CN1064684A - Peptides with insecticidal efficacy - Google Patents

Abstract

The present invention provides a spider venom from Diguetia canities High-efficiency insecticidal polypeptides that can be isolated in liquid, preparation and application of these insecticidal The method of the agent, and the DNA encoding these high-efficiency insecticidal polypeptides.

Description

The polypeptide that has desinsection to render a service

The present invention is relevant with the polypeptide that has desinsection to render a service.Special in following relevant: the polypeptide that has desinsection to render a service that can be separated to from the Diguetia spider poison, encode that these have desinsection to render a service the DNA of polypeptide, the method for production aforementioned polypeptides and control the method for invertebrate pests.

In recent years, the public has profoundly recognized the environmental hazard relevant with using artificial synthetic pesticide and to mammiferous toxicity.Therefore, these Utilization of pesticides promptly descend.Yet, the needs of effective insect control are not changed.This just impels the researchist to go to set up the method for new control insect.

The most widely used microbial pesticide is called for short B.t. later on from bacterium Bacillus thuringiensis().This bacteria preparation is used to control the multiple lepidopterous larvae of eating leaf.Japanese beetle and mosquito.The U.S. Patent number 4,797,279 that Karamata etc. delivered on January 10th, 1989 has been illustrated the heterozygosis bacterial cell of gene of the gene that contains coding B.t.Kurstaki delta-endotoxin and coding B.t.tenebrionis delta-endotoxin and their preparation method.These heterozygotes are highly resistant to the insect of B.t.Kurstaki strain sensitivity and to the insect of B.t.tenebrionis strain sensitivity.In general, these heterozygotes are better than the physical mixed mother for the viewed result of strain having useful insecticidal properties aspect the scope or above-mentioned two of insecticidal power level or insecticidal power.The insecticide mixtures that contains these microorganisms imposed in insect or their living environment with effective desinsection amount of heterozygote just can resist insect.

The derivative of another B.t. bacterium is published in the european patent application distribution and is numbered 0 325 400A1, belongs to Gilroy and Wilcox.This invention is with relevant to the virose heterozygosis virulent gene of lepidopterous insects.Especially, this invention contains the delta-endotoxin genes of heterozygosis.The toxin gene of the B.t.var.Kurstaki strain HD-1 of it some a B.t.var.Kurstaki HD-73 toxin gene and a part.The hybrid toxins gene (DNA) that this coding has anti-lepidopterous insects activated protein has been set forth in this invention.

European patent application issue number 0 340 948(belong to Wilcox etc.), also used the insecticidal properties of B.t. bacterium.This invention relates to the heterozygosis Pesticidal toxins, and they merge insect gastrodermis cell recognition B.t. gene region and lepidopteran toxin B chain in order to prepare the active heterozygosis B.t. toxin of the anti-lepidopterous insects of tool and produce.Invention is pointed out perhaps can insert in the plant or clone in the rhabdovirus with this heterozygosis B.t. gene to produce callable toxin.Perhaps, this host who contains heterozygosis B.t. gene can be used as sterilant, directly imposes in the living environment of target insect.

In the process of seeking the desinsection mixture, scorpion venom is considered to a kind of source of possible tool insecticidal properties mixture.Two kinds of insect selectivity toxin are separated from scorpion Leiurus quingquestriatus quinquestriatus venom, be published in Arch Biochem and Biophysics by zlotkin etc., 240:877-87(1985), exercise question is " activity and inhibition insect toxins all influence the sodium conduction and the common binding site is arranged in the scorpion venom ".About their chemistry and pharmacological properties studies show that the exciting fast paralysis of shrinking of a kind of toxin-induced fly larva, another kind of toxin-induced suppresses lax paralysis at a slow speed.The both influences the sodium conduction.

Canadian Patent 2,005,658 by delivering June 19 nineteen ninety such as Zlotkin, has been illustrated a kind of scorpion Leiurus quinquestriatus hebraeus Buthi-nas, effective insecticidal proteins of Buthidae of deriving from.In this invention venom by freeze-drying and portions from.Larva is had high toxicity and mouse is had minimum toxic that part of being further purified, and final product is called " Lqh P35 ".

Grishin(Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Seiences, Moscow 117988, GSP-1, " the toxin composition in Buthus eupeus and the Lucosa singoriensis venom " USSR) delivered disclosed four kinds of insect toxins that are separated to from scorpion Buthus eupeus venom.The separation and the evaluation of toxin composition in the poison spider Lucosa singoriensis venom of the blue Tula of tower also set forth in article.Former venom is nontoxic to insect.

Corresponding to the research and development of relevant multiple pesticidal mixture, the researchist is devoted to set up the generation killing gene and they is introduced the method for target insect.United States Patent (USP) numbering 4,879,236, deliver on November 7th, 1989 by Smith and Summers, related to a kind of like this method, produced the expression vector of a recombinant rhabdovirus in the genome of introducing rhabdovirus thereby a selected gene linked to each other with a rhabdovirus promotor.It can go out selected gene at insect cell inner expression.This method comprises that the bar-shaped viral DNA of cutting produces the part polyhedrin gene that section of DNA contains the polyhedrin gene or the polyhedrin promotor is arranged.In order to make up a recombinant transfer vector, above-mentioned dna fragmentation is inserted a cloning vector to be inserted selected gene in the cloning vector of this modified then, it is just under the control of polyhedrin promotor like this, and this recombinant transfer vector mixes with wild rhabdovirus DNA then to produce homologous recombination and selected gene integration to be gone into the genome of rhabdovirus.Rhabdovirus Autographa california(Ac MN-PV) and the associated polyhedrin promotor virus expression carrier that is found pairing energy selected gene of high expression in eukaryotic host cell be useful.

Inventors advise cloning on Ac MNPV expression vector by select producing to a certain specific insect or the virose proteic gene of various insects and it, and this expression vector can be used to control the system of insect.They advise that this carrier can be applied on the plant or animal that will protect.This recombinant virus can be invaded the intestines wall and be begun to duplicate after insect is taken in.Another kind of suggestion is it to be merged mutually with the polyhedrin structural order, so that after insect intestines neutral and alkali environment dissociated the polyhedron shell, toxic substance just was released.

Further make up killing gene and need their introducings the method for protection target biology to set forth by Cutler; be published in AG Biotech News vol.7(5): 3 17(1990), exercise question is " electroporation: be used to transform farm crop: by the success of mode crop confirmation ".This piece article points out that DNA can directly enter in the pollen of sprouting with electroporation, and pollen can relay and form seed in the lap waste, grows up to transformed plant then.This method successfully is applied to tobacco, may also can be successful in corn and trifolium.This method may be simpler than protoplastis electroporation, because final purpose is pollinate also " allow this work of doing of having spent " rather than regeneration plant.This program comprises collection pollen, allow it in germination fluid, sprout 30-60 minute, pollen tube will begin to stretch out pollen granule afterwards, the DNA that in containing the suspension of pollen, adds needs, shock by electricity, on pollen tube, punch, the DNA that flush away is unnecessary, the pollen that has changed is placed on the column cap of plant, waits by the time seed forms.This may be a kind of simple method of gene being introduced crop.

Another kind of transfer system is illustrated by Barnes and Edwards, is published in the United States Patent (USP) numbering 4,861,595 on August 29th, 1989.This invention related to that handled, complete on entity microorganism cells as transfer system, and albumen composition is sent in animal and human's body.This microorganism cells produces albumen in cell by a homologous gene earlier.Handling the proteic microorganism of this generation with chemistry or physical method again keeps cellularstructure complete simultaneously.The processed microbe of procedure operation generation is irreproducible in this way, but the activity of intracellular complexes does not obviously reduce.Because this cell is reproducible not, its stable cell walls can decompose in the Digestive tract zone of animal or human's class of needs, so this contrivance can regularly discharge involved product with fixing a point.Through suitable processing, the mixture that the proteic microorganism cells of this generation itself can not need purifying to produce as transfer system.Any protein, polypeptide that produces with microbial process, amino acid or mixture (comprising sterilant) can be as the parent materials of invention.

People such as Carbonell have inquired into the possibility of introducing the synthetic gene of neurotoxin in the coding scorpion venom with dna technique, article is published in Gene 73:409-18(1988) on, exercise question is " the insect specificity scorpion neurotoxin gene of encoding synthetic and with its trial of rhabdovirus vector expression ".Following possibility inquired in this piece article: improve the rhabdovirus pesticidal with the dna technique genome that the synthetic gene of neurotoxin in the scorpion Buthus eupens venom imports rhabdovirus of will encoding.Three kinds of methods that influence the toxin generation in order to the polyhe-dron promotor for the Ac MNPV expression system on basis discussed in this article.Singly be that the gene of expressing 36 codons just can produce a spot of toxin.A signal peptide be connected in also obtain on the toxin some the success.The N of polyhedron gene end and toxin gene are merged the albumen that can produce significant quantity.Yet the amount of detected polyhedron itself is 10 to 20 times of above-mentioned product amount.The restriction of this expression is considered to not be at transcriptional level but comprises translation and protein stability at post-transcriptional level.Do not find that this toxin product has the paralysis effect.

European patent application, issue number 0 431 829 has been illustrated following transgenic plant, in their cell, expresses a kind of insect specificity toxin of catching in the insect organism effectively, and its expression amount enough makes the selectivity insect that engulfs plant tissue poison.This special toxin of being illustrated is isolating from scorpion Androctonus australia venom.

Investigators also can be from spider venom extracting to toxin.Geren is at J.Toxi-col.-Toxin Reviews 5(2): 161-170(1986) delivered " neurotoxin of spider venom and necrotoxin ", the work of relevant neurotoxin and necrotoxin commented in article, and point out that the spider venom molecule can be used as the pattern of specificity insecticide.

United States Patent (USP) distribution numbering 4,925,664th, Jackson and Parks delivered in May 15 nineteen ninety.Illustrated the toxitherapy heart that obtains from spider Agelenopsis aperta and Hololena cur-ta and the method for neural aspect disease used.Thereby these toxin also can be effective as the hamper opposing insect or the relevant insect of special calcium channel or excitability amino acid receptor.

European patent application distribution numbering 0 395 357 has been illustrated the polyamines and the polypeptide that are separated to from spider Agelenopsis aperta venom, and polyamines and excitability amino acid neurotransmission thing be a word used in person's names mutually

These polypeptide and wherein a polyamine species hinder calcium channel in many organism viable cell.Mention available above-mentioned calcium channel hamper in the literary composition and control invertebrate pests.

European patent application issue number 0 374 940 has been set forth the toxin that is separated to from spider Hololena curta venom.These polypeptide can be used as sterilant, pharmaceutically also useful, for example, as a word used in person's names of calcium channel and L-glutamic acid

Thing.

Browers etc. are at Proc.Natl.Acad.Sci.84:3506-3510(1987) delivered article " identify and purifying spider Hololena curta venom in a kind of irreversible presynaptic membrane neurotoxin ", illustrated a kind of albumen neurotoxin that is separated to from spider Hololena curta venom, it can suppress the neuromuscular conduction of drosophila larvae.The author points out that this toxin hinders the calcium channel of the presynaptic membrane of fruit bat motor neuron.

People such as Quistad are at Toxicon 29(3): 329-336(1991) delivered article " paralytic and pesticidal toxin among the Funnel-web spider Hololena curta ", described from a peptide species and ten kinds of toxin of Hololena curta venom purifying and the influence after being injected into lepidopterous larvae.

People such as Stapleton are at J.Biol.Chem.265(4): 2054-2059(1990) publish an article " toxin: the nerve that from funnel-web spider Hololena curta venom, is separated to poisoning worm polypeptide ", illustrated the three peptide species neurotoxins that are separated to from spider Hololena curta venom and to the influence of cricket Acheta domestica.

Quick and Usherwood are at Pestic.Sci.20:315-317(1987) on publish an article " Extoncleol Summaries Pesticides Group and Physicochem-ical and Biophysical Panel Symposium Novel Approaches in Agrochemical Research " illustrated the toxin that exists in parasitic moth and some orb-web spider venoms, can cause quick paralysis after in being injected into insect body.The author points out that spider venom is the hamper of the acceptor of control ion selectivity membrane channel, and the open channel by glutamate receptor control in these passages and the locust skeletal muscle interacts.Article mentions that not only the lower molecular weight toxin in Ar-giope and Araneus spider venom also mentions a kind of toxin that is separated to from Joro spider Nephila clavata poison gland.

The low molecular weight factor that is separated to from the funnel-web spider venom that studies show that another is relevant with isolating spider venom toxin character can reversibly combine with calcium channel.Cherksey etc. on August 24th, 1989 delivered WO 89/07608, having illustrated these active low molecular weight factors can combine with the calcium channel reversibility, this is combined with enough specificitys and avidity disappears neuronic calcium conduction, and can be used to the purifying calcium channel structure.These venom are toxic to Mammals.

Jackson and Parks have discussed other application of spider venom, and article is published in Annu Rev Neurosci 12:405-14(1989), be entitled as " spider venom: the recent application on neurobiology ".The toxin that different taxa talked about in this piece article has very big heterology.It points out that experiment shows that calcium channel has specific specificity, and spider venom can be used as a word used in person's names of calcium channel Thing.The spider venom that comes into question is influential to vertebrates.This article also is used as may originating of agricultural application aspect insect specificity toxin to spider venom.

" separation and the biological activity thereof of cynapse toxin in the funnel web spider Agelenopsis Aperta venom " (" Insect Neurochemistry and Neurophysiology 1986 " the Borkovec and Gelmaneds. that publishes an article such as Adams, Humana Press, New Jersy, 1986).Article has been told about to be separated to from spider Agelenopsis aperta multiplely has a word used in person's names to insect cynapse conduction

The polypeptide toxin of effect.

United States Patent (USP) numbering 4,855,405 has been illustrated from Joro spider poison gland and has been obtained receptor inhibitor and working method thereof by delivering on Augusts 8th, 1989 such as Yoshioka.Desinsection promptly taking place by this mixture of liquid or solid delivery after by the insect contact renders a service.

