JPH09100299A - Insecticidal protein derived from larva of insect belonging to family myrmeleontidae - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new insecticidal protein having a specific amino terminal amino acid sequence, exhibiting extremely high insecticidal activity and useful as an active ingredient, etc., of insecticide from a poisonous liquid collected from large jaw of larva of insect belonging to the family Myrmeleontidae. SOLUTION: This new insecticidal protein is obtained from a poisonous liquid collected from large jaw of larva of insect belonging to the family Mymeleontidae and has an amino acid sequence in which amino terminal amino acid sequence is represented by the formula and exhibits extremely high insecticidal activity and is useful as an active ingredient of insecticide against insect pests. The protein is obtained by collecting larva of Myrmeleon bore, cutting the top of the large jaw with scissors, sucking a poisonous liquid by glass capillary from the cut part, cooling the collected poisonous liquid with ice, diluting the poisonous liquid with 25mM potassium phosphate buffer solution (pH7.5), centrifuging the solution to collect the supernatant and passing the supernatant through an anionic exchange column, eluting the absorbed ingredient to concentrate the supernatant, passing the concentrate through a hydrophobic column and eluting the absorbed ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an insecticidal protein derived from the larvae of Insects of the family Ephemeridae.

[0002]

BACKGROUND OF THE INVENTION Several insects utilize toxins for self-defense and predation. For example, melittin, apamin, kinin, etc. are known as insect-derived peptide toxins (Ann.Rev.Entomol., 27,33).
9-68,1982).

[0003]

Such insect-derived peptide-type toxins have the advantages that they have a high insecticidal activity against pests, are easily decomposed in the environment, and are less likely to remain. Therefore, if a peptide-based toxin derived from an insect having extremely high insecticidal activity is found, it will lead to the development of a new type of insecticide, which is highly expected in the future.

[0004]

Under the circumstances, as a result of intensive investigations by the present inventors, as a result, the amino-terminal amino acid sequence was obtained from a venom collected from the larva of the larva of the larva of the genus Ephemeridae. The protein having the amino acid sequence shown in No. 1 was successfully isolated and purified, and it was found that the protein has extremely strong paralytic insecticidal activity against pests, and the present invention was completed. That is, the present invention is a protein characterized by being obtained from a venom collected from the larva of the larva of the larva of the mosquito bug, and the amino terminal amino acid sequence being the amino acid sequence shown in SEQ ID NO: 1 (hereinafter referred to as the present invention. It is referred to as protein).

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail.

The protein of the present invention is characterized in that it is obtained from a venom collected from the larva of the larva of the larva of the mosquito insect, and the amino terminal amino acid sequence is the amino acid sequence shown in SEQ ID NO: 1.

The protein of the present invention can be obtained, for example, by the following method. (1) A venom is collected by cutting the tip of the large jaw of the larvae of the larvae of the insects of the family Ephemeridae, such as Crocous vulgare (Myrmeleon bore), with scissors and the like, and sucking with a glass capillary or the like from the cut portion. The collected venom is subjected to ammonium sulfate fractionation. In addition, as another method, larvae of the mosquitoes insects are added to, for example, water or a buffer solution and ground using a juicer mixer, a Waring blender, or the like,
Obtain a ground product. If necessary, after freezing by a method such as immersion in liquid nitrogen, the frozen tissue is ground at a low temperature such as in liquid nitrogen, and then suspended in water or a buffer solution to obtain a ground product. it can. The ground product is extracted by removing the residue by a method such as centrifugation. If necessary, the extract is fractionated with ammonium sulfate. (2) The fraction is, for example, DEAE-Sepharose (Se
It is purified by anion exchange resin column chromatography using pharose) or the like. The target substance is eluted by increasing the ionic strength of the eluate stepwise or continuously. For example, addition of sodium chloride or the like can be mentioned. (3) The elution fraction of the target substance is collected, fractionated with ammonium sulfate, and then purified by hydrophobic chromatography. The target substance is eluted by gradually or continuously decreasing the salt concentration of the eluate. For example, ammonium sulfate or the like can be used as the salt. (4) If necessary, the elution fraction of the target substance is collected, dialyzed, and further purified by molecular sieve chromatography such as gel filtration. Chromatography
In addition to the open column, a method of applying pressure such as FPLC and HPLC can be used. The target substance may be eluted using the activity of inducing paralysis in cockroaches as an index. The various operations from (1) to (4) above are performed, for example, in the Newborn Chemistry Experiment Course Protein I This is a method used in the purification of a normal protein described in Tokyo Kagaku Dojin (1990), edited by Japan Biochemical Society.
Further, if necessary, purification can be performed by combining the various operations described above or other methods used in ordinary protein purification. The amount of protein can be determined, for example, from Journal of Biological Chemistry (J. Biol. Chem.), Vol. 193, p. 265 (19).
51) It can be measured by the method described by Lowry et al.

