Lepidoptera larva venom protein extraction method

Abstract

The invention discloses a lepidoptera larva venom protein extraction method. The method comprises the following steps: S1, thorn cutting: taking lepidoptera larvae, and cutting off the poison thorns from the base parts without touching the tips of the poison thorns; s2, extraction: soaking the cut poison thorns in a PBS (Phosphate Buffer Solution) which is pre-cooled to 2-6 DEG C for 20-60 minutes, preferably 20-40 minutes; s3, performing low-temperature high-speed centrifugation to collect supernate: after soaking is completed, performing high-speed centrifugation in a centrifugal tube, and collecting the supernate to obtain the traditional Chinese medicine composition. The method provided by the invention can realize high-purity venom extraction based on a very small number of insect samples, and is especially suitable for the yellow thorn moth larvae of which toxins are difficult to extract by adopting a conventional method.

Description

Technical Field

[0001] This invention relates to the field of insect toxin extraction technology, specifically to a method for extracting venom proteins from lepidopteran larvae. Background Technology

[0002] Most lepidopteran larvae possess irritating stingers or hairs on their bodies. When these tiny stingers or hairs come into contact with human skin, they trigger a series of severe reactions: redness, swelling, pain, or itching (Jang et al., 2009; Areces-Berazain, 2022). In recent years, with the continuous development of urban green spaces and the increase in daily activity areas such as street trees and parks, people's opportunities to come into contact with lepidopteran larvae have been increasing, leading to a significant increase in the risk of caterpillar poisoning. Therefore, research on the toxins of lepidopteran larvae is crucial for targeted prevention and treatment in clinical practice. Furthermore, research on lepidopteran larval toxins can provide important supplementary information for revealing the complex mechanisms by which insects adapt to their environment and evolve over a long period.

[0003] Different toxin proteins have different functions and effects. For example, the paper published by Andrew A. Walke et al. (2021) (https: / / doi.org / 10.1073 / pnas.2023815118) reported on the larvae of the tussock moth (…). Doratifera vulnerans The venom of *Haemaphysalis contortus* contains 151 polypeptide toxins belonging to 59 families, most with a molecular weight less than 10 kDa. The core families include: Family 1: ACP-like peptides: specifically activate insect G protein-coupled receptors (GPCRs), presumably involved in insect physiological regulation related to ecological defense. Family 2: Cecropin-like peptides: possess pleiotropic defensive effects, including inducing pain in vertebrates, insecticidal activity, antibacterial activity, and inhibition of nematode development; key factors in the venom's analgesic and broad-spectrum defense. Family 3: ICK-like peptides: these peptides do not possess analgesic or insecticidal activity, but have a potent inhibitory effect on the development of *Haemaphysalis contortus* larvae without significant cytotoxicity. Different proteins in the venom may have different functions, playing multiple roles.

[0004] Research on the venom of common venomous species such as scorpions, snakes, and spiders mainly involves using electro-, mechanical, and chemical stimulation to induce venom spraying or secretion, or by dissecting their venom glands (Garb, 2014; von Reumont et al., 2014; Undheim et al., 2015; Walker et al., 2018). However, the extraction of insect biotoxins currently faces the following difficulties: (1) Most insects are small in size, making it difficult to obtain venom glands or venom-producing organs. During the experiment, the stinger is prone to breakage, leading to venom loss and low extraction content. Therefore, a large number of insect bodies are required for extraction; (2) Insects produce very little venom, making venom extraction difficult. During the extraction process, the venom is easily degraded or lost, resulting in undetectable levels; (3) Some insect species produce little venom and lack venom glands. These problems pose a great challenge to the collection of insect biotoxins, resulting in a serious lag in research on lepidopteran insect toxins.

[0005] Yellow-spotted moth ( Monema (Cnidocampa) flavescens The yellow-spotted caterpillar (Spodoptera exigua) is a pest belonging to the genus Spodoptera in the family Limacodidae of the order Lepidoptera. It is also known as the stinging caterpillar, hairy eight-pointed caterpillar, eight-pointed caterpillar, and thorny caterpillar. The larvae are yellowish-green to green, covered with poisonous bristles containing toxins. Contact with these bristles causes them to penetrate the skin and release venom, leading to redness, swelling, and stinging pain; in severe cases, it can cause systemic symptoms. Currently, there are few reports on the toxins of the yellow-spotted caterpillar larvae and their extraction methods. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned problems by providing a novel, convenient, and efficient method for extracting venom proteins from lepidopteran larvae, which can achieve high-purity venom extraction based on a very small number of insect samples.

[0007] To achieve its objective, the present invention employs the following technical solution: This invention provides a method for extracting venom proteins from lepidopteran larvae, comprising the following steps: S1. Cutting the spine: Take a lepidopteran larva, and cut the spine off from the base without touching the tip of the spine. S2. Extraction: Soak the cut stinger in PBS buffer pre-cooled to 2-6°C for 20-60 min, preferably 20-40 min; S3. Low-temperature high-speed centrifugation to collect the supernatant: After soaking, centrifuge at high speed in a centrifuge tube and collect the supernatant.