United States Patent (USP) numbering 4,918,107 was delivered by people such as Nakajima in April 17 nineteen ninety, and it is with following relevant: a kind of have glutamate receptor and suppress active mixture.A kind of insecticide mixtures for preparing the program of this mixture and contain this mixture.This mixture loads with the liquid or solid carrier and adds dispersion agent, directly imposes on plant or the animal that will protect.Very low consumption gets final product effective desinsection, and it is very low to Mammals and toxicity in fish, and is also minimum to the detrimentally affect of environment.

Also inquired into as biotic pesticide with rhabdovirus.Wild rhabdovirus is that as the major defect of biotic pesticide their effects are slow.The larva that engulfs wild rhabdovirus is generally dead within 5 to 7 days.The larva of being infected is a very long time continuation feed betwixt, destroys a large amount of farm crop.

Because the various problems relevant with some synthetic pesticide, the effectiveness that comprise toxicity, environmental hazard, is caused by resistance lowers, need the new invertebrates control method of development so hold with continuing, comprise that setting up the genetic engineering recombinant rhabdovirus produces the proteotoxin that infects the host of itself disabling quickly than rhabdovirus.

This invention provides a kind of new effective insecticidal peptide, the salt of acceptable polypeptide on (it is can being separated to from the Diguetia spider venom of basic purifying) and agricultural or the gardening.

This insecticidal peptide be considered to invertebrates particularly insect very high specificity is arranged.And, proved that this insecticidal peptide has utmost point effective insecticidal power to important pests on the agricultural thereby is considered to the invertebrate pests control epochmaking contribution in field.

In addition, the invention provides a kind of new DNA sequence, its insecticidal peptide in a kind of Diguetia spider venom of having encoded.

The present invention also provides a kind of new recombinant expression vector, and it has comprised the DNA sequence of the insecticidal peptide in one section encoding D iguetia spider venom, and this carrier can express described coded sequence effectively in transformant.

The present invention also provides a kind of new recombinant host cell, can express this polypeptide with the recon cell that DNA transforms or transfection obtains of the insecticidal peptide in the encoding D iguetia spider venom.

The present invention also provides a kind of new recombination rhabdovirus expression vector, and it can express the DNA sequence of the insecticidal peptide in the encoding D iguetia spider venom in host or host insect cell.

The present invention also provides a kind of method to produce insecticidal peptide in the Diguetia spider venom, and the polypeptide of producing.This method comprises following a few step: cultivate recombinant host cell, transform in this reconstitution cell or transfection the insecticidal peptide in a kind of DNA sequence encoding D iguetia spider venom, described carrier can be expressed said sequence effectively in transformant; And from the recombinant host cell of cultivating, separate above-mentioned insecticidal peptide.

The present invention also provides a kind of new transgenic plant, import the insecticidal peptide in the section of DNA sequence coding Diguetia spider venom in the kind system of this plant or in the generation earlier this plant, still remained with the proterties of this kind of expression DNA sequence by the offspring of sexual or these transgenic plant that vegetative propagation obtains.

The present invention also provides a kind of new method to control invertebrate pests, with the insecticidal peptide in the Diguetia spider venom of effective dose and on agricultural or gardening acceptable salt contact described insect.

The present invention also provides a kind of new method to control invertebrate pests, promptly with a kind of baculovirus of reorganization contact insect, this virus can express in the Diguetia spider venom insecticidal peptide and on agricultural or gardening acceptable salt.

The present invention also provides a kind of new sterilant to form insecticidal peptide in the Diguetia spider venom of the effective desinsection dosage that promptly has in the acceptable mediator and acceptable salt on agricultural or gardening thereof on a kind of agricultural or gardening.

The present invention also provides acceptable salt generation immune response on a kind of new antibody capable and the insecticidal peptide in the Diguetia spider venom and agricultural or the gardening.

The present invention also provides the existence of a kind of new method with above-mentioned antibody insecticidal peptide in detecting the Diguetia spider venom: obtain spider venom; Venom is contacted with the antibody that has certification mark; Detect the antibody labeling on the described polypeptide.

The present invention also provides a kind of novel method, comes the insecticidal peptide of purifying Diguetia spider venom with above-mentioned antibody.This method comprises following step: with antibody and solid support coupling; To contain the solution of described polypeptide and contact, described polypeptide is adsorbed onto on the antibody with antibody on the solid support; Described polypeptide is eluted from the antibody that is coupled at solid support; Collect the polypeptide of purifying.

The present invention also provides a kind of new dna probe that derives from the nucleic acid sequence of insecticidal peptide in the encoding D iguetia spider venom.

The existence that the present invention also provides a kind of new method to detect the nucleic acid sequence of insecticidal peptide in the encoding D iguetia spider venom comprises following a few step: the nucleic acid that obtains spider; With this nucleic acid contact with above-mentioned dna probe can with DNA bonded probe.

Fig. 1 is the RP HPLC collection of illustrative plates of Diguetia canities venom, uses 0.1% TFA to ethanoyl nitrous acid gradient, shows detected three composition parts in venom, and second section is represented the TBW active ingredient.

Fig. 2 has shown in the rat hippocampus section, detects DK9.2 in the cynapse transmission (bringing out the colony peak) on Schaffer side-CA1 pyramid synapse cell under the 1 μ m concentration.Colony's peak record (a) of data represented mean time is that to add preceding 5 minutes (b) be 15-20 minute gap after DK9.2 adds to DK9.2 among the figure.These records are identical, and this shows that under this concentration this test detects less than the activity of DK9.2 in rat CNS.

Fig. 3 is the pWR9 collection of illustrative plates, and the transfer vector of a kind of baculovirus has the gene of encoding D K9.2 precursor.

Fig. 4 feeds experimental result (1000,000PIB/gm feeding amount) to the continuous virus of new life's tobacco budworm.

Fig. 5 has shown the prevention of TTX or has reversed the ability that DK9.2 induces outburst.

A. definition.

" insecticidal peptide in the Diguetia spider venom " described in the literary composition comprises having the polypeptide that desinsection is renderd a service, and this polypeptide has the segment that desinsection is renderd a service, no matter the source how, and obtain as long as it can be from Diguetia separates with any known technology. For example, the source can be the insecticidal peptide that restructuring produces.

Described " expression vector " comprises the carrier that can express the DNA sequence that comprises herein, and such order can be operationally connected to other and go up in proper order in order to its expression. Although do not spell out always, it also implying these expression vectors can be in host's biology with chromosome outside or the form that is incorporated into the DNA on the chromosome copy. Clearly, the disappearance replication capacity is incited somebody to action so that they can't operate. Summary is got up, and " expression vector " is a definition on the function, any dna sequence dna, as long as it can effectively express with on specific dna segment be included in this definition. In general, carrier commonly used in recombinant DNA technology is " plasmid ", i.e. finger ring shape double-stranded DNA and not on chromosome. Because plasmid is the most frequently used carrier, so " plasmid " and " carrier " is used interchangeably. However, the present invention also should comprise other expression vector that known gradually, that have same function.

" recombinant host cell " refers to that the carrier that makes up with recombinant DNA technology transforms the cell of gained "

The spider of Diguetia section includes only now one and belongs to Diguetia. The kind of Diguetia mainly is found in Southwestern United Stares (comprising California) and Mexico. A lot of plants and the shrub of these little spiders comprise the irregular net that weaves expansion on the cactus. The kind of various Diguetia spiders as most of spiders, mainly is the predator, is easy to eat the insect that their hunt the most of kinds that arrive.

Although the present invention does not want to be limited in any theoretical reasoning, the uncommon semiotics of observing after to insect injection insecticidal peptide is very similar to veratridine, a kind of alkaline Na⁺Passage promoter, or the scorpion toxin that from Buthidae section and Buthus genus, is separated to-known presynaptic Na⁺Passage promoter is injected in the insect bodies. It is generally acknowledged that aforementioned polypeptides may introduce muscle or neurilemmal partial depolarization, may affect Na⁺Or K⁺Passage. Say that more single-mindedly above-mentioned insecticidal peptide has been induced the responsive nerve of tetradotoxin obstruction is repeated to discharge pulse. Neurilemmal Na to voltage-sensitive like this, probably⁺Passage is exactly the action site of these peptides.

B. isolated polypeptide from the Diguetia venom

Can be with any known method extracting venom from Diguetia spider cephalothorax body of gland. But, for avoiding the pollution of venom and extracting venom, preferably discharge venom with the electro photoluminescence spider, the collected at suction venom prevents because of the pollution that refluxes or hemolymph causes then. (referring to U.S.4,925,664)

After the electricity consumption method of " milking " obtains venom, by high pressure liquid chromatography (HPLC) (HPLC), or gel permeation chromatography, or any other effective separation means is isolated into different peptide components (toxin). Certainly, the final step separation is optimal with HPLC.

Like this, electricity consumption Milking Methods And Techniques, and the gel filtration of back, HPLC, or other relevant separation means, hydrophobic relevant laminate for example just may obtain the spider toxin of purifying. Certainly, use in the present invention other technical point also to be fine from toxin. Separating like this toxin that obtains is that available routine techniques is done the amino acid sequence analysis or measured insecticidal activity.

C. insecticidal peptide

The present invention has just provided a kind of insecticidal peptide from an one aspect, and the segment of insecticidal activity is arranged, and is separated to from the Diguetia spider venom in fact, makes acceptable salt on agricultural or the gardening.

Press preceding method and separate the also insecticidal peptide of purifying, can do the amino acid sequence analysis with any known method, for example the automatic amino acid sequence analyzer of n terminal amino acid sequence analysis.

Just as described in this paper, scope of the present invention also comprises other possible insecticidal peptide, that is to say, comprises other insecticidal peptide that is separated to from Diguetia three kinds that describe in detail except this paper. Introduce below some characteristics of the insecticidal peptide that from Diguetia, is separated to:

1) size: magnitude range is between 6200 to 7200 dalton, and length is 55-65 amino acid.

2) Bao Shou aminoterminal: SEQ ID NOS:1, preceding 5 amino acid whose 3 to 5 is the same.

3) SEQ ID NOS:1,3,5 have the sequence homology more than 40%.

4) SEQ ID NOS:1,3,5 have about 7 or 8 cysteine residues.

5) gene cluster: more than one toxin gene cluster on genomic dna exists, and this shows that these genes might derive from ancestors altogether.

Say more single-mindedly, now identified and separated three kinds of insecticidal peptides.First, DK9.2 is separated, and the amino-acid sequence that translation obtains according to cNDA shows to be exactly SEQ ID NO:1.Mass Spectral Data shows that two kinds of isozyme have conservative substituting on 26, and 26 of an isozyme is that another 26 of Threonines are glutamine.The glutamine isozyme is than about 27 awus of Threonine isozyme.Because of DK9.2 can refer to two isozyme or wherein any.DK9.2 molecular weight mass spectroscopy is about 6371-6391dalton, is single peak on the phase reversed-phase HPLC.Second, the DK11 molecular weight is 6700dalton, or with the 6740dalton of mass spectroscopy, is single peak at the phase reversed-phase HPLC.More definite theory, this HPLC component amino-acid sequence that translation obtains according to cDNA is shown in SEQ ID NO:3.Last is separated, OK12, and the molecular weight size is 7100dalton, or is according to the determined 7080dalton of mass spectrum, is single peak on the phase reversed-phase HPLC.And the amino-acid sequence of this component may be exactly SEQ ID NO:5.

Correspond to SEQ ID NOS:1, three kinds of peptide insecticidal activities of 3 and 5 are almost close.Possible other peptide has 55 to 65 amino acid, and with SEQ ID NO:1,3 and 5 have the sequence homology greater than 40%, and about 7 to 8 cysteine residues are arranged, and shows identical insecticidal activity.This peptide may natural existence or is synthesized by recombinant DNA technology.No matter the peptide that is natural or is re-combined into is included in the category of the present invention.

D. the insecticidal peptide coded sequence among the present invention determines.

Another aspect of the present invention provides the DNA sequence of insecticidal peptide in the encoding D iguetia spider venom.Use aforesaid partial amino-acid alphabetic data, promptly separable and definite these genes.There are many methods to obtain gene, for example: Fuqua, " a simple PCR method detects and the low abundance transcription product of clone " of S. etc., Biotechnique, Vol.9, No.2(Aug 1990); Frohman, " contest: the rapid amplifying of cDNA end " of M.A., PCR protocds, volumes such as Innis, Academic Press, Sam Diego, CA, (1990) and U.S. Patent No. 4,703,008 " coding Erythroprotein DNA sequence ".

DNA chain or its complementary strand that briefly, can synthesize one section decision amino-acid sequence.This synthetic dna molecular can come from genome or the DNA of homology is arranged with it from cDNA clone as probe.In general, 15 or more Nucleotide can be determined a homologous dna uniquely, that is to say comprising five amino acid at least in proper order.The DNA of the amino acid molecular that coding is determined is The more the better, because each amino acid all has six kinds of codons of as many as.Therefore, it is inappropriate using each dna probe individually, but several probe uses simultaneously, just degeneracy probe commonly used in this field.Though have only a dna molecular and the gene of being wished to get that homology is accurately arranged in probe mixture, other probe also may be discerned this gene uniquely, because only need the homology of higher degree.Just can not be cloned into gene so do not need synthetic all possible probes.In general, in probe library, need not to comprise the codon that is of little use in the biology.In fact, can only synthesize a kind of probe, in this probe each amino acid all be used the most frequently used amino acid, certainly, this method is not the total energy success.

A kind of method of determining gene order is polymerase chain reaction (PCR).Referring to United States Patent (USP) 4,683,195 and 4,683,202.In fact, as long as the two ends of known section of DNA order just can be synthesized this DNA with PCR method.With respect to the primer of two ends order or oligonucleotide probe synthetic after, just available PCR comes the DNA of synthetic mesophase.

In a method that obtains spider toxin gene with PCR, extracting RNA and carry out purifying from spider earlier.Make primer with the oligonucleotide of a deoxythymidylic acid tail RNA reverse transcription is become cDNA.The degeneracy probe for preparing foregoing coding venom albumen aminoterminal order then.This kind probe is carried out the PCR reaction with the oligonucleotide of aforementioned deoxythymidylic acid tail.Because the synthetic dna probe has specificity to required mRNA, so have only required cDNA to increase effectively, its product can be connected on some known carriers.However, be to be appreciated that in spider venom, to have gang's peptide, in this case, may make one or more cDNA that plants coding corresponding peptides amplifications in the PCR reaction with similar amino-acid sequence.Because relevant peptide also has insecticidal activity, so the gene of these peptides of encoding is also in category of the present invention.