The protein of the present invention has the following physicochemical properties and column properties. (1) Physicochemical properties (a) It is a protein having a molecular weight of about 165-1670,000. Molecular weight is measured by sodium dodecyl sulfate (SDS)
By -8% polyacrylamide gel electrophoresis (PAGE) (Staining with Coomassie Brilliant Blue R-250)
And the gel filtration method by Superdex 200HR. For example, in the former, 1.5 μg of protein
Based on the method of Aemmli (Nature, 227, 680-685, 1970) (see FIG. 1), the latter is a gel filtration carrier equilibrated with 25 mM potassium phosphate buffer (pH 6.0) containing 0.2 M potassium chloride. Superdex 200HR
(HR10 / 30, Pharmacia Biotech Co., Ltd.) packed column (diameter 10 mm × 300 mm) at an elution rate of 0.25 ml / min. As a molecular weight standard protein, ferritin (440,000), aldolase (158,000), lactate dehydrogenase (porcine heart, 142,000), albumin (bovine serum, 67,
000), albumin (egg white, 43,000), carbonic acid dehydratase (bovine erythrocyte, 29,000) and the like.
The molecular weight is calculated by comparison with these standard substances. (B) Precipitation in a 60% saturated ammonium sulfate fraction. Ammonium sulfate fractionation is performed as follows. 1 containing the protein of the present invention
3.9 g of powdered ammonium sulfate is gradually added to 10 ml of 25 mM potassium phosphate buffer (pH 6.0) containing 0% glycerol, and the mixture is allowed to stand at 4 ° C. for 30 minutes. Centrifuge (15,0
The precipitate is recovered by 100 g of 10 g), and the precipitate is suspended in 25 mM potassium phosphate buffer (pH 6.0) containing a small amount of 10% glycerol. The suspension was put into a dialysis tube (manufactured by Wako Pure Chemical Industries, Ltd.), and 25 mM potassium phosphate buffer solution (pH) containing 2 l of 10% glycerol was added.
Dialyze for 24 hours in 4) at 4 ° C. in 6.0). (2) Column characteristics In a 25 mM potassium phosphate buffer solution (pH 7.5), it is substantially adsorbed on an anion exchange resin column to give 250 mM.
Elute under the ionic strength of the potassium phosphate buffer solution (pH 7.5). For example, DEAE-Sephacel, an anion exchange resin equilibrated with 25 mM potassium phosphate buffer (pH 7.5) containing 10% glycerol.
(Sephacel) (manufactured by Pharmacia Biotech) was adsorbed using a column (diameter: 1.2 cm × 7 cm), and then stepwise with a 250 mM potassium phosphate buffer solution (pH 7.5) containing 10% glycerol. Elute the adsorbate (see Figure 2).

When the protein of the present invention is used as an active ingredient of an insecticide, it may be used as it is without adding any other component. For example, clays (kaolin clay, diatomaceous earth, synthetic hydrous silicon oxide, bentonite, fubasami clay, Acid clay etc.), talc, ceramics, other inorganic minerals (serisite, quartz, sulfur, activated carbon, calcium carbonate, hydrated silica, etc.), chemical fertilizers (ammonium sulfate, ammonium phosphorus, ammonium nitrate,
(Urea, ammonium chloride, etc.), solid carriers such as fine powder or granules, liquid carriers such as water, vegetable oils such as soybean oil and cottonseed oil, alkyl sulfates, alkyl sulfonates, alkylaryl sulfonates, alkylaryls Mixtures of ethers and their polyoxyethylene compounds, polyethylene glycol ethers, polyhydric alcohol esters, sugar alcohol derivatives, and other surfactants and other formulation auxiliaries, emulsions, wettable powders, water suspensions, and water It is also possible to formulate and use a flowable agent such as an emulsifying agent, a granule, a powder agent, a liquid agent, an ULV agent and the like.