[0008] Preferably, the method for extracting lepidopteran larval venom proteins of the present invention further includes step S4: S4. Preparation of freeze-dried powder: The obtained supernatant is freeze-dried to obtain freeze-dried powder crude powder.

[0009] Preferably, in step S1, ophthalmic scissors are used to cut the stingers, and the cut stinger clusters are placed in a low-temperature container at 2~6°C.

[0010] Preferably, in step S2, the amount of PBS buffer used is 1-3 mL (preferably 1.5-2.5 mL) of PBS buffer for soaking all the stingers of 1-5 lepidopteran larvae, wherein the PBS buffer is 0.5-5×PBS buffer (preferably 2×PBS buffer). In step S3, low-temperature high-speed centrifugation refers to centrifugation at 7000-12000 g for 2-8 min (preferably 3-5 min) at 2-6℃.

[0011] Preferably, in step S3, after aspirating the supernatant from the centrifuge tube, pre-cooled PBS buffer is added again, and steps S2 and S3 are repeated to extract the toxin repeatedly, for a total of 1 to 3 times. The lepidopteran larvae are middle to late instar larvae of the families Limacodidae, Lepidodidae, Saturniidae, Saturniidae, or Lymantidae, and the larvae have poisonous spines or hairs on their body surface.

[0012] Preferably, the freeze-drying is performed by rapidly freezing the supernatant in liquid nitrogen for 1-2 hours and then freeze-drying it.

[0013] Preferably, the method for extracting lepidopteran larval venom proteins of the present invention further includes step S5: S5, LC-MS / MS separation and purification S5.1 Sample Preparation: (1) Dissolve the lyophilized powder of lepidopteran larval toxin obtained in step S4 in ddH2O to prepare a venom solution with a concentration of 8~12 mg / mL. (2) Take the prepared venom solution and add it to a centrifuge tube. Dilute it with ultrapure water to 8-10 times its volume. Then add 0.8-1.2M ammonium carbonate solution to obtain diluted venom solution. The pH of the ammonium carbonate solution is 10.5-11.5, and the volume ratio of the ammonium carbonate solution to the venom solution before dilution with ultrapure water is 1:0.9-1.1. Then add an equal volume of modified reduction / alkylation mixture to the diluted venom solution, seal the centrifuge tube, and incubate at 36-38℃ for 30-60 min. After incubation, freeze dry to obtain the freeze-dried product. The modified reduction / alkylation mixture contains the following components by volume percentage: 97-98% acetonitrile, 1.7-2.3% iodoethanol, and 0.3-0.7% triethylphosphine. (3) Add protease solution to the freeze-dried product obtained in step (2) and perform enzymatic digestion; (4) After enzymatic hydrolysis, the liquid is drawn into an ultrafiltration tube for filtration, the filtrate is collected, and the liquid is lyophilized and used as the sample to be tested; preferably, ammonium bicarbonate solution is added to the filtrate obtained after ultrafiltration, and then the mixture is centrifuged, the filtrate is collected, and the liquid is lyophilized and used as the sample to be tested. S5.2, LC-MS / MS detection: The lyophilized sample was reconstituted with 0.08–1.2% (v / v) formic acid aqueous solution, and the mass spectra of the venom protein were obtained by LC-MS / MS. Mobile phase A was an aqueous solution containing 0.08–1.2% (v / v) formic acid, and mobile phase B was an acetonitrile solution containing 0.08–1.2% (v / v) formic acid, and gradient elution was performed.

[0014] Preferably, in step (3) of S5.1, the protease solution is a trypsin solution with a concentration of 0.04~0.06 wt%, and is added in an amount of 45~55 µL of 0.04~0.06 wt% trypsin solution to each 50 µg of the lyophilized powder crude toxin prepared in step S4 and the lyophilized product prepared in step (2) of S5.1; the trypsin solution is prepared using 45~55 mM ammonium bicarbonate solution; In step (4) of S5.1, after the enzymatic hydrolysis is completed, the liquid is drawn into an ultrafiltration tube, centrifuged at high speed, the filtrate is collected, and 45~55 mM ammonium bicarbonate solution is added. The ammonium bicarbonate solution is added at a ratio of 45~55 µL of 45~55 mM ammonium bicarbonate solution per 50 µg of the lyophilized powder crude toxin prepared in step S4 as the starting material. After centrifugation, the filtrate is collected, and after lyophilization, it is used as the sample to be tested.

[0015] The modified reduction / alkylation mixture comprises the following components by volume percentage: 97.5% acetonitrile, 2% iodoethanol and 0.5% triethylphosphine; wherein the 2% iodoethanol has an ethanol content of 40%~50% by volume and an effective iodine content of 18-22 g / L.