At last, can synthetic cDNA be cloned on the suitable carriers in proper order with traditional method, determine the order of base, the order of coded insect-killing polypeptide is as what provide in SEQ ID NO:2 and 4.The PCR product is translated into protein show that they have represented the all-cis preface of maturation protein.

Except the cDNA of DK9.2 mature protein coding region, the cDNA of its upstream also clones and checks order.（SEQ ID NO：7）。Complete mRNA translated show that the precursor of DK9.2 comprises a signal peptide, a precursor peptide and ripe toxin peptide.The effect of signal peptide is considered to guide the secretion of peptide, and signal sequence plays a crucial role to the location of nascent peptide.Usually, they provide topological (U.S.A.77 1496-1500(1980), thereby have been positioned in the cell institute's pilot protein or the extracellular for Blobel, G.Proc.Nat.Acad.Sci..This is that extracellular secretory protein is even more important for target site.This production for recombinant protein is also helpful, because can be easy to regard to the purifying expressing protein from extracellular substratum, and does not need broken cell purifying from total extraction liquid of cell.The signal peptide of DK9.2 precursor protein is made up of 17 amino acid:

Met-Lys-Val-Phe-Val-Val-Leu-Leu-Cys-Leu-Ser-Leu-Ala-Ala-Val-Tyr-Ala

Except signal peptide, the mRNA of DK9.2 upstream also has a precursor peptide.Its function is not clear, and 21 amino acid are arranged:

-Leu-Glu-Glu-Arg-Leu-Asp-Iys-Asp-Ala-Asp-Ile-Met-Asp-Ser-Pro-Ala-Asp-Met-Glu-Arg-

Order before these precursor peptides or the precursor peptide may be expressed and fold of great use the stability of DK9.2.These orders or wherein a part for example the gene of a structure may be also very important for the expression of other molecule.As a part of the present invention, the expression that we also will provide signal peptide that data prove other and precursor peptide also can be used for recombinating DK9.2.

E. recombinant expressed

The present invention also provides a recombinant expression vector, and it has comprised the insecticidal peptide in the section of DNA sequence coding Diguetia spider venom.Carrier can make coded sequence express in transformant.The present invention also provides the recombinant host cell that obtains with after desinsection DNA conversion or the transfection, makes host cell can express this polypeptide.The suitable DNA of coding desirable proteins has been arranged, just can produce this albumen with the recombinant technology in modern times.Coded sequence can obtain by cDNA or genomic dna, also can synthesize acquisition according to the nucleotide sequence of gene.If prepare DNA, can utilize host's codon preference with synthetic method.

Expression system must comprise the regulation and control order, and for example promotor preferably has enhanser and terminal regulation and control, and these are conventional in this field.Referring to the Molecu-lar Cloning a Laboratory Manual of Sambrook etc., second edition, Cold Spring Harbor Press(1989).

So, can in eucaryon or prokaryotic system, prepare desirable proteins, under many circumstances, can produce the form of different steps.

The most frequently used prokaryotic system still is E.Coli, though other system, for example B.Subtilis and Pseudomonas are also of great use.Regulation and control suitable in the prokaryotic system comprise composing type and inducible promoter in proper order, for example: the lac promotor, the trp promotor, hybrid promoter tac goes into phage PI promotor.Usually, foreign protein can form generation with fusion rotein or maturation protein in the host.When with maturation protein formal representation desired sequence, order often has a methionine(Met), and can not remove effectively.Described equally, herein albumen or peptide also have a Met as if producing with bacterium at the N end.But also should secrete in order to it at manipulable signal peptide of encoding sequence front construction, signal peptide is cut during secretion.

Now, existing many kinds of eucaryon hosts can be used to the express recombinant foreign protein.As host bacterium, eucaryon host also can be transformed expresses desirable proteins, secretes in order to it but usually need signal peptide.Eukaryotic expression system also has an advantage can shear intron exactly, and this is often to have in the genomic coded sequence of higher organism.Eukaryotic system also provides other treatment mechanism, for example glycosylation, oxidation or derive some amino-acid residue, conformation control or the like.

Eukaryotic system commonly used comprises yeast, insect cell, cells of mamma animals, birds cell and higher plant cell.Also row are incomplete for this.Each host type all has suitable promotor, and termination order and enhanser, for example: baculovirus polyhedrin body promotor.The same, promotor also is divided into to be formed and induction type.For example: in the mammals system, the MITT promotor can be induced by adding heavy metal ion.

The details of construction expression system is conventional in this field.Want a recombinant expressed albumen, earlier coding DNA is connected on the selected expression system, this system is transformed into suitable host and cultivates under certain condition, foreign gene is expressed, from culture, reclaim described insecticidal proteins then herein, can pass through lysing cell or other proper method.

Do some little modifications for the amino-acid residue on the protein, may cause increased activity.These modifications can be directed, for example by rite-directed mutagenesis, also may be accidental for example by common mutagenesis.The sudden change (common or refer in particular to albumen) that changes prlmary structure of protein is because change has taken place its nucleotide sequence of coding.These sudden changes comprise the equipotential sudden change specifically.The change of primary structure can be disappearance, increase or replacement." disappearance " is meant the disappearance of one or more amino-acid residues of polypeptide, and " increase " is meant in the polypeptide chain than wild-type and Duoed one or more residues." replacement " is meant that one or more amino-acid residues are replaced by other residue.Protein fragments is meant that its peptide order of a polypeptide is identical with the part of related polypeptide.

Useful " replacement " is meant what those were guarded, and the residue of also saying so is replaced by the residue of another same big class.The crowd knows that natural amino acid can be divided into acidity, alkalescence, neutral polarity or neutral nonpolar, and aromatic amino acid.General wish the codon replaced and the original code same class of amino acid of encoding.

Like this, usually, basic aminoacids Lys, Arg and His can mutual alternative; Acidic amino acid Asp and Glu can mutual alternative; Neutral pole acidic amino acid Ser, Thr, Cys, Gln, Asn can mutual alternative; The nonpolar fatty amino acid Gly of family, Ala, Val, Ile and Keu be each other guard (but because vary in size, Gly and Ala more recently, and Val, Ile and Leu are more recently.); Aromatic amino acid Phe, but Trp and Tyr mutual alternative.

Though Pro is a nonpolar amino acid, its some troubles because it to the effect of conformation, replace to Pro or Pro be replaced generally all infeasible, unless cause similar conformational change.Polare Aminosaeren comprises Ser, Thr, Gln, Asn can produce conservative variation, Met also on than low degree like this.And, though Ala, Gly and Ser belong to different big classes, also as if can mutual alternative, Cys also belongs to this class, perhaps can put the neutral pole acidic amino acid under.Having some inhomogeneous amino acid to replace also may be of great use.

Because the proteic recombined material among the present invention can provide, these albumen can obtain with recombinant technology or with automatic amino acid synthesizer.Because different hosts have different posttranslational modifications, can obtain the different modifying type of native protein.Because posttranslational modification, comprise glycosylation, amidation or fatization, or the variation of protein one-level, secondary or tertiary structure can make the albumen after the modification different with unmodified protein, and this also is contained in the category of the present invention.

The protein that is to be further noted that here to be said, SEQ ID NO:1 synthetic for example, more replaceable noncoding amino acid.Special residue comprises, omega amino acid for example, and formula is H ₂N(CH ₂) nCOOH, n is 2-6.They are neutral nonpolar amino acids, sarkosine (Sar) for example, t-butyl L-Ala (t-Bu Ala), t-butyl glycine (t-Bu Gly), N-methyl Isoleucine (N-Me Ile) and nor-leucine (Nleu).For example, phenylglycine alternative Trp, Tyr or Phe.Citrulline (Cit) and methionine sulfoxide (Mso) are neutral polar, and Cyclohexylalanine (Cha) is neutral nonpolar, and cysteic acid (Cya) is a tart, and ornithine (Orn) is alkaline.The conformation of Pro can be replaced oxyproline (Hyp) and be realized.

F. transgenic plant

What the present invention further provides is transgenic plant, and the DNA sequence introduced plant kind element of coding effective insecticidal peptide that can be separated to from the Diguetia spider venom in a large number, the expression proterties of DNA sequence is by syngenesis or monogony and by descendant inheritting.

According to the present invention, the gene of effective insecticidal peptide of encoding can pass through the genetic engineering method introduced plant, can be used as the effective means of control insect pest when vegetable cell produces polypeptide.Therefore, might produce the plant that stronger opposing insect ability is arranged than natural mutation.

The coding region that is used for transforming the insecticidal peptide of plant can be the total length or the activated part of gene.Yet this of coded polypeptide section genetic sequence is expressed and what produce must be the polypeptide that function is arranged in the vegetable cell that produces.It is believed that from the DNA of genome and cDNA and the synthetic DNA of coded insect-killing polypeptide and all can be used to transform.And making up gene can part clone with cDNA, and the part genomic clone is partly with synthetic gene and their various combinations.In addition, the DNA of coded polypeptide gene can contain part not of the same race and can not only derive from isolated polypeptide.

In addition, it is believed that this insecticidal peptide can combine with one or more other mixtures, in the transformed plant of expressing these mixture mosaic genes, produce beyond thought insecticidal properties.These other mixtures for example can comprise has the proteinase inhibitor of oral toxicity or the polypeptide that comes from Bacillus thuringiensis to insect.B.thuringiensis albumen causes the change of the saturating property of insect intestinal cells film potassium ion, and supposition can produce aperture on film.Other albumen that can form aperture also can use simultaneously with insecticidal peptide.The alpha-toxin of magainin, ce-cropin, attacin, meiittin, gramicidins, sodium channel protein and synthetic fragment, Staphylococcus aureus for example, apolipoprotein and fragment thereof, alame-thicin and multiple synthetic both sexes polypeptide.Lectin combines with cytolemma and strengthens endocytosis, is the common albumen that uses of the another kind of insecticidal peptide that can invent therewith, can make heredity go up the insect-resistance that changes plant.

The promotor of polypeptide gene can be used as expresses the mosaic gene order, yet other promotor also is useful.The effective plant promoter that comes in handy is the promotor of overexpression.Can operate the expression that the promotor that links to each other with the polypeptide genetic sequence in the system must be able to start this peptide so that plant transformed strengthens the resistance to insect.Having known can be to the plant promoter of the useful overexpression of the present invention.

The mosaic heredity order that contains the insecticidal peptide gene that manually is connected on the promotor can link to each other with suitable cloning vector and transform required plant.Generally, contain from duplicating and the regulation and control order that the kind compatible with host cell come with plasmid or virus (phage) carrier.Cloning vector generally contains replication origin and special gene, can generally be antibiotics resistance as table shape selective marker in transformed host cells.Behind the transformed host cell, can select conversion carrier by phenotypic markers.

Useful host cell can be prokaryotic organism, comprises that host bacterium resembles E.coli, Salmonella ryphimurium and serratia marcescens, or eucaryon host such as yeast or wire fungi.

The host cell that cloning vector and commentaries on classics thereof are stopped generally is used for increasing the copy number of carrier.Because copy number increases, the carrier that contains this polypeptide gene can be separated, and, give an example, can be genetic sequence introduced plant or other host cell of narration here.

There has been method to produce the plant of expression alien gene.For example, can transform plant tissue with the A.tumefaciens direct infection or with plant, plant tissue or co-culture of cells; Directly be transferred to protoplastis with foreign gene; Merge with PEG; Microinjection and micropellet bombardment.

With electroporation (see Ag Biotechnology News, Vol.7 is and 17(Sepet/oct 1990 P.3)) transformation of tobacco is proved.In this method, electroporation plant protoplast under the plasmid environment that contains the insecticidal peptide gene structure is being arranged.The electricimpulse of high strength of electric field reversibly makes the microbial film infiltration cause that plasmid enters.The plant protoplast that electroporation the is crossed cell walls of regenerating.Division forms callus.Screening is expressed the transformed plant cells of insecticidal peptide and can be finished with above-mentioned phenotypic markers.Foreign DNA can add protoplastis in any form, and for example: exposed wire, ring-type or super coiled DNA wrap in the DNA in the liposome, with salt compound DNA and and so on.

From then on used available Agrobacferium plant transformed cell reaches, and the whole plant of transformant regenerated also can transform the whole transformed plant that contains the insecticidal peptide gene of transfer with acquisition according to the present invention.But people such as D.M.Raineri have confirmed rice transformation, and article is published in Biotechnology vol.8, and pp33-38(Jan 1990) on, be entitled as " the purulence bacillus mediated transformation of paddy rice (oryzasatival.) ".

Another kind of method effective insecticidal peptide gene introduced plant is the A.tumefaciens infection plant cell of using by the insecticidal peptide gene transformation.Under suitable normal condition, formed cauline leaf, the root step-length of going forward side by side by plant transformed cell growth and become transformed plant.The insecticidal peptide gene order can import in the suitable vegetable cell, for example the Ti-plasmids by A.tumefaciens.Infect by A.tumefaciens, Ti-plasmids is imported into vegetable cell and is integrated into Plant Genome.

Ti-plasmids has two sections zones extremely important to producing transformant.One of them is transfer DNA (TDNA), causes that tumour forms.Another is called the territory, contaminated area, to forming tumour rather than keeping tumour very important.The TDNA zone that changes Plant Genome over to can increase its length by the gene order that inserts enzyme and not influence its transfer ability.The removal tumor inducting gene makes them no longer influence then, and this adorned Ti-plasmids can be used as the carrier that shifts gene structure of the present invention and introduces in the suitable vegetable cell.

The also available polyoxyethylene glycol of genetic material (PEG) shifts vegetable cell, and PEG and genetic material form sediment composite and absorbed by cell.

Transfer DNA enters plant also can be by injection protoplastis culturing cell and tissue, and the meristematic tissue of injection seedling and plant is finished.Can obtain transfer-gen plant and offspring thereof by ordinary method.