In these preparations, the protein of the present invention as an active ingredient is usually contained in an amount of 0.1% to 99% by weight, preferably 1 to 90%.

Examples of auxiliary agents for formulation such as sticking agents and dispersants are casein, gelatin, polysaccharides (starch powder, gum arabic, cellulose derivatives, alginic acid, etc.), lignin derivatives, bentonite, sugars, synthetic water-soluble polymers. (Polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acids, etc.) and the like, and examples of the stabilizer include PAP (isopropyl acid phosphate) and BH.
T (2,6-di-tert-butyl-4-methylphenol), a mixture of BHT (2-tert-butyl-4-methoxyphenol) and 3-tert-butyl-4-methoxyphenol, vegetable oil, mineral oil , Surfactants, fatty acids or esters thereof, and the like.

The thus obtained preparation is used as it is or after diluted with water or the like.

When the protein of the present invention is used as an active ingredient of an insecticide, the application amount of the active ingredient may be appropriately set depending on the target pest, the formulation form, the application method, the application time and the like.

The invention is further described in the following examples,
The present invention is not limited to this.

[0015]

【Example】

Next, formulation examples are shown. In addition, a part represents a weight part.

Formulation Example 1 Aqueous solvent 20 parts each of the protein of the present invention, crude product (I), crude product (II) or crude product (III), 3 parts of polyoxyethylene sorbitan monooleate, 3 parts of CMC and Water 7
4 parts are mixed to obtain each water solvent.

Formulation Example 2 Liquid formulation 0.1 part each of the protein of the present invention, crude product (I), crude product (II) or crude product (III) is mixed with 99.9 parts of water,
Obtain each solution.

Example 1 (Purification of toxins of larvae of Crocous vulgare) 700 second- and final-stage larvae of Myrmeleon bore were collected, and the tip of the larvae was cut with scissors. The venom was collected by blotting from the cut with a glass capillary. 3.17 ml of the collected venom solution was ice-cooled, and 2.46 times (v / v) was added with 25 mM potassium phosphate buffer (pH 7.5) containing 1 mM dithiothreitol (DTT) and 10% glycerol.
After diluting with v), centrifugation (6,700 × g, 10 minutes, 4 ° C.) was performed and the supernatant was separated (referred to as crude purified product (I)). A 25 mM potassium phosphate buffer solution (pH containing 1 mM DTT and 10% glycerol)
It was applied to a DEAE Sephacel column (diameter 1.2 cm, length 7 cm, Pharmacia Biotech) equilibrated with 7.5). After washing the column with the buffer used for equilibration, 2 mM containing 1 mM DTT and 10% glycerol
The adsorbed components were eluted by flowing a 50 mM potassium phosphate buffer solution (pH 7.5). Flow rate is 0.73 ml /
At min, the eluate was fractionated by 5.5 ml. The elution pattern is shown in FIG. Absorbance of elution fraction (280 nm)
Was monitored, and the peak fractions (fraction No. 20-26 in FIG. 2) eluted with 250 mM potassium phosphate buffer were collected (referred to as crude product (II)). This fraction was cut to a molecular weight of 10
It is concentrated by ultrafiltration under a nitrogen gas pressure using a 10,000 ultrafiltration membrane (Nippon Millipore) and further replaced with a 25 mM potassium phosphate buffer (pH 6.0) containing 10% glycerol. , And the volume was adjusted to 5.9 ml (referred to as crude product (III)). Next, 2.3 g of ammonium sulfate was added to completely dissolve it, and the mixture was allowed to stand at 4 ° C. for 30 minutes and then centrifuged (15,000 × g, 10 minutes). The supernatant was discarded, the precipitate was suspended in 25 mM potassium phosphate buffer (pH 6.0) containing 1.7 M ammonium sulfate and 10% glycerol, and the suspension was centrifuged (15,000 xg, 10 minutes). The supernatant was discarded and the precipitate was redissolved in 100 μl of 25 mM potassium phosphate buffer (pH 6.0) containing 10% glycerol. A 25 mM potassium phosphate buffer solution (pH 6.) containing 10% glycerol and saturated with ammonium sulfate was added to this solution.
0) 40 μl was added and immediately applied to a phenyl superose column (HR5 / 5, Pharmacia Biotech) equilibrated with 25 mM potassium phosphate buffer (pH 6.0) containing 1.7 M ammonium sulfate and 10% glycerol. The ammonium sulfate concentration of the buffer solution is 1.
After washing the column by applying a gradient from 7 M to 0 M, a 25 mM potassium phosphate buffer solution (pH 6.0) containing 10% glycerol was applied to the column. The flow rate was 0.3 ml / min. The elution pattern is shown in FIG. Fractions eluted at 27.0-28.1 minutes (Fig. 3) were collected (hereinafter referred to as purified product). All the above column chromatography was performed at 4 ° C.

