A plexomicoid polypeptide toxin ed79-wr and preparation method and application thereof

Abstract

The present application provides a kind of zootoxin Ed79-WR of zonatus sea anemone polypeptide and its preparation method and application, its amino acid sequence is ACRDTYFASSC (Acm) KAAVDGDRC (tBu) RKSNYKTGC (Acm) AKT C (tBu) GYCT.The linear peptide Ed79-WR is synthesized by using polypeptide solid-phase synthesis method (SPPS) in the present application, the linear peptide is purified, then it is obtained by site-specific oxidation to contain three disulfide bonds Oxidized peptide Ed79-WR (the connection mode of disulfide bond is C1-C6, C2-C4, C3-C5), and the product is prepared after oxidation and folding Liquid phase purification, high performance liquid chromatography analysis and mass spectrometry identification, obtain zonatus sea anemone polypeptide Ed79-WR.The synthetic oxidized peptide Ed79-WR is tested by lethal experiment in the present application to the insecticidal effect of grass caterpillar, and the zonatus sea anemone polypeptide toxin Ed79-WR can be applied to the preparation of insecticide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polypeptides, and particularly relates to a sea anemone polypeptide toxin Ed79-WR and a preparation method and application thereof. BACKGROUND

[0002] Sea anemones belong to the phylum Cnidaria and are one of the oldest poisonous animals. Sea anemones have rich pharmacological and biotechnological values and are marine drug resources, containing functionally complex and diverse peptide neurotoxins. Marine biological toxins are one of the fastest growing fields in the research and development of marine active substances and are a hotspot in the research of new marine drugs. Some marine biological toxins have been applied to neuroscience research tools or directly developed into marine drugs.

[0003] Modern pharmacological studies have shown that sea anemone toxins have insecticidal, hypoglycemic, antitumor, antibacterial, antihypertensive, analgesic, and central nervous system inhibitory effects. Based on the analysis of the transcriptome data of Exaiptasia diaphana in the early stage, a polypeptide sequence was screened from the widely studied ShK family, and a sea anemone functional polypeptide with three disulfide bonds was synthesized by site-directed oxidation. Through insecticidal experiments, it was clarified that the sea anemone polypeptide has certain insecticidal activity. This lays a foundation for the development of new insecticides, the development of new marine drugs, and provides strong support for human health and marine drug research. SUMMARY

[0004] In this study, Exaiptasia diaphana polypeptide toxin Ed79-WR was found from Exaiptasia diaphana by high-throughput transcriptome, which has six cysteines, forms three disulfide bonds, and its amino acid sequence is ACRDTYFASSC(Acm)KAAVDGDRC(tBu)RKSNYKTGC(Acm)AKTC(tBu)GYCT.

[0005] The application provides an Exaiptasia diaphana polypeptide toxin Ed79-WR and a preparation method and application thereof. The Exaiptasia diaphana polypeptide toxin Ed79-WR prepared by the method has high insecticidal effect, and lays a foundation for the research and development of new, efficient and safe biological insecticides.

[0006] The purpose of the application is achieved by the following technical solutions.

[0007] An Exaiptasia diaphana polypeptide toxin Ed79-WR has six cysteines, forms three disulfide bonds, and its amino acid sequence is ACRDTYFASSC(Acm)KAAVDGDRC(tBu)RKSNYKTGC(Acm)AKTC(tBu)GYCT. The connection mode of the disulfide bonds is C1-C6, C2-C4, and C3-C5.

[0008] A method for preparing the anemone polypeptide toxin Ed79-WR includes the following steps:

[0009] (1) Weigh the resin, soak it in DCM first, then wash it with DMF and DCM in sequence, add Fmoc-Ala(tbu)-OH, DCM and DIEA to react, then add methanol and DCM to react, wash with DMF, then add pyridine solution to react, and then wash with DMF to obtain the first resin.

[0010] (2) Add the second amino acid Fmoc-Cys-OH and a condensing agent to the first resin, then add DMF and DIC to react, wash with DMF, then add pyridine solution to react, and then wash with DMF to obtain the second resin;

[0011] (3) Repeat step (2) according to the above amino acid sequence until the peptide chain ends to obtain linear peptide resin;

[0012] (4) Add methanol to the linear peptide resin for rinsing and then dry it. Add cutting fluid for cutting, filter, add ice-cold ether, centrifuge and remove the upper layer of ice-cold ether to obtain the crude precipitated peptide.

