted onto a 100 mm plate inoculated with 5 × 10 ⁶ Sf9 cells,
And as a substrate, Blue-gal (Gibso BRL,
(Gaiselberg, MD). 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 seeded in T-25 flasks. Three days after infection, small supernatants were collected from six different isolates and used for the production of viral DNA. PCR amplification using virus-specific primers from the region surrounding the polyhedrin gene confirmed that 5 of the 6 virus isolates contained the appropriate size insert and were free of wild-type contamination. confirmed. 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
The biological activity of (DK9.2) was tested on intermediate forms of TBW larvae. The dose was calculated based on the A ₂₈₀ of the test solution. At a dose of 16 μg / g, the recombinant material caused significant muscle spasms within 2 hours after injection. Half of these larvae (3 or 6) became paralyzed and contracted severely after 48 hours, while the other three larvae continued to show low to moderate muscular calendula. 8 μg / g
Although 3 to 6 larvae were paralyzed even at the dose of 5.2 μg,
At a dose of / g, 2-6 larvae were paralyzed. These results confirm that recombinant DK9.2 is biologically active.

Example 11 Recombinant Baculovirus-In Vivo Test DK9.2 recombinant nuclear polyhydrosis virus (v
Biological activity of ACDK9.2)

1) Titration of virus preparation Virus stocks were assayed by plaque assay (Lur
ia) et al., "General Virology", 1
978, pp21-32; John Willy and &
Sands, titrated by New York. Titration was expressed in units of plaque forming units (PFU) per unit volume. In contrast, the dose was expressed as PFU / larva. IPFU is a preparation, a preparation in which all virions can infect one host cell (Lullia et al., Supra);
Functional virion virus). For example, 103 host cells have 106 PFU /
ml (ie 103 PFU / μl) could be infected by each microliter of the virus preparation.

2) Bioanalysis DK9.2 recombinant nuclear polyhydrosis virus (rNP
V) Biological activity vAcDK9.2 was tested in a series of dose response assays. In these analyses, TB
W (larvae) (average mass about 250 mg) were injected with vAcDK9.2 in tissue culture medium at various doses or with tissue culture medium alone (n = 10). 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 III. 500
At 0 PFU / larva and higher doses, DK
9.2 Representative symptoms of toxicity (muscle tremors and paralysis)
Began to appear approximately 24 hours after infection, and within 48 hours at least half of the test insects were disabled (ie, paralyzed or partially paralyzed). The lowest dose tested,
That is, 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 parent wild-type virus, Autographa California NPV (wt-AcMNPV)
The efficacy of vAcDK9.2 was determined in comparison with.
The purpose of these analyzes was to determine whether larvae infected with vAcDK9.2 became incapacitated in less time than larvae infected with an equal dose of Wt-AcMNPV.

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 vAcDK9.2 or Wt at ⁵ to 105 PFU / larvae of last instar larvae of tobacco and caterpillar (Heliosis virescens), larvae of cabbage and sucker (tricoprussia) and larvae of beetle (Spodoptera exigua)
-AcMNPV was injected. Control larvae were injected with tissue culture medium. The results summarized in Table III show that vA
cDK9.2 had Wt-AcM in all three.
Demonstrates that it is substantially more effective than NPV.

2) Housing analysis To test the activity of vAcDK9.2 upon ingestion,
The polyhedrin Negateibu (Pol ^-) was necessary to produce the recombinant in the form of oral infection. This is a method reported by other investigators on orally infected Pol-recombinant NPVs (eg, Kuroda et al., 1989 J. Virology 63 (4): 16).
77-1685, and Price et al., 1989 Pr.
oc Natl. Acad. Sci. 86: 1453-
1456) using vAcDK9.2 and wt-Ac
Achieved by co-occlusion with MNPV. SF-9 cells were co-infected with vAcDK9.2 and AcMNPV at a multiplicity of infection (MOI) of 10 and 2, respectively. At the same time, a second group of cells of SF-9 was
Infected with cMNPV alone (MOI = 2). 5 after infection
On the day, the cell inclusions are disrupted by cell disruption and differential centrifugation (eg Wood, 1980, Virology 104: 3).
92-399). 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. Polyhedral cell content (PIB) from mixed infections
Yield is 4.6 PIB / cell while pure wt
-The yield from 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 a non-agar-based insect diet at a concentration of 10 ⁵ PIB / g diet. Food is distributed in small containers,
The newborn TBW larvae were then fed (one per container,
n = 20). Control larvae received the same amount of untreated diet.
The larvae were fed ad Ibitum and the development of symptoms was observed periodically. Table I shows the results
I. No effect was observed 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 were dead or died. 100
After time, 100% of the recombinantly treated larvae had wt-Ac
Died or died compared to 75% when treated with MNPC. Therefore, in the breeding analysis, as in the injection analysis, vAcDK9.2-treated larvae were expressed in wt-Ac
It became incompetent in a significantly shorter time than the MNPV-treated larvae.