[0016] The samples were analyzed using a Dionex UltiMate 3000 liquid chromatography system and a Q Exactive mass spectrometer. The liquid chromatography conditions were as follows: C18 column with an inner diameter of 75 µm, a length of 15 cm, a particle size of 1.9 µm, and a pore size of 100 Å; liquid flow rate of 600 nl / min; mobile phase: phase A was 0.1% FA / H2O; phase B was an acetonitrile solution containing 0.1% (v / v) formic acid; chromatographic gradient: 0 min, 5% phase B; 8 min, 8% phase B; 16 min, 13% phase B; 39 min, 28% phase B; 50 min, 40% phase B; 51 min, 95% phase B; 55 min, 95% phase B; 56 min, 6% phase B; 60 min, 6% phase B; injection volume of 8 µl. The mass spectrometry conditions were as follows: spray voltage 2100 V, capillary temperature 320℃, full scan resolution 70000, scan quality range 300~1400, AGC value 3E6, IT time 60ms, two-stage scan, topN 20, resolution 17500, AGC value 5E4, IT time 80ms, and NCE 27.

[0017] The beneficial effects of this invention are as follows: (1) A high-efficiency extraction of biotoxins from small sample sizes was achieved for small-sized Lepidoptera larvae.

[0018] (2) Traditional methods of toxic extraction mainly involve squeezing the larvae to induce them to secrete venom, which is then collected. This method requires the larvae to be alive, but it is not very effective for extracting toxins from yellow-spotted moth larvae and is difficult to extract toxins. The method of this invention only requires that the larval venom not leak out, and the larval viability requirement is relatively low. It can also successfully extract a large amount of yellow-spotted moth larval toxin protein.

[0019] (3) It solves the problem of venom toxin protein extraction from larvae with low toxin production per unit, and allows toxin extraction and proteomics analysis to be performed on a single larva.

[0020] (4) The toxins extracted by the method of the present invention were confirmed by mass spectrometry to have more larval venom peptide sequences, and more types of toxic peptides could be obtained and discovered.

[0021] (5) The number of larvae required is greatly reduced. Compared with traditional toxin extraction methods, only a small number of larvae are needed, which provides a more convenient operation and more efficient application for subsequent medical testing for caterpillar poisoning.

[0022] (6) In the process of preparing samples for mass spectrometry, compared with existing methods, the conventional reducing alkylation reagent was adjusted to significantly increase the breaking ratio of disulfide bonds and ensure comprehensive and accurate determination of larval toxins.

[0023] (7) In the process of preparing samples for mass spectrometry, small molecule peptides are filtered through ultrafiltration tubes and the filtrate in the outer tube is retained together, which ensures that the molecular weight of most of the venom peptides is less than 10KD and avoids a large loss of venom peptides. Attached Figure Description

[0024] Figure 1 The process of extracting toxins from yellow-spotted moth larvae in Example 1 is shown in the diagram: An RNAase-free cryovial was placed on ice, and the venomous spines of the yellow-spotted moth were cut into the cryovial.

[0025] Figure 2 The extraction steps of Example 1, which involved cutting off the larval stingers and immersing them in 2×PBS buffer, are shown.

[0026] Figure 3 Preliminary experimental results of five reagents for a mouse paw injection pain test.

[0027] Figure 4 The results of the foot injection pain test in LCTX-Mf69-(99) mice.

[0028] Figure 5 The results are from a mouse paw edema experiment involving injection of a solution. Detailed Implementation

[0029] The present invention will be described below with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0030] Example 1: Extraction of toxins from yellow-spotted moth larvae I. Extraction Method Experimental insect: Yellow-spotted moth (Lymanid moth, Lepidoptera, Limacodidae) Monema flavescens Extract toxins from 6th instar larvae of Walker (a type of insect) by following these steps: (1) Removing the spines: Place the sterilized 2mL centrifuge tube on ice, and use ophthalmic scissors to cut off each cluster of spines of the yellow-spotted moth larva from the base. Throughout the process, ensure that the scissors do not touch the tip of the spine (to prevent the spine from breaking and leaking venom). Place the removed spine clusters into a 2mL centrifuge tube. The process is illustrated in the diagram below. Figure 1 As shown. One to five yellow-spotted moth larvae with their venomous stingers can be cut and placed in a centrifuge tube. Figure 2 The image shows the extraction of crude toxin from four yellow-spotted moth larvae after removing their spines and soaking them in 2×PBS buffer. Three parallel replicates were performed. During the procedure, care must be taken not to touch the tip of the spine when removing it, as the tip is brittle and easily breaks. If the spine breaks, the toxin will be lost, resulting in a lower extraction yield.