[0013] (5) The crude peptide was separated and purified by HPLC to obtain the pure peptide, namely the pure linear peptide Ed79-WR.

[0014] (6) Dissolve the linear peptide from step (5) in a methanol-water mixture, dilute with acetic acid, add methanol-iodine solution dropwise, and keep stirring to form the first disulfide bond;

[0015] (7) Add an equal volume of hydrochloric acid-methanol solution, then add methanol-iodine solution, and keep stirring to form a second disulfide bond;

[0016] (8) After separation and purification by HPLC, the product was freeze-dried to obtain the freeze-dried powder.

[0017] (9) Dissolve the freeze-dried powder in TFA / DMSO / anisole solution, stir at room temperature, then add TFA / DMSO / anisole solution and stir in a water bath.

[0018] (10) Add ice-cold ether, centrifuge and remove the supernatant ice-cold ether to obtain the crude precipitated polypeptide;

[0019] (11) The peptide was separated and purified by HPLC and identified by MS to obtain pure peptide, namely oxidized peptide Ed79-WR;

[0020] Further, in step (1), 0.6-1.0 mmol Fmoc-Ala(tbu)-OH, 10-15 mL DCM and 1-3 mL DIEA are added and reacted for 80-100 min, then 3-5 mL methanol and 8-12 mL DCM are added and the reaction is blocked for 25-35 min.

[0021] Furthermore, in steps (1) and (2), Fmoc is removed using a DMF solution containing 20% ​​piperidine by mass for 15–25 min.

[0022] Further, in step (2), 1.5-2.0 mmol of amino acids (Fmoc-Cys-OH) and 1.5-2.0 mmol of condensing agent (HOBT) are weighed, 5-15 mL of DMF is poured in, and then 1-3 mL of DIC is added and the reaction is carried out for 0.5-1.5 h.

[0023] Furthermore, in step (4), the cutting fluid is 95wt% TFA, 1wt% H2O, 2wt% EDT and 2wt% TIS, and the centrifugation is performed at 4℃, 12000r / min and 5min three times.

[0024] Furthermore, in step (5), the crude polypeptide is separated and purified by HPLC. The elution time of the linear peptide Ed79-WR is 22.035 min, and the pure polypeptide, namely the pure linear peptide Ed79-WR, is obtained.

[0025] Furthermore, in step (6), the methanol-water mixture is 50% methanol / 50% water, and then diluted with acetic acid to make the final concentration of the peptide solution 1 mg of peptide per milliliter of the 5% methanol / 5% water / 90% acetic acid mixture, and the methanol-iodine solution is 10 mg / mL, and stirred for 1 min.

[0026] Furthermore, in step (7), the hydrochloric acid-methanol solution is 50mM HCl / 50% methanol, the methanol-iodine solution is 10mg / mL (10-20 times excess), and the mixture is stirred for 1h.

[0027] Furthermore, in steps (8) and (11), mobile phase A (H2O) and mobile phase B (ACN) are separated and purified by HPLC at a flow rate of 5 mL / min, using linear gradient elution, and a detection wavelength of 220 nm.

[0028] Furthermore, the gradient elution procedure is as follows:

[0029]

[0030] Further, in step (9), the first added TFA / DMSO / anisole solution was 97.9% / 2% / 0.1%, 2 mg / mL, and stirred at room temperature for 40 min. Then, 40 μL / mg peptide was added to the TFA / DMSO / anisole (97.9% / 2% / 0.1%) solution, and stirred in a 70℃ water bath for 3 h.

[0031] Furthermore, in step (10), the centrifugation is performed three times at 4°C, 12000 r / min, and 5 min.

[0032] Furthermore, in step (11), the oxidized peptide is separated and purified by HPLC. The elution time of the oxidized peptide Ed79-WR is 17.805 min, and the pure polypeptide, namely the pure oxidized peptide Ed79-WR, is obtained.

[0033] This invention also proposes the application of the aforementioned anemone polypeptide toxin Ed79-WR in the preparation of insecticides. Furthermore, this invention relates to the application of the anemone polypeptide toxin Ed79-WR in the preparation of an insecticide for the armyworm.

[0034] The Chinese names of the reagents or raw materials used in this invention are shown in Table 1 below:

[0035] Table 1

[0036]

[0037]

[0038] Compared with existing technologies, the present invention has the following advantages:

[0039] 1. This invention utilizes high-throughput transcriptomics to discover the anemone polypeptide toxin Ed79-WR from the anemone *Exaiptasia diaphana*, which has six cysteine ​​residues that can form three disulfide bonds. Its amino acid sequence is ACRDTYFASSC(Acm)KAAVDGDRC(tBu)RKSNYKTGC(Acm)AKTC(tBu)GY CT.