With shooting device be called " particle gun " it squeezed into vegetable cell attached on the particle the another kind of method of foreign DNA order introduced plant cell is comprised DNA.Any plant tissue or organ all can be used as the target of this program.Include, but is not limited to: in embryo, top or other meristematic tissue, bud, the body or external somatocyte and reproduction organization cell.Transgenic cell and callus program is according to the rules selected.Induce target tissue according to conventional procedure, organizer embryo or regrowth form transfer-gen plant.Can select suitable procedure according to the plant species that uses.Change DNA over to cells,primordial with the little bullet of high pressure and obtained transgenic corns.The article report is seen Biotechnology, Vol.8, pp833-838(Sept 1990), be entitled as " heredity of rotaring gene corn plant filial generation and the expression of mosaic gene ".

Regeneration plant is chimeric for the foreign DNA of integrating.Become little or megaspore if contain the cell development of foreign DNA, the DNA of integration will be imported into sexual progeny.If containing the cell of foreign DNA is plant soma, can obtain non-chimeric transfer-gen plant by agamic ordinary method, can pass through the cutting or the external method of bud or stem in vivo by routine.Can select some program according to the plant species that uses.

Behind transformed plant cells or the plant, those give expression to the polypeptide transformed plant cells or plant can be selected by suitable phenotypic markers.These marks include but not limited to antibiotics resistance.Other phenotypic markers is all very conventional also to can be used for this invention.

Because multiple different conversion system is arranged, all vegetation types all can be transformed thereby be given expression to insecticidal peptide of the present invention in principle.

More and more evidences shows that in fact all plants all can include, but is not limited to from culturing cell or tissue regeneration: all main cereal are made kind, sugarcane, beet, cotton, fruit and other trees, beans and vegetables.Now, knowledge is confined to whether the available Agrobacterium of all plants transforms.The natural phant host species of Agrobacterium can be in vitro conversion.Monocotyledons, especially cereal and grass are not the natural hosts of Agrobacterium.Trial transforms till now also not success with Agrobacterium.Just there is being the evidence that increases to show that some monocotyledons can be transformed by Agrobacterium.Using becomes possible new experimental technique now, and cereal can be transformed.

Other can be belonged to by the Agrobacterium plant transformed and comprise Ipomoea, Passi-flora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, San-talion, Allium, Lilium, Nacissus, Ananas, Arachis, Phaseolus and Pisum.

Kind with kind between the regeneration of plant be different, but in general provide the conversion protoplastis suspension that contains multiple copied insecticidal peptide gene earlier.Can as natural embryo, induce protoplastis suspension cell to form embryo to ripe and sprouting stage.Nutrient solution generally contains each seed amino acid and hormone.Generally form cauline leaf and root simultaneously.Successful regeneration depends on the history of nutrient solution, genotype and cultivation.If these three variablees are controllable, then regeneration is fully repeatably.

Can selfing produce the selfing plant from the plastidogenetic ripe plant of plant transformed.The seed that this selfing plant produces contains the insecticidal peptide gene.These seeds can grow up to the plant of expressing insecticidal peptide.The suicide plant can, for example, form the heterozygosis plant of anti-worm.The selfing plant and another self-mating system that are anti-worm produce hybrid plant.

To the amphiploid plant, a common maternal insecticidal peptide (toxin) of using transforms, and another female parent is a wild-type.After the hybridization, first filial generation (F1) produces 1/2 toxin/wild-type: the separation of 1/2 toxin/wild-type.First filial generation heterozygote (F1) selfing produces second filial (F2).The gene isolation of F2 is 1/4toxin/toxin:1/2toxin wild type:1/4wild type/wild type.The genomic constitution of F2 in generation is that toxin/toxin is selected as insect tolerance plant.

As used herein, still can express under the prerequisite of insecticidal peptide of the present invention in mutation, mutation refers to that stable and heritable phenotype changes, but includes the heritable variation that the offspring is passed in sexual reproduction.In addition as used herein, sudden change refers to the variation that causes because of envrionment conditions such as radiation, or the heritable variation of transmitting through reduction division according to genetic development of a kind of proterties.In any case, this mutant plant must still be expressed polypeptide of the present invention.

In general, the next desirable insecticidal proteins of expressing in transformed plant of choosing must be safe to non-target insect and vertebrates.Select to make expression level that the expression system of enough desinsection effectiveness is arranged, therefore, obtain the technical feasibility of so genetically modified important agricultural crops, will in the The whole control pest system, increase new weapon, the harm of crop is not influenced environment simultaneously thereby reduce insect to the peasant.

G. polypeptide is as the application of sterilant

It is believed that insecticidal peptide of the present invention can be used to control invertebrate pests such as lepidopteran with the significant quantity contact insect of polypeptide.For the purpose of convenient, insect is the insect that refers to partially.

Control said insect with polypeptide contact invertebrate pests, its method as can be known, example has artificial embedding albumen oral by insect.Express the proteic recombinant host of the present invention, can be heated as Pseudomonas flubrescens and kill, allow then insect is oral to be controlled.

Certainly, also can use with the method for albumen control invertebrate pests of the present invention with other method of controlling insect.For example, operate transgenic plant and E.coli above-mentioned, to express other no vertebra toxin according to the variable of the insect type that will control and other existence.

The present invention thus also provide the insecticide mixtures content of peptides that contains polypeptide of the present invention have desinsection render a service or and its salt, be to load with acceptable carrier on agricultural or the gardening, can accept on the agricultural or on the gardening.

H. the effective antibody of insecticidal peptide

Another aspect of the present invention is the antibody of effective insecticidal peptide of the present invention.In the narration below, the immunological method that will can grasp with reference to various routines, detect with purifying can with the polypeptide of said antibody response here.

Be combined on the antibody with a part reaction thereby this molecule if antibody capable is special, just say the molecule combination therewith of this antibody capable.Determining that its " epitope " refers to can be that part of by antibody recognition and bonded in the polypeptide antigen.An antigen can have one or more decision bases.But the antigen induced animal produces can the antigenic therewith basic bonded antibody of decision.The above-described special antigen that should be meant will carry out immune response with the selectivity and the corresponding antibody of height, and not with multiple other antibody response that may cause by other antigen.

Here " antibody " (Ab) or " monoclonal antibody " (Mab) not only comprise complete molecule also comprise it can with antigen bonded fragment (for instance, as Fab and F(ba') ₂Fragment).Fab and F(ab') ₂Fragment lacks the Fc fragment of complete antibody, can eliminate from circulation quickly, combines less with the non-specific tissue of insect antibody.

Antibody of the present invention can be by any preparation of several different methods.The method for preparing this antibody is known very clearly, has a detailed description in the literature.As seen " Molecular Clonig a laboratory manul " Second ed.cold Spring Harbor Press of Sambrook etc. for example, Vol, 3 ch 18(1989).For example: expressing insecticidal peptide or its segmental cell is introduced animal, induce produce contain can with the blood plasma of polypeptide bonded polyclonal antibody.General with preparing and purifying makes it not contain that natural impurity obtains the insecticidal peptide fragment or with ordinary method synthetic insecticide polypeptide fragment.In order to produce more highly active polyclonal antiserum, the fragment of purifying or the natural fragment and/or the synthetic segmental mixture of synthetic fragment or purifying can be introduced animal.

Monoclonal antibody can prepare with hybridoma technology.Usually, this program comprises uses the insecticidal peptide antigen-immunized animal, and the splenocyte of extracting immune animal also merges with suitable myeloma cell.Any suitable myeloma cell line all can be used among the present invention.After the fusion, on suitable substratum, keep hybridoma, and by restriction dilution clone.Can combine to determine required clone with insecticidal peptide antigen by detecting secretory antibody at last.

If the peptide source is impure, the antibody capable that has only the part hybridoma to produce combines (antibody capable that other hybridoma produces combines with peptide impurity) with peptide.Like this, just be necessary the required clone of screening from hybridoma.In the process of screening, after venom sample and the insulation of hybridoma excretory antibody, whether the observation insecticidal activity is neutralized or weakens.In case determined required clone strain, just available proper method increases it to produce the single-minded monoclonal antibody of peptide.

For the Pesticidal toxins of purifying natural or reorganization, be necessary to use insecticidal peptide specificity antibody.Usually, this antibody is monoclonal antibody.After having obtained peptide specificity monoclonal antibody, it can be coupled on the solid support, use method isolated peptides from natural venom of immunoaffinity chromatography then.This method can obtain highly purified peptide does not have natural impurity.In this article, " no natural impurity " is meant does not have bonded impurity compound under the native state (that is to say other protein, fat, carbohydrate etc.).

At purifying after the peptide, (for example mouse or rabbit produce peptide specificity polyclonal antibody to induce to can be used to immune animal.

Suitably the dna probe of size is generally 10 to 50 Nucleotide, can derive from the encoding sequence of insecticidal peptide in the Diguetia venom.This probe can be used to detect the existence of the dna sequence dna of encoding D iguetia venom insecticidal peptide, with probe contact and detect with venom can with probe bonded DNA.

I. insecticidal microorganism from the heredity through transforming

Effective insecticidal peptide uses separately or is used with other insect toxins can be from the toxicity of intensity and degree enhancing such as microorganisms such as rhabdovirus (baculoviuses) and recombinant bacteria.

Comprise that those infect the cotton corn earworm of Heliothis Virescens(), the dark-coloured moth of Orgyia pseudot-sugata(Douglas fir), Lymantia dispar(gypsymoth), Auto-grapha Californica(autographa california), Neodiprion sertifer(Europe pine fly) and Lanspcyresia pomonella(blue or green apple moth) identify and as sterilant in some countries at interior several rhabdovirus.At least a to the insect tool optionally toxin gene introduce in its genome and can significantly strengthen the parasiticidal effectiveness of this class.

It is a kind of that to be highly suitable for recombinant expression vector of the present invention be as United States Patent (USP) 4,879, the rhabdovirus expression vector of the sort of type described in 236, this patent sets forth in detail its feature.See " the synthetic trials that reaches by this gene of rhabdovirus vector expression of the gene of a kind of insect specificity scorpion neurotoxin of encoding " such as Carbonell again, Gene, 73:409-418(1988).It is useful in an expression system that this carrier is estimated, in this system will in the Diguetia spider venom in a large number the dna encoding sequence clone of a kind of effective insecticidal peptide of separation and Extraction in a kind of Autographa Californica rhabdovirus (AcMNPV) expression vector, this rhabdovirus carrier is at United States Patent (USP) 4,879,236 and Miller etc., Science, 219,715-721, (1983), the middle description.This recombinant expression vector virus can be applicable to be harmful to the animal or plant that infects by insect.When virus is taken in by harmful insect, the intestines parietal cell that recombinant virus is just invaded insect begins to duplicate, effectively the insecticidal peptide gene just can be expressed in reproduction process, thereby causes insect wounded or disabled or dead in shorter time after taking in wild-type AcMNPV virus than insect.

A kind ofly estimated that equally useful recombinant virus is illustrated in european patent application 0,340, in 948.This kind hybrid virus of expressing DNA of the present invention can become the virus that has changed the insect host scope.As, can express a strand fusion protein product by the recombination that a DNA among the present invention and a species specificity insect intestinal cells identification protein gene constitute, this fusion protein product can make effective insecticidal peptide arrive the host insect target position under the guidance of identification protein gene product.

Press european patent application 0,325, method described in 400, multiple protokaryon and eukaryotic microorganisms can be transformed and express effective insecticidal peptide of its coding by a kind of recombinant toxin gene.

The recombinant bacteria that contains the plasmid that is loaded with protein gene of the present invention estimates to be applicable to the method among the present invention.Insect will be controlled in this insect by using this recombinant bacteria.Referring to example, United States Patent (USP) 4,797,279, in this patent sets forth in detail its feature.

Other examples that utilization is suitable for rhabdovirus of the present invention see Tomalski etc., " mediate the insect paralysis disease that a kind of expression of mite neurotoxin gene causes " by rhabdovirus, Na-ture, 352:82-85(1991) and Steuart etc., " a kind of improved structure that has the rhabdovirus sterilant of insect specificity toxin gene, " Nature 352:85-88(1991); McCutchen etc., " a kind of acquisition of expressing the recombinant rhabdovirus of insect selectivity neurotoxin: the possibility of pest control ", Biotechnology, 9:848-851(1991).

J. hybridization: dna sequence dna is as the probe of relevant molecule

Suitably greater than dna probe can be inferred from dna sequence dna of the present invention.This class probe can be used to wherein whether have with detection with the nucleic acid hybridization in other sources the DNA of the effective insecticidal peptide in the code book invention.Under non-rigorous condition, screen as oligonucleotide probe with its signal sequence or its cDNA fragment even its complete cDNA, can obtain and lps molecule other active polypeptide of the function homologous DNA of family as herein described.Can should include, but not limited to belong to together spider not of the same race with this good suitable nucleic acid source of finding the oligonucleotide probe hybridization of dna sequence dna, the relevant spider that belongs to, and same genus but the spider in the different places of production.

K. the evaluation of a promoter sequence

The present invention provides regulation and control DK9.2 synthetic promoter sequence in spider on the other hand.Do probe screening chromosomal DNA storehouse to analyze the structure of DK9.2 gene with ripe toxin cDNA.Chromosomal DNA is transcribed the analysis of recording the starting point upstream, show the promotor that has a supposition.The general dna sequence of the 421bp of this promoter region is shown in SEQ ID NO:8.The promotor of this supposition comprises the many essential adjustment signal that is present in usually in the upstream region that is right after transcription initiation site.(summary of promoter regulation signal is seen Meknight etc., 1982.The eukaryotic protein encoding gene transcribe control signal.Science217：316）。A typical TATA frame is positioned at transcribing of supposition and opens-30 places beyond the beginning site, and it seems that it is important that this frame starts the rna regulation synthetic.At upstream end more the palindromic sequence of two apart from each others is arranged, one of them comprise it is believed that to the RNA polymerase in conjunction with important same feeling CAAT box.By inference, this promotor or promoter region have use value to heterogeneic transcript and expression in plant, animal or bacterium, for example in the gene of a reorganization structure.

Embodiment

Following examples are in order to illustrating product concrete in the scope of the invention and method, but they do not limit range of application of the present invention.