[0031] (2) Soaking: Add 2×PBS buffer (phosphate buffer solution) that has been stored in a 4℃ refrigerator to a 2 mL centrifuge tube containing larval stingers, up to the 2 mL mark, and place on ice to soak for 30 min.

[0032] (3) Low temperature high speed centrifugation: After soaking, centrifuge at 10000 g for 4 min at 4℃ and then take the supernatant and store it in a cryovial.

[0033] (4) After the supernatant was aspirated by the low temperature high speed centrifugation in step (3), 2×PBS buffer that had been stored in a 4℃ refrigerator was added again to the 2 mL mark. The mixture was placed on ice and soaked for 30 min. After soaking, the supernatant was centrifuged at 10000 g for 4 min at 4℃ and then added to the cryopreservation tube in step (3).

[0034] (5) Freeze-drying: freeze the cryovial containing the supernatant in liquid nitrogen for 1 hour and then freeze-dry to obtain freeze-dried powder crude toxin.

[0035] Figure 2 Four yellow-spotted moth larvae were cut into the spiny clusters, yielding a total of approximately 1.095g of crude toxic powder.

[0036] Preparation method of 2×PBS buffer: Weigh 16.0g NaCl, 0.4g KCl, 2.84g Na2HPO4 and 0.54g KH2PO4 and dissolve them in 800mL distilled water. Adjust the solution to 7.4 with HCl and finally add distilled water to make up to 1L. II. The Influence of Experimental Conditions on the Extracted Crude Toxin In the method of this invention, temperature control during the experimental process has a significant impact on the experimental results. We conducted a comparative study on the temperature during the experimental process, setting up two comparison groups: a strictly controlled low-temperature group and a non-strictly controlled temperature group. The strictly controlled low-temperature group refers to the experimental operation being carried out at a temperature of 2-4℃ throughout the crude toxin extraction process, and the supernatant after soaking and centrifuging in 2×PBS buffer was freeze-dried at low temperature to obtain the crude toxin. The non-strictly controlled temperature group refers to the operation of each step at room temperature, at a temperature of approximately 20℃. The iBAQ (absolute quantification method based on peak intensity) results of the top 20 substances obtained by mass spectrometry of the crude toxins obtained by the two methods show (Table 1) that the iBAQ values ​​of the strictly controlled temperature group are significantly higher than those of the non-strictly controlled temperature group, indicating that the toxin content obtained under the 2-4℃ temperature condition is significantly higher than that obtained under the room temperature condition.

[0037] Table 1. iBAQ results of the top 20 crude toxicant mass spectrometry data.

[0038] Example 2: Detection of toxins using liquid chromatography coupled with tandem mass spectrometry For Example 1 Figure 2 The lyophilized powder crude toxin obtained in the experiment was subjected to mass spectrometry experiments. The experiments included two parts: sample preparation and LC-MS / MS detection. The specific operating steps are as follows: I. Sample Preparation: (1) Add about 4.5 mg of crude venom (lyophilized crude venom powder) to 450 µL ddH2O to dissolve the crude venom powder, and obtain a venom concentration of 10 mg / mL. Aliquot the dissolved venom and store it at -80℃.

[0039] (2) Take 5 µL of venom (50 µg crude venom) into a 1.5 mL centrifuge tube (RNAase-free), add 40 µL of ultrapure water (to make the final volume 45 µL), add 5 µL of 1M ammonium carbonate (adjusted pH=11 with ammonium hydroxide / ammonia water, prepared with mass spectrometry grade water, filtered through a 0.22 µm membrane), and then add 50 µL of modified reduction / alkylation mixture, wherein the modified reduction / alkylation mixture contains the following components by volume percentage: 97.5% acetonitrile, 2% iodoethanol (ethanol content (volume fraction) of 40%~50%, effective iodine content range of 18-22 g / L (2%)) and 0.5% triethylphosphine; seal the centrifuge tube and incubate at 37℃ for 60 min. After incubation, freeze-dry the sample.

[0040] In step (2), compared to the modified reduction / alkylation mixture, the unmodified traditional reduction / alkylation experimental method is as follows: first add 4 μL of the reducing component 1 M DTT, incubate at 37°C for 2 h, then add 15 μL of the alkylating component 1 M iodoacetamide solution, and incubate at room temperature in the dark for 1 h.

[0041] (3) Add 50 µL of 0.05 wt% trypsin (Promega, catalog number V5113) solution (prepared with 50 mM ammonium bicarbonate solution) to the lyophilized sample obtained in step (2) and digest it at 37°C for 12 h.

[0042] (4) After enzymatic hydrolysis, the liquid is aspirated into an ultrafiltration tube (10 KD), centrifuged at 12000 g at room temperature for 20 min, the filtrate is collected, and 50 µL of 50 mM ammonium bicarbonate solution is added. The mixture is then centrifuged again (the addition of ammonium bicarbonate solution and centrifugation ensures that as many peptides as possible are eluted from the ultrafiltration tube). The filtrate is collected in an RNase-free tube and lyophilized to obtain the sample for mass spectrometry detection. After enzymatic hydrolysis, the toxin is converted into peptides smaller than 10 KD. These peptides are collected by ultrafiltration and used for LC-MS / MS detection.