[0040] 2. In this invention, a linear peptide Ed79-WR was synthesized using solid-phase peptide synthesis (SPPS). The linear peptide was purified by HPLC, and then the synthesized linear peptide was oxidatively folded using a stepwise method. After site-directed oxidation via disulfide bonds, an oxidized peptide Ed79-WR containing three disulfide bonds was obtained. The disulfide bond linkage was C1-C6, C2-C4, and C3-C5. The oxidized product was purified by HPLC and identified by mass spectrometry to obtain the anemone polypeptide toxin Ed79-WR, i.e., the oxidized Ed79-WR.

[0041] 3. The anemone polypeptide toxin Ed79-WR prepared by the method of this invention has a highly efficient insecticidal effect, which can lay the foundation for the development of new, efficient and safe biological insecticides. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of HPLC analysis of the linear peptide Ed79-WR.

[0043] Figure 2 This is a schematic diagram of the mass spectrometry identification of the linear peptide Ed79-WR.

[0044] Figure 3 This is a schematic diagram of HPLC analysis of the oxidized peptide Ed79-WR.

[0045] Figure 4 This is a schematic diagram of the mass spectrometry identification of the oxidized peptide Ed79-WR.

[0046] Figure 5 This is a schematic diagram of the insecticidal effect of oxidative peptide Ed79-WR on the armyworm. Note: The experimental group is compared with the blank control group and the negative control group. * indicates a significant difference (*p<0.05; **p<0.01; ***p<0.001). Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0048] This invention utilizes high-throughput transcriptomics to discover the anemone polypeptide toxin Ed79-WR from the anemone *Exaiptasia diaphana*. Ed79-WR has six cysteine ​​residues that can form three disulfide bonds. Its amino acid sequence is ACRDTYFASSC(Acm)KAAVDGDRC(tBu)RKSNYKTGC(Acm)AKTC(tBu)GY CT, with disulfide bond connections of C1-C6, C2-C4, and C3-C5.

[0049] This invention also provides a method for preparing the anemone polypeptide toxin Ed79-WR, the method comprising the following steps:

[0050] 1. Materials and Methods

[0051] 1.1 Experimental Materials

[0052] Chromatographic grade trifluoroacetic acid (TFA) and chromatographic grade acetonitrile (ACN) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; a water analytical C18 column (5 μm, 4.6 mm × 250 mm) was purchased from Waters Corporation, USA; and an Elite preparative C18 column (10 μm, 10 mm × 250 mm) was purchased from Dalian Elite Analytical Instruments Co., Ltd.

[0053] 1.2 Experimental Apparatus

[0054] CEM fully automated microwave peptide synthesizer (LibertyBlue, USA); reversed-phase high-performance liquid chromatography (ThermoFisher, Germany); triple quadrupole liquid chromatography-mass spectrometry (Shimadzu, Japan); benchtop freeze dryer (Saif, China).

[0055] 1.3 Experimental Methods

[0056] 1.3.1 A method for preparing a mantle anemone polypeptide toxin Ed79-WR, comprising the following steps:

[0057] (1) Weigh 2-cl resin with a degree of substitution Sd = 0.2-1.4 mmol / g, soak it in DCM first, then wash it with DMF and DCM in sequence, add 0.6-1.0 mmol Fmoc-Ala(tbu)-OH, 10-15 mL DCM and 1-3 mL DIEA and react for 80-100 min, then add 3-5 mL methanol and 8-12 mL DCM, block the reaction for 25-35 min, wash with DMF, and then wash with DMF solution with a piperidine mass fraction of 20% for 15-25 min to obtain the first resin;

[0058] Specifically, in this embodiment, 2-Cl resin with a degree of substitution Sd = 0.4 mmol / g was weighed, first soaked in DCM, then washed sequentially with DMF and DCM, and then 0.8 mmol of Fmoc-Ala(tbu)-OH, 12 mL of DCM and 2 mL of DIEA were added and reacted for 90 min. Then, 4 mL of methanol and 10 mL of DCM were added, the reaction was blocked for 30 min, washed with DMF, and then washed with a DMF solution with a piperidine mass fraction of 20% for 20 min to obtain the first resin. In other embodiments, the purpose of the present invention can be achieved by adjusting within the scope of the present invention.