Material and method

Spider is by Spider pharm, Inc of Black Canyon city, AZ, and the evaluation of being undertaken kind by the said firm.Diguetia canities spider venom is extruded with electrical method, adopts sfgd. to guarantee wherein not have the pollution of saliva and hemocyte simultaneously.Toxin purifying-original venom (being stored in-80 ℃) is melted, before chromatography with its thorough mixing and be dissolved in 0.1% the trifluoroacetic acid (TFA).Original venom uses Beckman System Gold 126 solvents and 168 photorectifier bundle detectors with reverse liquid chromatography (LC) (RPLC) method branch.Acetoxyl nitrous acid or Virahol add TFA as the positively charged ion coupling agent.Use the following pillar that contains same matrix in the purifying: Dynamax 300ARPC ₁₈Post (25cm * 4.6mm i.d., granular size 12 μ m), Vydac 300A C ₁₈Analytical column (25cm * 4.6mm i.d., granular size 5 μ m), and Vydac 300A C ₁₈Preparation property post (25cm * 10mm i.d., granular size 5 μ m) partly.Peak value is sought in monitoring at 220nm light wave place, and carries out fraction collection with GILSON208 trace Fraction Collector.All collection units all behind frost drying, are stored in-80 ℃.Fast atom bombardment MS analysis (FAB-MS)-under standard FAB condition, on the VG of Univ.of ILLinois instrument ZAB-SE mass spectrograph, measure mass spectrum (xenon, resolving power 1000, acceleration voltage 8KV, 40 ℃ of internal temperatures).Sample (about 1ug) is dissolved in 4ml and contains 25% water-soluble TFA(1%) thioglycerin in analyze.

Embodiment 1

To be dissolved in 0.1% trifluoroacetic acid (TFA) with the original venom of 25 μ l lyophilizes that preceding method obtains in Diguetia canities, at preparation property Vydac RP C partly ₁₈On the post with reverse liquid chromatography classification, with the flow velocity wash-out of per minute 3.5ml, use linear gradient elution liquid, its composition is water-soluble 0.1%TFA and 50% nitrous acid dissolved 0.1%TFA, the ratio of the two is 85: 15 during the beginning wash-out, and 50: 50 ratio finishes wash-out after 180 minutes.

The collection unit residence time (approximately the number of minutes)

1 3.2

4 29.6

6 48.2

8 57.1

9 62.8

10 66.4

11 68.7

12 71.4

13 77.1

16 126.1

17 131.4

Each collection unit is all removed eluent and water successively through frost drying, and sample is stored in-80 ℃, and each collection unit sample all was dissolved in the 25 μ l physiological buffer saline solutions when desinsection was identified.The insecticidal activity of some collection units is by to tobacco budworm(Heliothis Virescens) test of the effect of " TBW " be confirmed (table I)

The TBW larva is used 50 μ l Hamilton needle cylinder injections, the 3 μ l test solns of having adorned 33gauge syringe needle and PB 600 continuous application of samples, and each collection unit is tested with five larvas.Injecting method is, syringe needle is inserted into belly side midline (near arbitrary foreleg place), with the Small angle injection in order to avoid destroy internal organ.Every the insect in injection back is all put into a container that contains synthetic food.Regularly observe; Inject and began to measure paralysis symptom in back 24 hours.When calculating dosage, use the mean body weight of 0.3gm as TBW.One group of control larvae is only injected the physiological buffer saline solution.

Paralysis symptom develops gradually, and a tangible muscle spasm stage is arranged before this.Under the serious situation, spasm produced between the 15-30 after the injection minute, and strengthened gradually until larva and lose motility fully, and it is lasting more than 48 hours to tremble sometimes.This phenomenon also can betide has sometimes only injected sublethal dose and the final situation of bringing back to life of larva.The severity of trembling is the most reliable index of toxicity.Shrink if be applied to complete paralysis and/or health, larva just can not be brought back to life.

The table I

The original venom RPLC of Diguetia canities collection unit is right

The toxicity of TBW (the every gram of 7.5WVE/ insect) ^*

Paralysis percentage ratio paralysis percentage ratio

Collection unit is 24hr at the beginning

1 0 0

4 0 0

6 0 0

8 100 100

9 100 100

10 100 100

11 100 100

12 100 100

13 0 0

16 0 0

17 0 0

Contrast 00

Paralysis is defined as insect is being changeed by side or is overturning and can not recover the symptom of self normality.

* WVE, original venom equivalent amount is meant the amount of any toxin of the original venom that normal presence is extruded in 1 μ l; Obtain 5WVE from 25 μ l parting liquids and be equivalent to 20% of recovery sample.

Embodiment 2

The purifying of Diguetia canities collection unit 9

The main component of the active collection unit 9 of TBW obtains one-component by chromatographyization once more, and (the per minute component is a visible band in the SDS-DAGE electrophoresis, molecular weight is about 6, and 500Dalton), chromatography is at Vydac RP C ₁₈Carry out on (25cm * 10mm i.d) post,, under the 220nm wavelength, monitor with speed wash-out under the linear solvent gradient (1.0%/min) of Virahol/0.1% trifluoroethanol of 3.5ml/min.

The detection at peak and fraction collection are collected two parts as shown in example 1.First goes out (collection unit 9.1) at 21.81 minutes wash-outs, and second peak goes out (collection unit 9.2) at 22.39 minutes wash-outs.

Collection unit 9.1 and 9.2 is successively removed eluent and water by frost drying respectively, concentrates to stay sample 9.1 and 9.2.The collection unit degree of purity of production is estimated to reach 99% at least after the lyophilize.From the original venom of 25 μ l, estimate approximately to be contained the sample 9.2 of 6 μ g pure proteins.Sample 9.2 is injected into tobacco badworm shown in example 1 testing its insecticidal activity, method be in five experimental insects every all inject the 6.3 μ g samples 9.2 that are dissolved in the buffering saline solution, only inject with five larvas in addition and cushion saline solution and organize in contrast.Check insect after 24 hours and 48 hours.Those all processing insects are all benumbed and are contrasted insect thereupon dying and but shown normally in the time of 24 hours.There is not any variation in the time of 48 hours.

Analyze this polypeptide by fast atom bombardment mass spectroscopy(FABMS) and show that its molecular weight is 6371 ± 2.The-terminal amino acid sequence analysis of sample 9.2 is got in fact consistent with shown in the SEQ ID No:1 amino acid/11-33 of its partial amino-acid order.

At H.Virescens(TBW) in the half-dead dosage LD of Diguetia toxin 9.2 ₅₀Be approximately 1.0nmol/gm. its produce use among symptom and the embodiment 1 original venom produced similar.

Embodiment 3

The purifying of Diguetia Canities collection unit 11

With mode described in the similar example 2, the collection unit 11 that is separated in the original venom of Diguetia Canities in the example 1 obtains a component through reverse liquid chromatography (LC) purifying again.This component is successively removed eluent and water and is obtained a kind of hangover through frost drying and is called sample 11.This sample detects it to tobac-co budworm vigor by example 2 described methods with the dosage of 5.0WVE/g.After 24 hours, present paralysis symptom through the insect 80% of sample preparation, 60% presents paralysis symptom after 48 hours.The control group insect is then acted normally.

Show that through the fast atom bombardment mass spectroscopy(FABMS) analysis this polypeptide molecular weight is 6740 ± 2, record the partial amino-acid order of sample 11, see SEQ ID NO:3 amino acid/11-29 with end amino acid sequence analysis method.

Embodiment 4

The purifying of Diguetia Canities collection unit 12

With the mode in the similar example 2, the collection unit 12 that is separated in the original venom of Diguetia Canities in the example 1 obtains a component through reverse liquid chromatography (LC) purifying.This component is successively removed eluent and water obtains spissated sample through lyophilize, claims sample 12.This sample detects its insecticidal activity to tobacco budworm by example 2 described methods.80% presented palsy after 48 hours after the insect 100% that sample 12 is handled presents paralysis symptom in 24 hours.The control group insect is then acted normally.

Analyzing this polypeptide through fast atom bombardment mass spectroscopy(FABMS), to record its molecular weight be 7080 ± 2.Its partial amino-acid order of-terminal amino acid analysis revealed is seen SEQ ID NO:5.

Limited materials supply do not allow accurately to demarcate toxicity; Therefore sample 9.2 detects toxicity on dosage (nmol/mg) level than sample 11,12 difference high 39% and 67%.The dosage of selecting is to arrive no effect dosage level in order to cover minimum lethal dose, but is not all to achieve the goal at every turn.However, the result shows that these polypeptide have closely similar toxicity.Sample 9.2 may have strong slightly toxicity than other two samples, but difference may be less than 3 times.Minimum 100% lethal dose of these polypeptide (sample 9.2,11,12) is between 3.0 to 5.0nmol/gm, and this compares with commercial sterilant and has more superiority; As teflubenzuron, its medial lethal dose (SAW) is 1.0nmol/gm.

Embodiment 5

The encoding gene of the sample 9.2,11 that separation is separated in Diguetia Canities venom.

The separation of the first step: RNA

Collect Diguetia Canities spider from the field.Spider alive is freezing and taking-up cephalothorax under liquid nitrogen.Press Chomczynski and Sacchi, Analytical Biochemisfry, 162, the extracting RNA in cephalothorax of method 156(1987).Polyadenylic acid courier RAN(mRNA) through few d(T) and the Mierocrystalline cellulose chromatography (Pharmacia LKB, Sweden) and purifying.

Second step: the cDNA's is synthetic

(Bethesda Research Lab-oratories, under effect MD), the operational degree reverse transcription that provides by the manufacturer synthesizes cD-NA to messenger RNA(mRNA) at mouse white corpuscle viral reverse transcriptase.In the reaction mixture of 20 μ l, contain by cDNA synthetics box (Borbringer Mamnbeim, the enzyme buffer liquid that 1N) provides, 50 μ g mRNA, 2 RNaseH of unit, 30ngd(T) the NotI primer (promega, Madison, WI), four kinds of each 1mM of deoxynucleoside triphosphate, and 100 μ g reversed transcriptive enzymes.Reaction mixture continued incubations 10 minutes at 42 ℃ then 37 ℃ of incubations 1 hour.Add ethanol sedimentation again, be dissolved at last in the 20 μ l water.

The 3rd step: primer synthetic

The primed DNA order mixture of one degeneracy

The codon that uses Drosophila to be inclined to use designs one section by the 1st to the 8th amino acid whose degenerated primer DNA sequence mixture of SEQ ID NO:1 codified.This primer is synthetic by the Howard Hughes Medical Institute of University of Utah contract equipment.

The 4th step: amplification

Use the heat-stable DNA polymerase to carry out the DNA cloning that is instructed by primer, this method is published in Science by Saikki etc. at first, and 239,487(1988).In actual applications, we (Perkin Elmer Cetus adds 5 μ l Diguetia cephalothorax cDNA as template in the polymerase chain reaction thing that CA) provides containing GeneAmp Tm DNA cloning instrument cases.Contain meaningful in the amplification reaction system and each 2 μ M of antisense primer, four kinds of each the thermally-stabilised reorganization of 100 μ M and 4 units TaqI polymerases of deoxynucleoside triphosphate.Be reflected on the DNA thermal cycler that Perkin Elmer Cetus company produces and carry out.From relevant toxin gene family with similar-terminal amino acid order by using the gene of tight annealing temperature (58 ℃) selective amplification coding sample 9.2.Amplification has produced single section of DNA under such condition, and agarose gel electrophoresis shows its big or small 275bp. of being under more not tight condition, and then the gelose detected through gel electrophoresis goes out the amplified production more than five, and its size does not wait to 360bp from 200bp.These different PCR products that obtain under stringent condition more not may be encoded on some amino-acid sequences, the polypeptide close with sample 9.2 on the molecular weight size and on the insecticidal activity.In these relevant polypeptide in advance in respect of sample 11 and 12, because its-terminal amino acid order is as SEQ ID NO ₈: shown in 1,3 and 5 to each other and closely similar with sample 9.2.

The 5th step: the clone of PCR product

By the height stringent condition and more not the PCR product that obtains of stringent condition through Centri-con-100(Amicon) the molecular size separation system removes the primer that does not participate in and obtains purifying.Products therefrom is used restriction enzyme NotI(MBR again, Milwawkee, and WI) enzymolysis, this enzyme can cut in the downstream of primer (3' end) and produce a sticky end.Carrier PKC(Sfratagene, LaJolla is CA) through EcoRV(US Biochemical) and the NotI double digestion produce unidirectional cloning site.Carrier transforms compex DH5 α F' with inserting after fragment is connected.Transformant is used ³²The intermediate segment probe screening of P mark, the clone who selects uses the intermediate segment probe and does the further evaluation of primer measurement miniprep dna order (US Biochemical's Sequerase Ver-sion 2.0) do.

Two clones' cDNA inserts segmental global DNA order shown in SEQ ID NO:2 and 4.Only the former is the clone from tight PCR reaction gained, and two kinds of cD-NA insertion fragments all can be found in the clone who is obtained by more not tight PCR reaction conditions.SEQ ID NO:2 encoded polypeptides amino-acid sequence is seen SEQ ID NO:1, and this polypeptide has the terminal order of the N-consistent with sample 9.2.The molecular weight of this polypeptide is 6377.9Daltons according to estimates, supposes that this polypeptide all (halfcystines) all exists with the form of intramolecular disulfide bond (alkylamino) under natural form, and then molecular weight is 6369.7D.Therefore, it seems that this polypeptide be same a part with sample 9.2.The latter determines that through mass spectroscopy its molecular weight is 6371 ± 2.See SEQ ID NO:3 by SEQ ID NO:4 encoded polypeptides amino-acid sequence.This polypeptide has a N-terminal order identical with sample 11.Therefore, this polypeptide it seems with sample 11 are same polypeptide.

Embodiment 6

To mammiferous toxicity

The original venom of 5 μ l that foregoing method obtains from D.Canities if divide three injections (IP) in mouse peritoneum, will cause dead mouse.The mouse of handling originally with the control mice indistinction of only injecting saline solution.But injection back 10 to 15 minutes is handled mouse and is just become many moving, shows unique jump attitude; The exercise not harmony that the short period of time then occurs, expiratory dyspnea and spasm are certainly producing symptom by dead about 2 minutes.

With about 4.2,0.9, the dosage of 1.2mg/kg injected sample in mouse peritoneum does not all have effect and produces in 9.2,11,12,24 hours respectively.