[0043] II. LC-MS / MS Detection Add 15 µL of 0.1% (v / v) formic acid aqueous solution to 5 µL of lyophilized venom (50 µg crude venom) for redissolution. Obtain the mass spectrum of venom protein using liquid chromatography-tandem mass spectrometry (LC-MS / MS) (without desalting). Use mobile phase A, which is an aqueous solution containing 0.1% (v / v) formic acid, and mobile phase B, which is an acetonitrile solution containing 0.1% (v / v) formic acid, for gradient elution. The elution method is shown in Table 2 below. Table 2

[0044] Samples were analyzed using a Dionex UltiMate 3000 liquid chromatography system and a Thermo Scientific Q Exactive mass spectrometer: The liquid chromatography conditions were as follows: a Dionex U3000 series C18 column with an inner diameter of 75 µm, a length of 15 cm, a particle size of 1.9 µm, and a pore size of 100 Å was used; the liquid flow rate was 600 nl / min; the mobile phase was: phase A was 0.1% FA / H2O; phase B was an acetonitrile solution containing 0.1% (v / v) formic acid; the chromatographic gradient was: 0 min, 5% phase B; 8 min, 8% phase B; 16 min, 13% phase B; 39 min, 28% phase B; 50 min, 40% phase B; 51 min, 95% phase B; 55 min, 95% phase B; 56 min, 6% phase B; 60 min, 6% phase B; and the injection volume was 8 µl. The mass spectrometry conditions were as follows: spray voltage 2100 V, capillary temperature 320℃, full scan resolution 70000, scan quality range 300~1400, AGC value 3E6, IT time 60ms, two-stage scan, topN 20, resolution 17500, AGC value 5E4, IT time 80ms, NCE 27. Mass spectrometry data analysis: Raw mass spectrometry data were processed using MaxQuant software (version 1.3.0.1), with the *Tabarella spp.* genome database as a reference for retrieval. Peptide retrieval employed the Andromeda search algorithm, and the protein identification parameters were set as follows: initial precursor ion mass tolerance was 6 ppm, fragment ion mass tolerance was 20 ppm; initial search tolerance was set to 20 ppm, master search tolerance was set to 6 ppm; high-energy collision dissociation (HCD) fragment ion mass tolerance was set to 20 ppm. Cysteine ​​carbamylation was set as a fixed modification, while N-terminal acetylation and methionine oxidation were set as variable modifications. The minimum peptide length was set to 6 amino acids, allowing a maximum of 2 missed cleavage sites. The false discovery rate (FDR) for both peptides and proteins was set to 0.01. The protein list was filtered to remove proteins identified from the reverse database and common contaminating proteins; each identified protein must contain at least one unique peptide. For comparing protein abundance, the intensity-based absolute quantification (iBAQ) algorithm in MaxQuant software was used.

[0045] In performing mass spectrometry detection, this invention has made improvements based on previous detection methods: (1) Small molecule peptides are filtered and retained using an ultrafiltration tube in the early stage of mass spectrometry, ensuring that the molecular weight of most short venom peptides is less than 10KD, thus avoiding the loss of small molecule peptide toxin molecules compared with traditional methods. (2) Compared with traditional methods, the reduction alkylation ratio has been adjusted, which greatly increases the disulfide bond breaking ratio and ensures comprehensive and accurate determination of larval toxins.

[0046] In this experiment, the effects of the traditional and the modified reduction / alkylation mixtures of this invention on the experimental results were compared. Except for the difference in the reduction / alkylation mixtures used, the experimental settings were identical. The iBAQ results represent the abundance (i.e., content) of the protein, as shown in Table 3. The iBAQ results of the Top 20 data from mass spectrometry analysis of the crude toxins obtained by both methods show that the modified reduction / alkylation mixture significantly increased the content of the identified toxin proteins.

[0047] Table 3 Comparison of the effects of different ratios of reduction / alkylation mixtures on the experiment

[0048] The obtained mass spectrometry results were searched against the transcriptome database, yielding 127 results. After matching with the genome, a total of 99 polypeptide gene sequences were finally obtained. Using the detailed annotation information of the sequences in different databases (Uniprot database, Nr database, PFAM database), the 99 gene sequences were annotated using eggNOG 5.0. The detailed annotation results are shown in Table 4. Among them, LCTX-Mf69-(99) is a new toxin that has not been reported before, indicating that the toxin extraction and mass spectrometry detection method of the present invention can obtain more comprehensive information on the types of insect toxins.