[0059] (2) Add 1.5-2.0 mmol of amino acid (Fmoc-Cys-OH) and 1.5-2.0 mmol of condensing agent (HOBT) to the first resin, add 5-15 mL of DMF, then add 1-3 mL of DIC and react for 0.5-1.5 h, then add DMF solution containing 20% ​​piperidine by mass and wash for 15-25 min to obtain the second resin;

[0060] Specifically, in this embodiment, 1.8 mmol of amino acid (Fmoc-Cys-OH) and 1.8 mmol of condensing agent (HOBT) were added to the first resin, 10 mL of DMF was added, and then 2 mL of DIC was added to react for 1.0 h. Then, a DMF solution containing 20% ​​piperidine by mass was added to wash for 20 min to obtain the second resin.

[0061] In other embodiments, adjustments within the scope of the invention can achieve the objectives of the invention;

[0062] (3) Repeat step (2) according to the above amino acid sequence until the peptide chain ends to obtain linear peptide resin;

[0063] (4) Add methanol to the linear peptide resin for rinsing and then dry it. Add cutting solution (95wt% TFA, 1wt% H2O, 2wt% EDT and 2wt% TIS) for cutting. After filtration, add ice ether and centrifuge at 4℃ and 12000r / min for 5min. Repeat the centrifugation 3 times and remove the upper layer of ice ether to obtain the crude precipitated peptide.

[0064] (5) The crude peptide was separated and purified by HPLC. The mobile phase was A (H2O), the mobile phase was B (ACN), the flow rate was 5 mL / min, and the linear gradient elution was performed. The elution program is shown in Table 2 below. The detection wavelength was 220 nm. The pure peptide, namely the pure linear peptide Ed79-WR, was obtained.

[0065] (6) Dissolve the linear peptide from step (5) in a 1:1 mixture of methanol and water, then dilute with acetic acid to achieve a final peptide concentration of 1 mg per mL of the mixture (5 v / v% methanol + 5 v / v% water + 90 v / v% acetic acid). Add 10 mg / mL methanol-iodine solution dropwise (add the methanol-iodine solution dropwise, stopping once a light yellow color appears and persists; approximately 10 μL of methanol-iodine solution is needed per milligram of peptide). Stir for 1 min to form the first disulfide bond.

[0066] (7) Add an equal volume (i.e., the same as the total volume in step 6) of 50mM HCl / 50% methanol hydrochloric acid-methanol solution, then add 10mg / mL of methanol-iodine solution (approximately 0.2mL of methanol-iodine solution needs to be added for each milligram of peptide), and keep stirring for 1 hour to form the second disulfide bond;

[0067] (8) The product was separated and purified by HPLC. The mobile phase was A (H2O) and the mobile phase was B (ACN). The flow rate was 5 mL / min. The linear gradient elution was performed. The elution program is shown in Table 2 below. The detection wavelength was 220 nm. The product was then freeze-dried.

[0068] (9) The lyophilized powder was dissolved in a TFA / DMSO / anisole (v / v / v = 97.9% / 2% / 0.1%) solution at 2 mg / mL and stirred at room temperature for 40 min. Then, 40 μL / mg peptide was added to the TFA / DMSO / anisole (v / v / v = 97.9% / 2% / 0.1%) solution and stirred in a 70℃ water bath for 3 h.

[0069] (10) Add ice-cold ether, centrifuge at 4°C and 12000r / min for 5min, repeat centrifugation 3 times, remove the upper layer of ice-cold ether, and obtain the crude precipitated polypeptide.

[0070] (11) The mobile phase A (H2O) and mobile phase B (ACN) were separated and purified by HPLC at a flow rate of 5 mL / min and linear gradient elution. The elution program is shown in Table 2 below. The detection wavelength is 220 nm. Then MS was used for identification to obtain the pure peptide, namely oxidized peptide Ed79-WR.

[0071] Table 2 Gradient elution program

[0072]

[0073] 1.3.2 Insect Injection Method

[0074] The insect injection method was used with an insect moth weighing approximately 180 mg. The method is briefly described below, referring to existing techniques:

[0075] The anemone polypeptide toxin Ed79-WR obtained in this invention, namely oxidized peptide Ed79-WR, was dissolved in 0.7% NaCl to concentrations of 2.5 nM, 5 nM, 10 nM, 15 nM, and 20 nM. 5 μL of each solution was injected into the abdomen of *Arbuscular myxostreatus*. *Arbuscular myxostreatus* injected with no liquid was used as a blank control, and 5 μL of 0.7% NaCl solution injected into *Arbuscular myxostreatus* served as a negative control.