With the dosage (about 1mg/kg) of every about 30 μ g of mouse to mouse (about 28gm) brain inner room injected sample 9.2.All do not find any effect in back 48 hours up to injection.Other has the about 125 μ g(4.2mg/kg of injection in the mouse peritoneum) sample 9.2, also do not have effect as seen.

In order to identify that this venom is to vertebrate toxicity.Original venom carries out toxicity test through the latter incorporated collection unit of reverse liquid chromatography (LC) in mouse, collection unit 2 contains the budworm(TBW to tobacco) great-hearted composition.The dosage that is equivalent to the original venom of 25 μ l is injected collection 1 and 2 no any effects in mouse peritoneum.Collection unit 3 then generation is viewed the same with the original venom of injection, and these results show that insecticidal active ingredient and vertebrates objectionable constituent are visibly different in the composition of Diguetia Canities venom.

Embodiment 7

Electric physiological data

DK9.2 is with concentration research cynapse transduction (exciting colony hook connects) in the Schaffer collateral-CA1 cone cell cynapse of rat hippocampus section of 1 μ M.Be shown in and data representedly among Fig. 2 connect record (a) DK9.2 with time averaging colony hook and add preceding 5 minutes record, (b) DK9.2 adds the record of back during 15-20 minute.These records can be that eclipsed shows, under this concentration, DK9.2 does not have this analytical method to central nervous system in rat can detected vigor.Can detect the diversified effect of different Mammals ionic channels and neurotransmitter receptor with this analytical method.（T.V.Dunwiddie，The Use of In Vitro Brain Slices in Neuropharmacology，In Eletophysiological Techniques in Pharmacology，edited by H.M.Geller，Alan R.Liss，Inc.，New York，1986）。For example, stop the molecule of sodium channel inactivation,, can cause colony's hook cipher obvious expansion of phase at last as some scorpion toxin.(Kaneda M, MyamaY, Ikemoto Yand Akaike N., Scorpion foxin prolongs an lnacfiva-tion phase of the voltage-dependent Sodium current in rat iso-lafed single hippocampal pleurons, Brain Res, 487:192-195,1989; Alan L.Mueller, Natural prochucts Sciences, Inc., Unpublished obseruations), comparatively speaking, DK9.2 has activity to insect trial target (housefly) under low 100 times concentration.It is to stop the mode of insect sodium channel inactivation to play a role with a kind of supposition.

Embodiment 8

The cDNA upstream order of encoding D K9.2 precursor

In order to obtain the cDNA upstream order of encoding D K9.2, synthesized one section intermediate segment oligonucleotide corresponding to 159-178 nucleotide residue on the antisense strand of SEQ ID NO:2DNA order, at the 5' of this primer end an EcoRI point of contact is arranged.The plain cDNA of the strand venom of 10 μ l adds some IUDR acid residues with terminal deoxynucleotidyl transferase (Bethesda Research Laboratories) at its 3' end.The enzyme and the 500 μ M dGTP that contain 14 units in the reaction solution of 20 μ l were 37 ℃ of incubations 15 minutes.Sample is dissolved in behind ethanol sedimentation in the 20 μ l water.

By one to amplification downstream/or similar anchored pcr technology of ripe toxin cDNA sequential grammar, by the special primer guiding upstream region of gene dna sequence dna that increased.Contain adopted primer is arranged (primer of dcc ending) and each 2 μ M of nonsense primer in the amplification reaction solution, deoxynucleoside triphosphate is 100 μ M respectively, and the thermally-stabilised reorganization Tag polymerase of 4 units.Temperature variation is as follows: 94 ℃ 2 minutes, 37 ℃ 2 minutes, 37 ℃ 1 minute, this circulation repeats twice, in the circulation of back the second step annealing temperature is brought up to 54 ℃.This circulation repeats 32 times altogether.

4% fine jade that exists at EB refers to that sugared gel electrophoresis shows, anchor PCR produces the fragment of a 380bp.This fragment is used ethanol sedimentation then with the E.coli big fragment of DNA polymerase I (Klenow fragment) (Molecular Biology Resources, Madison WI), cuts with restriction enzyme EcoRI enzyme behind the dissolution precipitation.Endonuclease bamhi in the presence of 1mMATP by T ₄The proteolytic enzyme phosphorylation is connected on the P Blue scripts KS carrier by EcoRI and EcoRV double digestion then.Subclone Sequenase (Sequenase) 2.0(USB) analyzes the double-stranded DNA order.The cDNA translation that contains mature polypeptide toxin gene upstream region shows that DK9.2 is former body protein form synthetic.Upstream cDNA sees SEQ ID NO:6 in proper order.On the gene of coding precursor protein a segment signal sequence and preceding polypeptide gene fragment (SEQ ID NO:7) are arranged, remove this section and just can produce the mature polypeptide toxin of getting from spider venom.Sequence is essential for spider generation and secretion DK9.2 in signal sequence and the preceding polypeptide by inference.

Embodiment 9

The structure of recombinant rhabdovirus

The signal sequence of one section lepidopterous insects (Jones et al., Molecular Cloning Regulation and Complete Sequence of a Hemocyanin-Related Juvenile Hormore-Supressible Proteim From Insect Hemolymqhs, J.Biol.Chem, 265:8596(1990)), use Rossi, et al(J.Biol.Chem, 257:9226(1982)) method is made up by two sections synthetic oligonucleotide and forms.Obtain the single-chain fragment that two sections 48 Nucleotide are formed via the ion exchange chromatography purifying, but they there are 11 complementary bases at the 3' end.When these two fragments are having when annealing in the presence of four kinds of deoxynucleoside triphosphates and the DNA polymerase I Klenow fragment, can synthesize a double-stranded product, this product is through the hydroxyapatite chromatography purifying, use Aat II enzymolysis again, the fragment of generation can be inserted into the upstream of cloning in the DK9.2cDNA of PKS-DK9C.Screening obtains being inserted with the subclone of signal sequence and surveys its DNA sequence.

Dna sequencing confirms to have the fusion product of this two cDNA order not change the reading frame.Downcut this complete synthetic gene, through the appropriate reconstruction rear clone to a kind of rhabdovirus transfer vector P BlueBac(Vialard, J., et al., J, Vinrology 64:3-50(1990)) the NheI point of contact in.The mensuration of subclone order has been proved the correct insertion of recombination.The mensuration of the DNA sequence of plasmid WR9 is confirmed should synthetic " Caterspider " gene (pre DK9.2) to be inserted into rhabdovirus transfer vector P BlueBac(Fig. 3) in.The screening process has been accelerated in the application of P BlueBac carrier because be inserted in the rhabdovirus genome recombination can with the beta-galactosidase gene coexpression, so whether growth the time can change by color and to discern on indicator medium.

Use the method that Summers and Smith set up, preceding 9.2 the recombinant rhabdovirus of encode can be with 1 μ g AcMNPV viral DNA and 2 μ g plasmid DNA mixing cotransfection Spodoptera frugiperda bacterial strain Sfq acquisition.After the transfection 4 days, the diluent of cell conditioned medium coated be connected to 5 * 10 ⁶On the 100mm flat board of Sf9 cell, on cover one deck and contain Bluo-gal substrate (Gibco BRL, Gaithersburg, sepharose MD).In 5 to 6 days, recon can be identified because of it is sky-blue.The reorganization plaque is chosen with Pasteur pipet micro sample adding appliance, with 1ml nutrient solution wash-out, this elutriant infects the Sf9 cell that grows in the T-25 flask again, collects on the small volume please in order to the preparation viral DNA from the bacterium training liquid that has infected six different plaques after three days.The Auele Specific Primer of using coat protein (Polyhedrin) the gene peripheral region of virus carries out pcr amplification, confirms to have in these 6 viral plaques 5 to contain the insertion fragment of suitable size and do not have any wild-type pollution.Then preparation becomes the recombinant virus stock solution of spot number to be used in the body and in vitro tests through titration.

Embodiment 10

The biologic activity of reorganization DK9.2

The reorganization sample 9.2(DK9.2 that separation obtains in the self-contained serum tissue nutrient solution) biologic activity detects carries out in instar TBW larva.Dosage calculates on the A280 of detected solution absorption value basis.Adopt the dosage of 16 μ g/g, recombinant products can cause clear and definite muscle spasm in injection in back 2 hours, have half larva (6 3 of merely hitting) benumb and serious contraction after 48 hours, and other 3 larvas still shows light to moderate muscle spasm.The dosage of 8 μ g/g also makes 3 paralysis in the larva, and the dosage of 5.2 μ g/g then only makes two paralysis.The result proves that reorganization DK9.2 has biological activity.

Embodiment 11

The body build-in test of recombinant rhabdovirus

The A.DK9.2 reorganization nuclear type polyhedron disease virus (biologic activity of vAcDK9.20

1) titration of viral prepared product

Virus the stock solution titration of plaque analytical method (Luria et al., " General Virol-ogy ", 1978, pp.21-32; John, Willey and Sons, New York).Titre represents that with the number (pfu) that per unit volume storage liquid can form spots such as biting dosage is then represented with the pfu/ larva.If each virion all can successfully infect a different host cell, pfa just be equivalent to a ripe virion (Luria et al., ibid).For example, 103 host cells can be the viral prepared product infection (i.e., 130PFU/ μ l) of 106PFU/ml by 1ml content.

2) bioanalysis experiment

DK9.2 reorganization nuclear type polyhedron disease virus (rNPV), the biologic viability of vAcDK9.2 can detect through a series of dose response analysis experiments.In this experiment, TBW larva (the about 250mg of average quality) is injected the tissue culture medium that contains various dose vAcDK9.2 or simple tissue culture medium (n=10, promptly 10).Toxin injection experiment shown in example 1, the larva of handling is incubated at respectively in the container that is placed with food, regularly observes.The synthesis result of these two virus infection analysis experiments sees Table II.Adopt 5000pfu/ larva and bigger dosage, the toxic classical symptom of DK9.2 (muscle trembles and spasm) children after injection began to occur in 24 hours.Half is tested insect and has lost motility (i.e. paralysis or half paralysis) at least in 48 hours.Minimum proof load 50pfu/ larva was induced in 48 hours and trembles and spasm, and at least 70% larva loses motility in 96 to 120 hours.

The biology motility of B.vAcDK9.2 and wild-type NPV relatively

Further vAcDK9.2 and its parent's wild-type virus Auto-grapha Carforica NPV(Wt-AcMNPV have been compared in research) effectiveness.These purposes of analyzing experiment are compared with the larva that is subjected to wait dosage Wt-AcM-NPV to infect by the larva of vAcDK9.2 infection in order to determine, whether lose motility in shorter time.

1) experiment is analyzed in injection

The biologic viability of vAcDK9.2 and wt-AcMNPV compares in a series of injection experiments.To the end tobaco budworm(Heliothis virescens in age) larva, Cabbage Looper(Tr ichoplusiani) larva and beet army worm(Spodopte exigua) vAcDK9.2 or the wt-AcMNPV of larva injection 5 * 10pfu/ larva; Control larvae is only injected tissue culture medium.The result shows that on all three species, vAcDK9.2 determines to have stronger effectiveness than wt-AcMNPV, the results are summarized in the table III.

2) feed the analysis experiment

But in order to detect the polygonal plain negative (pol of a kind of oral cavity infection of the essential preparation of the vigor of vAcDK9.2 after ingesting ^-) recon.The pol that builds up tool oral cavity infection power that this purpose can be delivered according to other investigators ^-Reorganization NPV ₈Method, reach through its retention of vAcDK9.2 and wt-AcMNPV.(as Kuroda et al., 1989, J.Virology 63(4); 1677-1685 and Price et al., 1989.Proc Natl Acad, Sci 86 1453-1456).The SF-9 cell is respectively 10 and 2 by the multiple infection coefficient (MOI) of vAcDK9.2 and wt-AcMNPV infection simultaneously.Simultaneously, another group SF-9 cell is only by wt-AcMNPV(MOI=2) infect.Infect after 5 days, difference is centrifugal behind the smudge cells, collects inclusion body (e.g., Wood, 1980, Vivology 104:392-399).Inclusion body is counted on hematimeter.The cell of those coinfections vAcDK9.2 and wt-AcM-NPV is than the cell that only infects wt-AcMNPV, the less while of inclusion body that produces is less, polygonal inclusion body (PIB) output that is obtained by the former is the 4.6PIB/ cell, and latter's inclusion body output is the 33.3PIB/ cell.

In order to compare the biologic viability of wt-AcMNPV and vAcDK9.2, used a kind of food to mix analytical procedure in the experiment.The PIB of blended or anti-wild-type is with 10 ⁵The concentration of PIB/gm is incorporated in a kind of insect food that does not contain agar, and this food is assigned in the small vessels, and the TBW larva that is given birth to is put into container (one in each container, totally 20).Control larvae gives equivalent undressed food.Allow the larva ad libitum access, regularly observe the development of its symptom.The results are shown in Table II.Begin no any effect in 48 hours, benumbed but feed with the larva that mixes PIB more than 50% after 72 hours; Wild-type virus does not cause any effect at this moment.After 96 hours, feed more than 90% to mix the PIB larva and benumbed, and the larva that feeds with wild-type PIB has only 10% death or dying.After 120 hours, last group of larva 100% is dead or dying, then one group of only 75% death or dying of larva.Therefore, analyze result of experiment as injection, analyze in the experiment in nursing, the larva that vAcDK9.2 handles is handled larva in the remarkable time devitalization power that shortens than wt-AcMNPV.