[0167]

[Table 2]

[0168]

[Table 3]

Example 12 Promoter of Gene Encoding DK9.2 A genomic library was prepared in order to evaluate regulators of the gene encoding DK9.2. Harman (H
errmann) and Frischa (Frischa)
uf) (“Methods
in Enzymology ", Vol. 152, Academic Press, Incorporated, pp1
80-183) to prepare DNA from spiders. This DNA was partially digested with Sau 3A and TLS-55 rotor (Beckman Company,
Limited) at 25,000 rpm for 16 hours,
Fractionated by centrifugation on a 10% -38% sucrose gradient. A pooled DNA fraction of 25-45 kb was prepared and inserted into the Xho I digested cosmid vector using the partial filling method. 1/4 of binding mixture
To Gigapak gold (registered trademark) (Strat
, LaJolla CA) and 3 × 10 ⁹ bp of spider DNA after transformation.
We have the above equivalent. This library consists of 25 15
Plated on 0 mm Petri dishes and mounted on nylon filters for probing with radiolabeled DNA. Colonies 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. Sotheso blotting of the DNA of the Eco RI digested cosmid showed that the 3.0 kb fragment hybridized to both the internal and C-terminal probes. The double-stranded sequence of cosmid cDK2 using the three primers previously identified confirmed the isolation of the gene for DK9.2. And this sequence has the amino-terminal oligonucleotide (which has a high degree of homology to all peptides that are effective in killing Diguetia) with cosmid DN
Starting at a composite site on A, it suggests that another (or other) gene from this family could be on the same adjacent piece of DNA.

To gain access to the promoter region of the DK9.2 gene, we synthesized an oligonucleotide corresponding to the pre / signal sequence 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.
The primers of the antisense strand were then used to 5 'of the cDNA encoding the signal peptide.
Genomic DNA was sequenced in the region directly upstream of the end.

The DNA arranged upstream of the signal sequence on the genomic DNA is composed of the starting methionine codon to bp-11.
Suggested one other intron. Precursor DK
Oligonucleotide primers corresponding to the 5 'region of the 9.2 cDNA were synthesized and used to prime the PCR reaction to determine 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 the 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 section of this promoter, can be used for transcription / translation of other eukaryotic genes in bacteria, viruses, plants or animals.

Example 13 Neurophysiological Studies Relevant to the Mode of Action of DK9.2 A neurophysiological study was performed to determine the mode of action of DK9.2. Recordings from the neuromuscular junction and peripheral nerves of maggots show that DK9.2 repeats bursts (burs) in nerves sensitive to tetrodotoxin blocking.
t) Indicates that a discharge is induced. 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. It is also known as scorpion Leiurus quinquestriatus.
s) is at least 50 times more potent than mammalian toxin 4.

The experimental animals used in this study were house flies (M
usca domestica).
Larvae were fixed with insect pins and the back was opened by 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. This preparation was washed with a large amount of an aqueous solution of insect sodium chloride consisting of the following (mM) NaCl (140), K
Cl (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 stimulation threshold gradually increased until 6-7 muscle contractile fibers were observed. Then, a recording intracellular microelectrode was pierced into this fiber,
This is connected to an intracellular preamplifier, and EXcitat as a response to nerve stimulation is connected.
ory Post Synaptic Potenti
als (EPSP) was monitored. A suction electrode was attached to an AC preamplifier to record the rising electrical activity of the peripheral nerve fibers. The signal was amplified 100-fold and the output was filtered at 0.3 and 1 kHz. All records were digitized, displayed (Mac) 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. D
In the presence of K9.2, a single stimulus resulted in a high frequency discharge of EPSP. In addition to the stimulus-induced discharge, spontaneous burst discharges also appeared, which lasted several minutes. The duration of the burst was typically more than 1 second, but 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 were performed in aqueous sodium chloride solutions containing high concentrations of sucrose (300 mM), which suppressed contraction but did not affect larval electrical activity. Similar results were obtained using either normal or high concentration aqueous sodium sucrose chloride solutions.

The burst discharge observed after DK9.2 treatment was similar to that observed in the presence of the scorpion venom alpha-toxin. The protein is known to interact with voltage-sensitive sodium channels in the neural membrane. Therefore, the burst discharge 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 was easily blocked by 1 μMTTX, rendering the prepared sample inactive for subsequent treatment with 100 nM DK9.2. A similar experiment is also shown in FIG. 6, where the burst is triggered by DK9.2 and is reversed by subsequent application of TTX. In this experiment, the start of the burst removes the electrode from the muscle,
Puncture artifacts occur 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 nerves of maggots. These recordings are composed of ascending sensory nerve activity, as well as the naturally occurring reverse activity of motor nerve fibers. At 70 nM, DK
9.2 results in an increase in nerve discharge unaffected by subsequent treatment with aqueous sodium chloride solution, but 0.4
Easily blocked by μTTX. Also, DK
Application of TTX before 9.2 will prevent the appearance of bursts.

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

[0179] The last group in the experiment was performed in a similar manner.
pion Leiurus quinquestria
tus) (LgTX4). Preparative samples (n = 4) were 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. DK9.
A slight increase in the effective threshold concentration of 2 is probably due to inactive LgTX
4 due to the weak blocking effect. These experiments demonstrate that DK9.2 is at least 50 times more active on insect nerves than LgTX4.