[0076] 1.3.3 Data Processing

[0077] All data were statistically analyzed and processed using GraphPad Prism7 software. The data between the control group and the experimental group were analyzed using t-tests. * indicates a significant difference (p<0.05), ** indicates an extremely significant difference (p<0.01), and *** indicates an extremely significant difference (p<0.001).

[0078] 2 Results

[0079] 2.1 Synthesis and Oxidative Folding of Peptides

[0080] The linear peptide Ed79-WR from the sea anemone was synthesized using solid-phase peptide synthesis (SPPS). The linear peptide was purified by HPLC, and its molecular weight was determined by mass spectrometry to be 4259.087 Da (see [link to SPPS]). Figure 2 The synthesized linear peptide was subjected to a one-step oxidative folding method using site-directed oxidation, and the final oxidative folding product was purified by HPLC and identified by mass spectrometry. The actual molecular weight of the linear peptide Ed79-WR was 4259.087 Da, and the molecular weight of the oxidized peptide Ed79-WR was 3999.0836 Da (see...). Figure 4 The difference between the two is approximately 260 Da, proving that three disulfide bonds were correctly formed.

[0081] 2.2 Isolation and purification of oxidized peptides

[0082] The linear peptide Ed79-WR was analyzed using analytical HPLC, and the results are as follows: Figure 1 As shown, the elution time of the linear peptide Ed79-WR was 22.035 min. Preparative HPLC was used to separate and purify the three-step oxidized Ed79-WR, followed by analytical HPLC analysis. The results are shown below. Figure 3 As shown, the elution time of the oxidized peptide Ed79-WR was 17.805 min.

[0083] This invention utilizes a site-directed oxidation method to perform a three-step oxidative folding of the synthesized linear peptide Ed79-WR sample, and then purifies the oxidative folding final product by HPLC and identifies it by mass spectrometry to obtain the anemone polypeptide toxin Ed79-WR, namely the oxidized peptide Ed79-WR, with the sequence ACRDTYFASSC(Acm)KAAVDGDR C(tBu)RKSNYKTGC(Acm)AKTC(tBu)GYCT. It has 6 cysteine ​​residues forming 3 disulfide bonds, with the disulfide bond configurations being C1-C6, C2-C4, and C3-C5.

[0084] 2.3 Lethality Test

[0085] Experimental results are as follows Figure 5 As shown, the mortality rate of armyworms in both the blank control and negative control groups was 0, indicating that the injection method is feasible for evaluating the insecticidal effect of oxidative peptide Ed79-WR. The mortality rate of armyworms increased with increasing dose of oxidative peptide Ed79-WR, showing significant differences compared to the control group. A low dose of 2.5 nM oxidative peptide Ed79-WR was already lethal to insects, with a mortality rate of 13.34%, while a high dose of 20 nM oxidative peptide Ed79-WR achieved a mortality rate of 76.67%. The mortality rate increased with increasing oxidative peptide dose, and the median lethal dose (LD50) was calculated to be 11.16 nM.

[0086] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A suite of sea anemone polypeptide toxins Ed79-WR, characterized in that, The sequence is ACRDTYFASSC(Acm)KAAVDGDRC(tBu)RKSNYKTGC(Acm)AKTC(tBu)GYCT, with six cysteines, forming three disulfide bonds, and the connection mode of the disulfide bonds is C1-C6, C2-C4, and C3-C5.