[0049] The amino acid sequence of LCTX-Mf69-(99) is as follows (SEQ ID NO.1): MSKIAIIFVLAVLVCYIEGFQTIVGGPTDGAPRQARWAEYGPDEHSSSESHSSFSSETIVDGQTVSKKSGGETVSNVDGQETRTHYGDFF.

[0050] Table 4

[0051] Example 3: Extraction of toxins from the yellow-spotted moth using conventional methods Traditional methods for extracting insect toxins typically involve pressing the insect's body to release the venom, then collecting the venom and extracting the toxins. This embodiment uses a traditional method to extract toxins from the larvae of the yellow-spotted moth, and the specific procedures are as follows: (1) Toxin Extraction: Toxins were collected using a clean sealing film fitted over a 20 mm × 30 mm wire loop with an extended wire handle. The sealing film was gently pressed onto the larvae of the yellow-spotted moth, and the larvae were gently pressed with the wire. However, the larvae of the yellow-spotted moth rarely expel venom droplets by pressing, unlike other insects. 20 µL of water was added to the sealing film to rinse the venom (the venom expelled by the yellow-spotted moth larvae is very little and almost invisible to the naked eye). The rinsing solution was then transferred to a 1.5 mL tube for recovery. The rinsing solution from the sealing films of multiple larvae was collected into a test tube. The collected rinsing solution was then transferred to a 10 kDa ultrafiltration tube, and 8 M urea was added to bring the volume to 200 μL. After repeated pipetting and aspiration, the tube was centrifuged at 12000 g for 20 min. The process was repeated three times with the addition of 8 M urea. The waste liquid in the outer tube was discarded. (2) Add 4 μL of 1 M DTT and 150 μL of 8 M urea, and incubate at 37°C for 2 h. (3) Add 15 μL of 1 M iodoacetamide and incubate at room temperature in the dark for 1 h. Centrifuge at 12000 g at room temperature for 20 min. (4) Add 200 μL of 8 M urea and centrifuge three times, then add 200 μL of 50 mM ammonium carbonate and centrifuge four times. (5) Add 200 μL of 25 μg / mL trypsin, seal the tube opening with sealing film, and incubate at 37℃ for 24-36 h. (6) Centrifuge at 12000 g at room temperature for 20 min and collect the lower filtrate; add 40 μL of 50 mM ammonium carbonate and centrifuge at 12000 g for 20 min. (7) Freeze-concentrate at 4℃ until the sample is completely dry, add 200 μL of 0.1% TFA solution, vortex and then briefly elute. Filter the supernatant using a C18 desalting column and collect the eluent. (8) After desalting the peptides, they were lyophilized and then resuspended in 30 μL of 0.1% formic acid solution. 15 μL of the supernatant was taken and separated into peptides using liquid chromatography. (9) MaxQuant 1.6.10 software for analyzing mass spectrometry data.

[0052] The above method yielded zero types of toxic peptides, and factors and enzymes unrelated to toxic peptides, such as EF1-a, CAD, and IDH, were identified, indicating that the traditional method is not suitable for extracting toxins from the larvae of the yellow-spotted moth.

[0053] Example 4: Verification of the pain function of toxic proteins in yellow-spotted moth larvae The yellow tussock moth larval toxin proteins LCTX-Mf69-(99), LCTX-Mf2-(10) and LCTX-Mf55-(84) identified in Example 2 were used in mouse experiments and were artificially synthesized for use in experiments.

[0054] The amino acid sequence of LCTX-Mf2-(10) is as follows (SEQ ID NO.2): MSKIAIIFVLAVLVCYIEGGPIDGPARQSRSPGYGNAPGSHSSSVSQSSFSSETVVNGKTVSKKFGGETVRNVDGHETRSRYGNY.

[0055] The amino acid sequence of LCTX-Mf55-(84) is as follows (SEQ ID NO. 3): MLQAGLGCILLTFLFVLPTTAGEDQVEREKRGVDLGLQRGYSGAQLAKLKMGLEAANNPNGPGRRRRDTQNLEREKRGIDLGLQRGYSGAQLAKLKMALEAANNPNGPGRRRRDTQNLEREKRGIDLGLQRGYSGAQAARLKMGLAAANNPNGPGRK.

[0056] We conducted a mouse paw injection experiment to verify the pain response of these three proteins: by injecting the toxin protein solution, capsaicin solution, and PBS solution into the paws of mice, we counted the number of times the mice licked their paws and the thickness of their paws within five minutes to verify the pain-inducing and edema-inducing toxin protein components in the yellow-spotted moth toxin protein.

[0057] I. Solution Preparation (1) Preparation of experimental group solutions: The solid powders of toxin proteins LCTX-Mf69-(99), LCTX-Mf2-(10) and LCTX-Mf55-(84) were respectively prepared into solutions with a volume of 2 ml and a final concentration of 260 µM using 1×PBS solution.