The dose response analysis experiment % paralysis percentage ratio of table II vAcDK9.2 in TBW

Dosage becomes spot number/larva	24 hours	48 hours	72 hours	96 hours	120 hours
						5.1×10 5	0	80	100	100	100
5.1×10 4	0	35	95	100	100
						5.1×10 3	0	25	75	90	100
5.1×10 2	0	0	55	85	100
						5.1×10 1	0	0	20	65	95
Contrast	0	0	0	0	0

Table III vAcDK9.2 and wt-AcMNPV injection TBW.CL and BAW larva effect are relatively

Processing mode	Kind		24 hours	48 hours	72 hours	96 hours	120 hours	168 hours
									vAcDK9.2	TBW 2	0	0	100	100	100	100
wt-AcMNPV	TBW	0 3	0	0	0	0	0 4
								Contrast	TBW	0	0	0	0	0	0
vAcDK9.2	CL 5	0	100	100	100	100	N/A
								wt-AcMNPV	CL	0	0	0	0	100	N/A
Contrast	CL	0	0	0	0	0	N/A
								vAcDK9.2	BAW 6	0	70	90	100	100	100
wt-AcMNPV	BAW	0	0	0	0	0	100
								Contrast	BAW	0	0	0	0	0	30

1. every larva injecting virus 5 * 10 ⁵Pfu; Control group is only injected the tissue culture medium (TCM) of equivalent volumes

2.n=8; The mean body weight of every larva is 300mg

3.8 the permanent disability that produces when only 2 in the larva are because of injection is dead, does not therefore do further analysis

4. the larval mortality of wt-AcMNPV processing in the 9th day is 100%

5.n=10; Every larva mean body weight is 165mg

6. to cAcDK9.2, n=20; To wt-AcMNPV, n=10 and contrast; Every larva mean body weight is 200mg

Embodiment 12

The promotor of DK9.2 gene

For obtaining the adjusting component of DK9.2 gene, made up a chromogene library.Adopt the scheme of Herrmarn and Frischanf (to see Methods in Enzymslogy, Vol, V52, Acaclemic Press, Ine 1987,99,180-183) preparation spider DNA is partially digested with Sau3A, use TLS-55 rotary head (Beckman Co. then, Ltd) in 10%-38% sucrose density gradient system with 25, centrifugal 16 hours of 000rpm rotating speed, fraction collection again.Merge the DNA collection unit of 35-45Kb, dna fragmentation is inserted in the XhoI site of powder carrier with partially filled method.Connect 1/4 in the product and use Gigak gold ^TM(Stratagene, LaJolla CA) are packaged in the phage particle, obtain being equivalent to surpassing 3 * 10 through transfection ⁹The spider DNA of bp.This gene pool is laid on 25 150mm flat boards, print again to nylon leaching film, screen the purpose bacterium colony with 4 kinds of probes such as the radiolabeled intermediate segment of end mark oligonucleotide of the end mark oligonucleotide of the radiolabeled cDNA of encoding D K9.2, its N-terminal of encoding, its C-terminal of encoding.Have clay and above all probes of a code cDNA all to hybridize, the DNA of its EcoRI endonuclease bamhi shift results of hybridization show one of them 3.0kb fragment can with intermediate fragments and C-terminal probe hybridization.Measure the gene that the double-stranded sequence of clay cDK2 confirms to be separated to DK9.2 with aforesaid three primers, and another or some genes of having pointed out this toxin family are positioned at the possibility on the same contiguous dna fragmentation, because this gene N-terminal oligonucleotide (it has high homology with the effective polypeptide of all Dignetia desinsections) inserts in a plurality of sites of cosmid DNA.

Be to obtain the promoter sequence of DK9.2 gene, obtain earlier one section with the sense strand consistent synthetic oligonucleotide of signal sequence of going forward.Pcr amplification product between the primer of this primer and other gene specific of the present invention is positioned signal sequence for show outside the N-terminal upstream greater than 3, the 000bp place.Primer on the antisense strand is used to measure the DNA sequence of the cDNA5' end upstream region that is right after the coded signal peptide.

This signal sequence shows to the DNA sequence of upstream, beyond the initial methionine password-and there is another intron at the 11bp place.Synthesized regional one section consistent Oligonucleolide primers, be used to cause a PCR reaction to determine the size of intervening sequence between transcription initiation site and translation initiation site with the cDNA5' of DK9.2 precursor.One 1, the amplified production of 000bp has confirmed the existence of this intron and has shown its size.Transcription initiation site upstream DNA sequence conclusive evidence wherein comprises a promotor, the dna sequence dna of this promoter region is seen SEQ Listing NO:8, and it comprises a plurality of essential adjustment signals that being positioned in other eukaryotic promoters is right after the transcription initiation site upstream region that are prevalent in.This promotor or wherein the part fragment be used in and transcribe or translate other eukaryotic genes in bacterium, virus, plant or the animal.

Example 13

Neurophysiology research about the DK9.2 mode of action

Neurophysiology research is to be easy to nerve (paroxysm) discharge repeatedly of being blocked by tetraodotoxin for perineural record that the mode of action of illustrating DK9.2, maggot neuromuscular engage shows that DK9.2 brings out, therefore, this detoxifying function site neu sodium-ion channel that may be voltage-sensitive.Further studies show that DK9.2 sustainable exciting peripheral nerve when reaching 10nM threshold values concentration.In addition, its effectiveness is 50 times of No. 4 Mammals toxin of scorpion at least.

Used laboratory animal is a housefly third phase instar in this research.Larva is fixed with the insect nail, separates section's cutter hara kiri with medical surgical, flattens the larva beam, and fixing with nail, removes internal organ and exposes body wall muscle tissue.Outside neural around and brain joins it is cut out, remove decerebrate, immerse then in the insect saline solution, composition is (mM): NaCl(140), KCl(5), CaCl ₂(0.75), MgCl ₂(4), NaHCO ₃(5) and HEPES(5), pH7.2.

One stimulation is accepted electrodes and is conducted in order to the record neuromuscular on arbitrary suitable nerve trunk.Slowly strengthen and irritate threshold values till a filament contraction observing muscle 6 or 7.Insert in this root fiber in born of the same parents then and write down microelectrode, this electrode connects in the born of the same parents that are used to detect the exciting post-synapse electromotive force (EPSP) of replying neural stimulus and gives amplifier.

For record peripheral nerve fiber rise electroactive, recording electrode is attached to an interchange and gives on the amplifier, signal is exaggerated 100 times, 0.3 and 1Hz under export, all digitizings in the instrument system of MacLab equipment computer of record are arranged, and show, analyze.

Used neuromuscular to link prepared product in the initial experiment of relevant DK9.2 effect.If giving the peripheral nerve single irritates and then causes body wall muscle once single EPSP to occur.When having DK9.2 to exist, the high efficiency discharge of EPSP is led in single stimulation.Except stimulating the discharge that causes, spontaneous burst firing also occurs and continue several minutes.One paroxysm process often continued more than 1 second, and EPSP frequency at this moment is greater than 30Hz, and the appearance of burst firing causes the body wall myocyte shrinks strongly, and this makes and keeps writing down in the born of the same parents very difficulty.Because the contraction that the moving electricity of paroxysm causes is discharged muscle with electrode.Therefore most experiments are to carry out in the salts solution that contains high concentration sucrose (300mM), and sucrose can suppress to shrink and not influence electroactive.The result who obtains with saline solution common or high concentration sucrose is similar.

Observed phenomenon was similar when observed burst firing and scorpion alpha toxin existed after DP9.2 handled, and known scorpion toxin α acts on the voltage-sensitive ionic channel of neuron membrane.The electricity this as seen, burst firing can be prevented by specificity sodium-ion channel co-inhibitor tetraodotoxin (TTX).The burst firing ability that TTX stops or reverse DK9.2 to bring out is shown among Fig. 5.In Fig. 5 A, EPSP is prevented rapidly by 1 μ M TTX, makes prepared product insensitive to the processing of subsequently 100nM DK9.2.Similarly experiment sees among Fig. 5 B, brings out burst firing by DK9.2 earlier in this experiment, and the TTX that is added subsequently then reverses.In this experiment, the generation of burst firing makes electrode be evicted from muscle, makes that introducing one when attempting to write down again thrusts the illusion record.In case find a suitable record site, then detect burst firing about 2 minutes, its activity stops in the several seconds after at this moment this prepared product is presented at TTX and handles.

For fear of the difficulty that causes intracellular recording because of Muscle contraction, the extracellular recording repeated experiments is carried out in above-mentioned experiment on the maggot peripheral nerve.The result is, the reverse activation of associated movement fiber, and the affective neuron activity rises to some extent.When the concentration of DK9.2 can cause the enhancing of neural discharge during at 70nM, and the influence that not handled by saline solution subsequently, but 0.4 μ M TTX can suppress this reaction rapidly.In addition, before DK9.2, add the appearance that TTX can stop burst firing.

The purpose of setting about further research is for the susceptibility of definite insect nerve to DK9.2, and compares its effectiveness with the standard scorpion toxin.DK9.2 tests its effect on the nervus peripheralis prepared product, begin to use 1nM, approximately increases concentration later in per 5 minutes until the rising of observing its vigor.In this prepared product, 1nM is adiaphorous to 5nM DK9.2, but when reaching 10nM, once bringing out vigor on this prepared product after of short duration time of lag.Five neural prepared products are in order to determine DK9.2 in the supraneural effective valve concentration of insect, and it the results are summarized in the table five.

The table IV

The concentration number of processes produces the shared ratio % of response insect

Response

1nM 2 0 0

2nM 1 0 0

5nM 3 1 33

10nM 3 3 100

Use similar methods, in the end determine scorpion Leiurus quingues-triatus toxin 4(LqT * 4 in one group of experiment) to the susceptibility of insect nerve.Prepared product (4) o'clock does not have effect being subjected to LqT * 4 that concentration reaches 500nM.These find to equate with former result of study, promptly find LqT * 4th, specific action Mammals sodium channel.When these prepared products were handled at follow-up DK9.2, DK9.2 concentration need reach 10-30nM and just produce reaction.Rising slightly of effective threshold concentration of DK9.2 may be due to the weak restraining effect of CqT * 4 of inactivation.These experimental results show that DK9.2 is stronger at least 50 times than LqT * 4 to the effect of insect nerve.

The table V

Sequence number	Content
			1	DK 9.2 amino-acid sequences
2	DK 9.2cDNA encoding sequence
		3	DK 11 aminoacid sequences
4	DK 11cDNA encoding sequence
		5	DK 12 aminoacid sequences
6	The global cDNA sequence of encoding D K 9.2
		7	The aminoacid sequence of the signal/homing sequence of DK 9.2 precursors
8	The dna sequence dna of DK 9.2 promotors

Sequence is single

(1) generalized case:

(ⅰ) applicant: Karen J.Krapcho; Bradford Carr

Van Wagenen;J.R.Hunter Jackson

(ⅱ) invention title: effective insecticidal peptide

(ⅲ) sequence number: 8

(ⅳ) address:

（A）ADDRESSEE：Woodcock，Washburn，Kurtz，Mack-iewicz&Norris

（B）STREET：One Liberty Place-46th Floor

（C）CITY：Philadelphia

（D）STATE：Pennsylvania

（E）COUNTRY：U.S.A.

（F）ZIP：19103

(ⅴ) form of computers:

(A) media model: DISKETTE, 3.5INCH, 1.44Mb STORAGE

(B) computer: IBM PS/2

(C) operating system: PC-DOS

(D) software: WORDPERFECT 5.0

(ⅵ) present application materials:

(A) application number:

(B) enrollment time:

(C) classification:

Application materials (ⅶ):

(A) application number: 662,373

(B) enrollment time: March 1,1991

(ⅷ) lawyer/proxy's situation:

(A) name: John W.Caldwell, Esp.

(B) registration number: 28,937

(C) work code name: FMC-0054

(ⅸ) communication situation:

(A) phone: (215) 568-3100

(B) fax: (215) 568-3439

(2) SEQ ID NO:1 situation:

(ⅰ) sequence signature:

(A) length: 56 amino acid

(B) classification: amino acid

(D) sterie configuration: the unknown

(ⅹ ⅰ) sequence content: SEQ ID NO:1:

Ala?Lys?Asp?Gly?AsP?Val?Glu?Gly?Pro?Ala?Gly?Cys?Lys?Lys?Tyr

5 10 15

Asp?Val?Glu?Cys?Asp?Ser?Gly?Glu?Cys?Cys?Xaa?Lys?Gin?Tyr?Leu

20 25 30

Trp?Tyr?Lys?Trp?Arg?Pro?Leu?Asp?Cys?Arg?Cys?Leu?Lys?Ser?Gly

35 40 45

Phe?Phe?Ser?Ser?Lys?Cys?Val?Cys?Arg?Asp?Val

55 55

(2) SEQ ID NO:2 situation:

(ⅰ) sequence signature:

(A) length: 275 base pairs

(B) classification: nucleic acid

(C) chain: two strands

(D) sterie configuration: the unknown

(ⅹ ⅰ) sequence content: SEQ ID NO:2

GCC AAG GAC GGC GAC GTC GAG GGG CCT GCG 30

Ala Lys Asp Gly Asp Val Glu Gly Pro Ala

1 5 10

GGC TGC AAG AAA TAC GAC GTA GAG TGC GAC 60

Gly Cys Lys Lys Tyr Asp Val Glu Cys Asp

15 20

AGT GGA GAG TGC TGC MMSAAG CAG TAC CTG 90

Ser Gly Glu Cys Cys Xaa Lys Glu Tyr Leu

25 30

TGG TAC AAG TGG CGA CCC CTG GAT TGC CGA 120

Trp Tyr Lys Trp Arg Pro Leu Asp Cys Arg

35 40TGC CTA AAG AGC GGT TTC TTC AGC AGC AAG 150

Cys Leu Lys Ser Gly Phe Phe Ser Ser Lys

45 50

TGC GTT TGC AGA GAC GTG TAGATTTGAA ATGAAATTCG 188

Cys Val Cys Arg Asp Val

55

TGTTCTTTTT TGGTTGTAGA TGACCTAATG AAACAACTGA 228

CATGAATAAA ACAAAATTGA ATGAATTGAA AAAAAAAAAA 268

AAAAAGC 275

(2) SEQ ID NO:3 situation:

(ⅰ) sequence signature:

(A) length: 58 amino acid

(B) classification: amino acid

(D) sterie configuration: the unknown

(ⅹ ⅰ) sequence content: SEQ ID NO:3:

Ala?Lys?Asp?Gly?Asp?Val?Lys?Gly?Pro?Ala?Gly?Cys?Met?Lys?Tyr

5 10 15

Lys?Ser?Gly?Asp?Cys?Arg?Gly?Lys?Thr?Cys?Cys?Asp?Gln?Gln?Tyr

20 25 30

Leu?Trp?Tyr?Lys?Trp?Arg?Asn?Leu?Ala?Cys?Arg?Cys?Phe?Thr?Val

35 40 45

Glu?Val?Phe?Lys?Lys?Asp?Cys?Trp?Cys?Asn?Asp?Ile?Ser

50 55

(2) SEQ ID NO:4 situation:

(ⅰ) sequence signature:

(A) length: 204 base pairs

(B) classification: nucleic acid

(C) chain: two strands

(D) sterie configuration: the unknown

(ⅹ ⅰ) sequence content: SEQ ID NO:4

(2) SEQ ID NO:5 situation:

(ⅰ) sequence signature:

(A) length: 62 amino acid

(B) classification: amino acid

(C) chain:

(D) sterie configuration: the unknown

(ⅹ ⅰ) sequence content: SEQ ID NO:5:

Ala?Lys?Asp?Gly?Asp?Phe?Glu?Gly?Pro?Pro?Gly?Xaa?Leu?Lys?Met

5 10 15

Gly?Glu?Leu?Xaa?Lys?Gly?Gly?Thr?Xaa?Xaa?Thr?Lys?Val?Tyr?Lys

20 25 30

Tyr?Trp?Lys?Trp?Arg?Lys?Leu?Glu?Cys?Leu?Gly?Lys?Asn?Asp?Gly

35 40 45

Trp?Phe?Lys?Lys?Lys?Phe?Ile?Cys?Asp?Glu?Arg?Xaa?Asn?Pro?Xaa

50 55 60

Xaa?Xaa

(2) SEQ ID NO:6 situation:

(ⅰ) sequence signature:

(A) length: 359 base pairs

(B) classification: nucleic acid

(C) chain: two strands

(D) sterie configuration: the unknown

(ⅹ ⅰ) sequence content: SEQ ID NO:6

(2) SEQ ID NO:7 situation:

(ⅰ) sequence signature:

(A) length: 38 amino acid

(B) classification: amino acid

(C) chain:

(D) sterie configuration: the unknown

(ⅹ ⅰ) sequence content: SEQ ID NO:7:

Met?Lys?Val?Phe?Val?Val?Leu?Leu?Cys?Leu?Ser?Leu?Ala?Ala

-35 -30 -25

Val?Tyr?Ala?Leu?Glu?Glu?Arg?Leu?Asp?Lys?Asp?Ala?Asp?Ile

-20 -15

Met?Leu?Asp?Ser?Pro?Ala?Asp?Met?Glu?Arg

-10 -5 -1

(2) SEQ ID NO:8 situation:

(ⅰ) sequence signature:

(A) length: 421 base pairs

(B) classification: nucleic acid

(C) chain: two strands

(D) sterie configuration: the unknown

(ⅹ ⅰ) sequence content: SEQ ID NO:8

Claims (69)

CLAIMS 1. A polypeptide having effective insecticidal activity isolated in large quantities from the venom of Diguetia spiders or an agriculturally or horticulturally acceptable salt thereof. 2. Polypeptide according to claim 1, characterized in that the spider venom is isolated from Diguetia canities. 3. The effective insecticidal polypeptide according to claim 1, characterized in that, the molecular weight determined by mass spectrometry is about 6371-6397D, and it moves as a single peak in reverse high performance liquid chromatography, and thus can be used in agriculture or horticulture. Accepted salts. 4. The effective insecticidal polypeptide according to claim 1, which has a sequence substantially identical to SEQ ID NO:1. 5. The effective insecticidal polypeptide according to claim 1, characterized in that, the molecular weight determined by mass spectrometry is about 6740D; it moves as a single peak in reversed high performance liquid chromatography, and is therefore agriculturally or horticulturally acceptable salt kind. 6. The effective insecticidal polypeptide according to claim 1, which has an amino acid sequence substantially identical to SEQ ID NO:3. 7. The effective insecticidal polypeptide according to claim 1, characterized in that the molecular weight determined by mass spectrometry is about 7080D; it moves as a single peak in reversed high performance liquid chromatography, and is therefore agriculturally or horticulturally acceptable salt kind. 8. The effective insecticidal polypeptide according to claim 1, which has an amino acid sequence substantially identical to SEQ ID NO:3. 9. A useful isolated DNA sequence, characterized in that the DNA sequence encodes a potent insecticidal polypeptide which can be isolated in large quantities from the venom of Diguetia spiders. 10. The DNA of claim 9, wherein the spider venom is from Diguetia canities. 11. The DNA sequence with application value as claimed in claim 9, characterized in that its sequence is shown in SEQ ID NO:6. 12. The DNA sequence with application value as claimed in claim 9, characterized in that its sequence is shown in SEQ ID NO:4. 13. The DNA sequence with application value as claimed in claim 9, characterized in that its DNA sequence has been shown in SEQ ID NO:2. 14. A recombinant expression vector, characterized in that it has a DNA sequence that can encode a large number of effective insecticidal polypeptides isolated from the venom of Diguetia spiders, and is also characterized in that the vector can realize said expression in transformed cells. Representation of coding sequence. 15. The recombinant expression vector according to claim 14, wherein the spider venom is from Diguetia canities. 16. The recombinant expression vector according to claim 14, wherein the DNA sequence encodes an effective insecticidal polypeptide; the polypeptide is characterized in that its molecular weight is 6371-6397D as determined by mass spectrometry analysis, and it is 6371-6397D in reverse high performance liquid chromatography. Movement with a single peak. 17. The recombinant expression vector according to claim 14, wherein the DNA sequence actually encodes the polypeptide represented by SEQ ID NO:1. 18. The recombinant expression vector according to claim 14, wherein the DNA sequence encodes an effective killing polypeptide; the polypeptide is characterized in that its molecular weight is determined to be 6740D by mass spectrometry analysis, and it moves as a single peak in reverse high performance liquid chromatography. . 19. The recombinant expression vector according to claim 14, characterized in that the DNA sequence substantially encodes the polypeptide shown in SEQ ID NO:3. 20. The recombinant expression vector according to claim 14, wherein the DNA sequence encodes an effective insecticidal polypeptide; the polypeptide is characterized in that mass spectrometry analysis shows that its molecular weight is 7080D, and it can be detected as a single The peak moves. 21. The recombinant expression vector according to claim 14, characterized in that the DNA sequence substantially encodes the polypeptide shown in SEQ ID NO:5. 22. The recombinant expression vector according to claim 14, characterized in that the DNA sequence referred to has actually been clearly shown in SEQ ID NO:2. 23. The recombinant expression vector according to claim 14, wherein the DNA sequence referred to is actually shown in SEQ ID NO:4. 24. The recombinant expression vector of claim 14, wherein the DNA sequence referred to is actually shown in SEQ ID NO:6. 25. The recombinant expression vector of claim 14, wherein the transformed cells are plant cells susceptible to insect infestation. 26. A transgenic plant, characterized in that a DNA sequence encoding a high-efficiency insecticidal polypeptide that can be isolated in large quantities from the venom of Diguetia spiders has been transferred into the germplasm cells of the indicated plant or the ancestors of the indicated plant, so that the indicated DNA The traits expressed by the sequences can be passed on to the next generation by the plant through sexual or asexual transmission. 27. A recombinant baculovirus expression vector, capable of expressing a DNA sequence in a host or an insect host, the DNA sequence encodes a high-efficiency insecticidal polypeptide that can be isolated in large quantities from the venom of Diguetia spiders. 28. The recombinant baculovirus expression vector according to claim 27, characterized in that, the expression vector referred to is the baculovirus genome, and the DNA sequence referred to is inserted in the polyhedrin gene and is regulated by the transcription of the baculovirus polyhedrin promoter . 29. A recombinant host cell transformed or transfected with a DNA sequence, characterized in that the DNA sequence encoding a large amount of isolatable and effective insecticidal polypeptide from the venom of Diguetia spider exists in a manner to express the indicated polypeptide in the host cell. 30. The recombinant host cell of claim 29, wherein the spider venom is obtained from Diguetia canities. 31. The recombinant host cell according to claim 29, characterized in that the DNA sequence codes for a high-efficiency insecticidal polypeptide; the characteristic of the polypeptide is that mass spectrometry analysis shows that its molecular weight is 6371-6397D, and in reverse high performance liquid phase Moves as a single peak during chromatography. 32. The recombinant host cell of claim 29, wherein said DNA sequence substantially encodes the polypeptide shown in SEQ ID NO:1. 33. The recombinant host cell as claimed in claim 29, characterized in that, the said DNA sequence encodes an effective insecticidal polypeptide; the characteristic of the polypeptide is that mass spectrometry analysis shows that its molecular weight is 6740D, and it is 6740D in reverse high performance liquid chromatography. moves as a single peak. 34. The recombinant host cell of claim 29, wherein said DNA sequence substantially encodes the polypeptide shown in SEQ ID NO:3. 35. The recombinant host cell according to claim 29, characterized in that the DNA sequence codes for an effective insecticidal polypeptide; the characteristic of the polypeptide is that mass spectrometry analysis shows that its molecular weight is 7080D, and it is 7080D in reverse high performance liquid chromatography. moves as a single peak. 36. The recombinant host cell of claim 29, wherein said DNA sequence substantially encodes the polypeptide shown in SEQ ID NO:5. 37. The recombinant host cell of claim 29, wherein said DNA sequence is substantially as set forth in SEQ ID NO:2. 38. The recombinant host cell of claim 29, wherein said DNA sequence is substantially as set forth in SEQ ID NO:4. 39. The recombinant host cell of claim 29, wherein said DNA sequence is substantially as set forth in SEQ ID NO:6. 40. A method for producing high-efficiency insecticidal polypeptide, characterized in that, (a) Cultivating recombinant host cells, characterized in that the recombinant expression vector of the host cell transformed or transfected contains a DNA sequence encoding an effective insecticidal polypeptide that can be isolated in large quantities from the venom of Diguetia spiders; the expression vector is characterized in that, the vector enables expression of the indicated coding sequence in transformed cells, and (b) recovering the indicated effective insecticidal polypeptide from the culture medium of the recombinant host cell. 41. The method of claim 40 resulting in recombinantly produced polypeptides. 42. The method according to claim 40, wherein the DNA sequence codes for an effective insecticidal polypeptide, and the characteristic of this polypeptide is that its molecular weight is 6371-97D as shown by mass spectrometry analysis, and when reversed high performance liquid chromatography Moves as a single peak. 43. The method of claim 40, wherein said DNA sequence actually encodes the polypeptide shown in SEQ ID NO:1. 44. The method according to claim 40, characterized in that the DNA sequence codes for an effective insecticidal polypeptide, and the characteristic of this polypeptide is that its molecular weight is 6740D as shown by mass spectrometry analysis, and it appears as a single The peak moves. 45. The method of claim 40, wherein said DNA sequence substantially encodes the polypeptide shown in SEQ ID NO:3. 46. The method according to claim 40, characterized in that the DNA sequence referred to encodes an effective insecticidal polypeptide, which is characterized by mass spectrometry analysis showing that its molecular weight is 7080D, and a single peak in reversed high performance liquid chromatography move. 47. The method of claim 40, wherein said DNA sequence substantially encodes the polypeptide shown in SEQ ID NO:5. 48. The method of claim 40, wherein the DNA sequence referred to is actually shown in SEQ ID NO:2. 49. The method of claim 40, wherein the DNA sequence referred to is actually that set forth in SEQ ID NO:4. 50. The method of claim 40, wherein the DNA sequence referred to is actually set forth in SEQ ID NO:6. 51. A method of controlling invertebrate pests, characterized by contacting the pests with an effective amount of a polypeptide isolatable from Diguetia spider venom, or an agriculturally or horticulturally acceptable salt . 52. The method of claim 51 wherein the spider venom is obtained from Diguetia canities. 53. The method of claim 51 wherein the pests are insects. 54. The method of claim 51 wherein the pest is of the order Lepidoptera. 55. A method of controlling invertebrate pests characterized by contacting said pests with a recombinant baculovirus expressing an effective amount of a polypeptide isolatable from the venom of the Diguetia spider. 56. A pesticidal mixture characterized by a pesticidally effective amount of the polypeptide according to claim 55 bound to an agriculturally or horticulturally acceptable carrier. 57. An antibody that strongly immunoreacts with a polypeptide isolated from the venom of a Diguetia spider. 58. The antibody of claim 57, wherein the spider venom is obtained from Diguetia canities. 59. A detection method, using the antibody of claim 57 to detect the presence of an effective insecticidal polypeptide that can be isolated in large quantities from the venom of Diguetia spiders, characterized in that, (a) Obtaining spider venom (b) contacting the spider venom with the indicated detectably labeled antibody, and (c) A labeled antibody to detect the polypeptide to which the linkage refers. 60. The method of claim 59 wherein the spider venom is obtained from Diguetia canities. 61. A DNA probe derived from a DNA sequence encoding a potent insecticidal polypeptide isolated in large quantities from the venom of Diguetia spiders. 62. A method of detection for the presence of DNA encoding a potent insecticidal polypeptide which is largely isolatable from the venom of Diguetia spiders, characterized by: (a) Obtaining spider nucleic acid (b) contacting said venom with the DNA probe of claim 61, and (c) detecting a referent probe that hybridizes to a referent nucleic acid. 63. A purification method, using the antibody in claim 57 to purify the effective insecticidal polypeptide that can be isolated in large quantities from the venom of Diguetia canities, characterized in that, (a) Attaching the antibody to a solid support (b) contacting a solution containing the indicated polypeptide with the indicated antibody attached to the indicated solid support so that the indicated polypeptide present in the solution binds to the indicated antibody. (c) eluting the indicated polypeptide bound to the indicated antibody attached to the indicated solid support. (d) Collecting the previously washed polypeptides. 64. A sequence in front of the precursor that is homologous to the amino acid sequence encoded by SEQ ID NO: 7 or a part thereof, and guides the folding and secretion of the mature protein. 65. A genetic construct having homology to the amino acid sequence encoded by SEQ ID NO: 7 can be operatively linked to a peptide. 66. A promoter sequence homologous to SEQ ID NO: 8 or a portion thereof capable of directing gene expression in a recombinant construct. 67. A recombinant construct having homology to the sequence of SEQ ID NO: 8 or a portion thereof, operatively linked to the expressed gene. 68. The insecticidal polypeptide referred to here includes: (a) is about 55 to 65 amino acids in size (b) More than 40% homology with SEQ ID NO: 1, 3, 5 (c) has about 7 to 8 photocystine residues. 69. The source of polypeptide referred to in claim 68 is Diguetia spider venom.

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