2. The method of claim 1, wherein the preparation of the sea anemone polypeptide toxin Ed79-WR is characterized by, It comprises the following steps: The resin is weighed, soaked in DCM first, then washed with DMF and DCM in turn, Fmoc-Ala(tbu)-OH, DCM and DIEA are added for reaction, then methanol and DCM are added for reaction, washed with DMF, then pyridine solution is added for reaction, then piperidine-DMF solution is added for washing, to obtain the first resin; In the first resin, the second amino acid Fmoc-Cys-OH and 1-hydroxybenzotriazole are added, then DMF and DIC are added for reaction, washed with DMF, then pyridine solution is added for reaction, then piperidine-DMF solution is added for washing, to obtain the second resin; According to the amino acid sequence of claim 1, step (2) is repeated until the end of the peptide chain, to obtain a linear peptide resin; Methanol is added to the linear peptide resin of step (3) for flushing and drying, a cleavage solution is added for cleavage, after filtration, ice ethyl ether is added, the upper liquid ice ethyl ether is removed after centrifugation, to obtain a precipitated polypeptide crude product; The polypeptide crude product of step (4) is separated and purified by HPLC, to obtain a pure linear peptide Ed79-WR by elution; The linear peptide of step (5) is dissolved in a mixed solution of methanol-water, then diluted with acetic acid, to obtain an acetic acid mixed solution, methanol iodine solution is added dropwise, stirring is maintained, to form a first disulfide bond; Then hydrochloric acid-methanol solution is added, then methanol iodine solution is added, stirring is maintained, to form a second disulfide bond; After separation and purification by HPLC, freeze-drying is performed, to obtain a freeze-dried powder; The freeze-dried powder is dissolved in a TFA-DMSO-anisole solution, stirring is performed at room temperature, then a TFA-DMSO-anisole solution is added, stirring is performed in a water bath; Ice ethyl ether is added, the upper liquid ice ethyl ether is removed after centrifugation, to obtain a precipitated polypeptide crude product; The polypeptide crude product of step (10) is separated and purified by HPLC, to obtain a pure oxidized peptide Ed79-WR.

3. The method of claim 2, wherein the preparation of the sea anemone polypeptide toxin Ed79-WR is characterized by, In the reaction system of step (1), the substitution degree Sd of the resin is 0.2-1.4 mmol / g, 0.6-1.0 mmol Fmoc-Ala(tbu)-OH, 10-15 mL DCM and 1-3 mL DIEA are added in proportion for reaction for 80-100 min, then 3-5 mL methanol and 8-12 mL DCM are added for reaction for 25-35 min.

4. The method of claim 2, wherein the preparation of the sea anemone polypeptide toxin Ed79-WR is characterized by, In step (2), in the reaction system, in the first resin obtained in step (1), 1.5-2.0 mmol amino acid Fmoc-Cys-OH and 1.5-2.0 mmol 1-hydroxybenzotriazole are added in proportion, 5-15 mL DMF is poured, then 1-3 mL DIC is added for reaction for 0.5-1.5 h.

5. The method of claim 2, wherein the preparation of the sea anemone polypeptide toxin Ed79-WR is characterized by, In step (4), the cleavage solution is prepared by 95% TFA, 1% H2O, 2% EDT and 2% TIS by mass percentage; in step (4) and step (10), the centrifugal condition is 11000-13000 r / min at 3-5℃ for 4-6 min, and the centrifugal is repeated for 2-4 times.

6. The method of claim 2, wherein the preparation of the sea anemone polypeptide toxin Ed79-WR is characterized by, In step (6), the volume ratio of methanol and water in the mixed solution of methanol and water is 1:0.8-1.2, the volume ratio of acetic acid and the mixed solution of methanol and water is 8.5-9.5:1, and 0.8-1.2 mg of peptide is contained in each milliliter of the mixed solution of acetic acid; the concentration of the methanol iodine solution is 9-11 mg / mL, 9-11 μL of the methanol iodine solution is added dropwise per milligram of peptide, and the stirring time is 0.5-2 min.

7. The method of claim 2, wherein the preparation of the sea anemone polypeptide toxin Ed79-WR is characterized by, In step (7), the hydrochloric acid-methanol solution is prepared by 45-55 mM HCl and methanol in a volume ratio of 1:0.8-1.2; the concentration of the methanol iodine solution is 8-12 mg / mL, and 0.15-0.25 mL of the methanol iodine solution is added per milligram of peptide.

8. The method of claim 2, wherein the preparation of the sea anemone polypeptide toxin Ed79-WR is characterized by, In step (5), step (8) and step (11), the mobile phase A for HPLC separation and purification is H2O, the mobile phase B is ACN, the flow rate is 4-6 mL / min, and the detection wavelength is 220 nm.

9. The method of claim 2, wherein the preparation of the sea anemone polypeptide toxin Ed79-WR is characterized by, In step (9), the volume ratio of TFA, DMSO and anisole in the TFA-DMSO-anisole solution is 97.8-98.0:1.9-2.1:0.1; 0.4-0.6 mL of the TFA-DMSO-anisole solution is added per milligram of the freeze-dried powder before room temperature stirring, 35-45 μL of the TFA-DMSO-anisole solution is added after room temperature stirring, the room temperature stirring time is 35-45 min; the water bath stirring temperature is 65-75℃, and the stirring time is 2-4 h.

10. Use of the lemafiin polypeptide toxin Ed79-WR in the preparation of an insecticide for killing Spodoptera exigua.

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