[Sequence Listing] SEQ ID NO: 1 shows the amino acid sequence of DK9.2. SEQ ID NO: 2 is the cryptic cDN of DK9.2
A sequence is shown. SEQ ID NO: 3 shows the amino acid sequence of DK11. SEQ ID NO: 4 shows the coding cDNA sequence of DK11. SEQ ID NO: 5 shows the amino acid sequence of DK12. SEQ ID NO: 6 shows the complete cDNA encoding DK9.2.
SEQ ID NO: 7 shows the amino acid sequence of the signal / leader sequence of precursor DK9.2. SEQ ID NO: 8 shows the DNA sequence of promoter region DK9.2.

List of Sequences 1) General Information (i) Applicant: Karen J. Klapco; Bradford Cal Bangwagenen; J.R. Hunter Jackson (ii) Title of the Invention: Insecticidally Effective Peptide ( iii) Sequence number: 8 (iV) Destination of communication (A) Destination: Woodcock, Washbaum, Kurtz, Makiebitz and Norris (B) Street: One Library Place 4
6th floor (C) City: Philadelphia (D) State: Pennsylvania (E) Country: U.S.A. S. A. (F) Postal code: 19103 (v) Computer-readable form (A) MEDIUM TYPE: DISKETTE,
3.5 INCH, 1.44 Mb STORAGE (B) COMPUTER: IBM PS / 2 (C) OPERATING SYSTEM: PC-DO
S (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) Application date: March 1, 1991 (viii) Agent / agent information: (A) Name: John W. Caldwell (B) Registration number: 28,937 (C) Reference / docking number: FMC-0054 (ix) Telephone Communication information: (A) Telephone: (215) 568-3100 (B) Telefax: (215) 568-3439

(2) Information of SEQ ID NO: 1 (i) Sequence characteristics: (A) Length: 56 amino acids (B) Type: amino acids (C) Topology: unknown (xi) Sequence description: SEQ ID NO : I:

(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 ( xi) Sequence description: SEQ ID NO: 2:

(2) Information of SEQ ID NO: 3 (i) Sequence characteristics: (A) Length: 58 amino acids (B) Type: amino acid (C) Topology: unknown (xi) Sequence description: SEQ ID NO: 3 :

(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 (Xi) Sequence description: SEQ ID NO: 4:

(2) Information of SEQ ID NO: 5: (i) Sequence characteristics: (A) Length: 62 amino acids (B) Type: amino acid (C) Topology: unknown (xi) Sequence description: SEQ ID NO: 5:

(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 (Xi) Sequence description: SEQ ID NO: 6:

(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 (xi) Description of the sequence: SEQ ID NO: 7:

(2) Information of SEQ ID NO: 8: (i) Characteristics of sequence: (A) Length: 421 base pairs (B) Type: nucleic acid (C) Strand property: double (D) Topology: unknown (Xi) Description of sequence: SEQ ID NO: 8:

[Brief description of the drawings]

FIG. 1 shows the R of all Diguetia canis toxins.
R using a 0.1% TFA: acetonitrile gradient showing P HPLC and showing three groups of components tested in mice.
P HPLC. Fraction 2 represents the TBW active ingredient.

FIG. 2 shows synaptic transmission (excited population spikes) at Schaffer colleteral CA-1 pyramidal cell synapses in rat hypocampal slices.
1 shows DK9-2 tested at 1 μM. The data drawn are (a) 5 minutes before DK9.2 addition and (b) D
15 represents the time averaged population spike record for 15-20 minutes after K9.2 addition. 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 genetic map of a baculovirus conversion vector pWR9 carrying a gene encoding preDK9.2.

FIG. 4 shows neonate tobacco caterpillars (1000,0
00FIB / gm diet) continuous virus supply analysis (fe
eding assay).

FIG. 5 shows DK9.2-induced bursting (bur)
Figure 4 shows the ability of TTX to block or reverse sting.

FIG. 6 shows the ability of TTX to induce a burst by DK9.2 and then apply TTX to reverse that bursting.

────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Karen, Joan Clapcho, Utah, USA 84109 Salt Lake City, San Rafael Avenue 3840 (72) Inventor Bradford, Cal Vanwaagenen Utah, United States 84102 Salt Lake City 1070 East 300 South Apartment 507 (72) Inventor John, Randolph, Hunter Jackson 84109, Utah, USA Salt Lake City Gilroy Road 3661 (58) Fields studied (Int. Cl. ⁶ , DB name) C07K 14/435 C12N 15/12 BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. A peptide having an amino acid sequence represented by SEQ ID NO: 7 or a partial amino acid sequence thereof, wherein the peptide directs secretion and / or folding of a mature peptide as a prepro sequence. 2. A peptide having the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence of a portion thereof,
The peptide secretes and / or folds the mature peptide
A peptide characterized in that it is operatively linked to the peptide as a prepro sequence that directs only the sequence .