Example 2 (Analysis of toxin) The amount of protein in the purified product obtained in Example 1 was Bradford.
(Anal. Biochem., 72, 248-254, 1976).
The purified product containing 1.5 μg of protein was prepared according to the method of Laemmli (Na
ture, 227, 680-685, 1970) and analyzed by sodium dodecyl sulfate (SDS) -8% polyacrylamide gel electrophoresis (PAGE). The dyeing method was Kumagee Brilliant Blue R-250. The migration pattern is shown in FIG. The purified product was detected as a single band on the gel at a position of 167 kDa. Further, the purified product was applied to a Superdex 200HR column (HR10 / 30, Pharmacia Biotech Co., Ltd.) and eluted with a 25 mM potassium phosphate buffer solution (pH 6.0) containing 0.2M potassium chloride to perform gel filtration chromatography. , Was eluted at a position corresponding to a molecular weight of 165 kDa. Therefore,
This protein was presumed to be a monomer. Furthermore, when the purified product was subjected to an amino acid sequencer and the amino terminal amino acid sequence was analyzed, Ser-Tyr-Glu-Asn was found.
-The amino acid sequence of Asp (SEQ ID NO: 1) was confirmed.

Test Example (Bioassay) Adult male German cockroaches at room temperature 27 ± 2 ° C. and humidity 70 ±
The animals were bred under the condition of 5% and used for the assay of paralytic insecticidal activity. The test solution of the purified product was 25 mM potassium phosphate buffer (pH: 1 mM DTT and 10% glycerol).
Prepared in 6.0). As a control, Juro Spider Poison 3
An aqueous solution of (JSTX-3, Wako Pure Chemical Industries) and tetrodotoxin (TTX, Wako Pure Chemical Industries) was tested. The test solution was injected between the third and fourth abdominal sections of the German cockroach, placed in a transparent cup and observed in a breeding room. When the cockroach was on its back and could not return to its normal posture, it was determined to be paralyzed. At least 30 cockroaches per sample were tested using 3 cockroaches per concentration and varying the concentration of the test solution. The minimum amount of protein required to paralyze all three cockroaches used at one concentration within 10 minutes after injection (n
g protein / insect) was taken as the MPD value. The test was performed at least 3 times and the average was calculated. No paralytic activity was observed in the solution containing no toxin. The protein of the present invention has an MPD value of 40 and, as shown in Table 1, has a molar ratio of 3670 times that of JSTX-3 and 1 of TTX.
It showed 30 times higher activity.

[0022]

[Table 1] ────────────────────────────────── Test sample Paralytic insecticidal activity (mol / INSECT ) ────────────────────────────────── Protein of the present invention 2.4 × 10 ⁻¹³ JSTX-3 8. 8 × 10 ^-10 TTX 3.1 × 10 ^-11 ───────────────────────────────────

[0023]

INDUSTRIAL APPLICABILITY A protein having extremely strong paralytic insecticidal activity against pests has been successfully isolated and purified, and the protein can be provided as an active ingredient of an excellent insecticide.

[0024]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Sequence characteristics Fragment type: N-terminal fragment Origin Biological name: Crocous Myrmeleon bore Sequence characteristics Characterization method: E Sequence Ser Tyr Glu Asn Asp 5

[Brief description of the drawings]

FIG. 1 shows a pattern of SDS-PAGE analysis of a purified product. The migration direction is from top (-) to bottom (+).

FIG. 2 is a chromatogram of column chromatography using a DEAE Sephacel column. Fraction number 20-26
Was collected as the active fraction. There was no activity in the other fractions.

FIG. 3 is a chromatogram of column chromatography using a phenyl superose column. 27.0-2
The fraction eluted at 8.1 minutes was collected as the active fraction. There was no activity in the other fractions.

Claims (1)

[Claims] 1. A protein which is obtained from a venom collected from the larva of the larva of the larva of the larva of the genus Ephemeridae, and whose amino terminal amino acid sequence is the amino acid sequence shown in SEQ ID NO: 1.