[0058] (2) Preparation of positive control solution: We selected capsaicin (CAS Registry No. 404-86-4) as the positive control for this experiment. We took 15.27 mg of capsaicin powder and added 1 mL of pure DMSO to dissolve it into a 50 mM stock solution. Then we took 20 µL of the stock solution and added 1×PBS to make up to a total volume of 2 mL. We vortexed the solution to obtain 2 mL of 500 µM capsaicin working solution (DMSO final concentration 1%).

[0059] (3) Preparation of negative control solution: We selected 1×PBS solution as the negative control solution for this experiment.

[0060] II. Foot Injection Pain Pre-test 25 µL of the solution was drawn up with a syringe and injected into the right hind paw of a mouse. The mouse was then placed in a transparent glass enclosure, and the number of times the mouse licked its paw was recorded using a camera over five minutes. The experiment was repeated three times for each of the three proteins in the experimental group, as well as the solutions in the negative and positive control groups. The statistical results of the number of times the mouse licked its paw are shown in Table 5. Significance analysis is as follows: Figure 3 As shown.

[0061] Table 5. Number of times mice licked their paws within five minutes of paw injection (preliminary experiment)

[0062] We found that mice injected with LCTX-Mf69-(99) protein solution showed a significant increase in paw licking frequency within five minutes compared to mice injected with PBS, with an injury effect comparable to capsaicin. The LCTX-Mf2-(10) group showed a slight increase in paw licking frequency compared to the PBS group, but this was not significant. No significant change was observed with LCTX-Mf55-(84). Therefore, we believe that LCTX-Mf69-(99) protein, which has the highest content in the yellow-spotted moth toxin, is the main factor causing pain after yellow-spotted moth stings.

[0063] III. Supplementary Verification Experiment of Foot Injection Pain Test To verify the reliability of the experiment, we performed 10 replicate experiments on the LCTX-Mf69-(99) protein, and the results are shown in Table 6. Figure 4 As shown.

[0064] Table 6. Number of times mice licked their paws within five minutes of paw injection.

[0065] The experimental results are consistent with the preliminary experimental results in Table 5.

[0066] IV. Verification of Foot Injection Edema Experiment 25 µl of the experimental solution was drawn into a syringe and injected into the right hind paw of a mouse. The mouse was then placed inside a transparent glass enclosure. After 15 minutes, the mouse was removed, and the change in the thickness of the mouse's paw sole was measured and recorded using a ruler. The results are shown in Table 7. Figure 5 As shown: Table 7. Foot plantar thickness in mice (mm)

[0067] We found that the thickness of the foot of mice in the LCTX-Mf55-(84) group was significantly higher than that in the PBS group, but slightly lower than that in the capsaicin group. The thickness of the LCTX-Mf69-(99) group and the LCTX-Mf2-(10) group did not increase significantly compared with the PBS group. The experiment shows that LCTX-Mf55-(84) may be an important reason for tissue edema caused by the yellow tussock moth toxin.

Claims

1. A method for extracting venom proteins from lepidopteran larvae, characterized in that, Includes the following steps: S1. Cutting the spine: Take a lepidopteran larva, and cut the spine off from the base without touching the tip of the spine. S2. Extraction: Soak the cut stinger in PBS buffer pre-cooled to 2-6°C for 20-60 min, preferably 20-40 min; S3. Low-temperature high-speed centrifugation to collect the supernatant: After soaking, centrifuge at high speed in a centrifuge tube and collect the supernatant.

2. The method for extracting venom protein from lepidopteran larvae according to claim 1, characterized in that, It also includes step S4: S4. Preparation of freeze-dried powder: The obtained supernatant is freeze-dried to obtain freeze-dried powder crude powder.

3. The method for extracting venom proteins from lepidopteran larvae according to claim 1, characterized in that: In step S1, ophthalmic scissors are used to cut off the stingers, and the cut stinger clusters are placed in a low-temperature container at 2~6℃.

4. The method for extracting venom proteins from lepidopteran larvae according to claim 1, characterized in that: In step S2, the amount of PBS buffer used is 1-3 mL (preferably 1.5-2.5 mL) of PBS buffer for soaking all the stingers of 1-5 lepidopteran larvae. The PBS buffer is 0.5-5×PBS buffer (preferably 2×PBS buffer). In step S3, low-temperature high-speed centrifugation refers to centrifugation at 7000-12000 g for 2-8 min (preferably 3-5 min) at 2-6℃.

5. The method for extracting venom proteins from lepidopteran larvae according to claim 1, characterized in that: In step S3, after aspirating the supernatant from the centrifuge tube, add pre-cooled PBS buffer again, and repeat steps S2 and S3 to extract the toxin repeatedly, repeating the extraction 1 to 3 times. The lepidopteran larvae are middle to late instar larvae of the families Limacodidae, Lepidodidae, Saturniidae, Saturniidae, or Lymantidae, and the larvae have poisonous spines or hairs on their body surface.

6. The method for extracting venom proteins from lepidopteran larvae according to claim 2, characterized in that: The freeze-drying process involves rapidly freezing the supernatant in liquid nitrogen for 1-2 hours followed by freeze-drying.

7. The method for extracting venom protein from lepidopteran larvae according to claim 2, characterized in that: It also includes step S5: S5, LC-MS / MS separation and purification S5.1 Sample Preparation: (1) Dissolve the lyophilized powder of lepidopteran larval toxin obtained in step S4 in ddH2O to prepare a venom solution with a concentration of 8~12mg / mL. (2) Take the prepared venom solution and add it to a centrifuge tube. Dilute it with ultrapure water to 8-10 times its volume. Then add 0.8-1.2 M ammonium carbonate solution to obtain diluted venom solution. The pH of the ammonium carbonate solution is 10.5-11.5, and the volume ratio of the ammonium carbonate solution to the venom solution before dilution with ultrapure water is 1:0.9-1.

1. Then add an equal volume of modified reduction / alkylation mixture to the diluted venom solution, seal the centrifuge tube, and incubate at 36-38℃ for 30-60 min. After incubation, freeze dry to obtain the freeze-dried product. The modified reduction / alkylation mixture contains the following components by volume percentage: 97-98% acetonitrile, 1.7-2.3% iodoethanol, and 0.3-0.7% triethylphosphine. (3) Add protease solution to the freeze-dried product obtained in step (2) and perform enzymatic digestion; (4) After enzymatic hydrolysis, the liquid is drawn into an ultrafiltration tube for filtration, the filtrate is collected, and the liquid is lyophilized and used as the sample to be tested; preferably, ammonium bicarbonate solution is added to the filtrate obtained after ultrafiltration, and then the mixture is centrifuged, the filtrate is collected, and the liquid is lyophilized and used as the sample to be tested. S5.2, LC-MS / MS detection: The lyophilized sample was reconstituted with 0.08–1.2% (v / v) formic acid aqueous solution, and the mass spectra of the venom protein were obtained by LC-MS / MS. Mobile phase A was an aqueous solution containing 0.08–1.2% (v / v) formic acid, and mobile phase B was an acetonitrile solution containing 0.08–1.2% (v / v) formic acid, and gradient elution was performed.

8. The method for extracting venom protein from lepidopteran larvae according to claim 7, characterized in that: In step (3) of S5.1, the protease solution is a trypsin solution with a concentration of 0.04~0.06 wt%, and is added in an amount of 45~55µL of 0.04~0.06 wt% trypsin solution to each 50µg of the lyophilized powder crude toxin prepared in step S4 and the lyophilized product prepared in step (2) of S5.1; the trypsin solution is prepared using 45~55 mM ammonium bicarbonate solution; In step (4) of S5.1, after the enzymatic hydrolysis is completed, the liquid is drawn into an ultrafiltration tube, centrifuged at high speed, the filtrate is collected, and 45~55mM ammonium bicarbonate solution is added. The ammonium bicarbonate solution is added at a ratio of 45~55 µL of 45~55 mM ammonium bicarbonate solution per 50µg of the lyophilized powder crude toxin prepared in step S4 as the starting material. After centrifugation, the filtrate is collected, and after lyophilization, it is used as the sample to be tested.

9. The method for extracting venom protein from lepidopteran larvae according to claim 7, characterized in that: The modified reduction / alkylation mixture comprises the following components by volume percentage: 97.5% acetonitrile, 2% iodoethanol and 0.5% triethylphosphine; wherein the 2% iodoethanol has an ethanol content of 40% to 50% by volume and an effective iodine content of 18-22 g / L.

10. The method for extracting venom protein from lepidopteran larvae according to claim 7, characterized in that: Samples were analyzed using a Dionex UltiMate 3000 liquid chromatography system and a Q Exactive mass spectrometer: The liquid chromatography conditions were as follows: C18 column with an inner diameter of 75 µm, a length of 15 cm, a particle size of 1.9 µm, and a pore size of 100 Å; liquid flow rate of 600 nl / min; mobile phase: phase A was 0.1% FA / H2O; phase B was an acetonitrile solution containing 0.1% (v / v) formic acid; chromatographic gradient: 0 min, 5% phase B; 8 min, 8% phase B; 16 min, 13% phase B. 39 min, 28% B phase; 50 min, 40% B phase; 51 min, 95% B phase; 55 min, 95% B phase; 56 min, 6% B phase; 60 min, 6% B phase, injection volume 8 µl; The mass spectrometry conditions were as follows: spray voltage 2100 V, capillary temperature 320℃, full scan resolution 70000, scan quality range 300~1400, AGC value 3E6, IT time 60ms, two-stage scan, topN 20, resolution 17500, AGC value 5E4, IT time 80ms, and NCE 27.

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