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CN-121974972-A - Extraction method of venom protein of lepidoptera larva

CN121974972ACN 121974972 ACN121974972 ACN 121974972ACN-121974972-A

Abstract

The invention discloses a method for extracting venom protein from lepidoptera larvae. The method comprises the following steps of S1, cutting, namely, taking lepidoptera larvae, cutting the toxic thorns from a base without touching the tip of the toxic thorns, S2, leaching, namely, soaking the cut toxic thorns in PBS buffer solution precooled to 2-6 ℃ for 20-60 min, preferably 20-40 min, S3, centrifuging at a low temperature and high speed, collecting supernatant, namely, centrifuging at a high speed in a centrifuge tube after soaking is completed, and collecting supernatant. The method can realize high-purity venom extraction based on a very small number of insect samples, and is particularly suitable for the yellow moth larvae which are difficult to extract toxins by adopting a conventional method.

Inventors

  • ZHANG AIBING
  • LI JING
  • DONG ZHAOMING
  • LIU WENYUE
  • YAN XIAOLONG
  • HAN YING

Assignees

  • 首都师范大学
  • 西部(重庆)科学城种质创制大科学中心

Dates

Publication Date
20260505
Application Date
20251218

Claims (10)

  1. 1. The extraction method of the venom protein of the lepidoptera larva is characterized by comprising the following steps of: S1, cutting, namely taking lepidoptera larvae, and cutting the toxic thorns from the basal part without touching the tip of the toxic thorns; S2, leaching, namely soaking the sheared poison thorns in PBS buffer solution precooled to 2-6 ℃ for 20-60 min, preferably 20-40 min; s3, centrifuging at a low temperature and a high speed to collect supernatant, namely centrifuging at a high speed in a centrifuge tube after soaking is completed, and collecting supernatant to obtain the finished product.
  2. 2. The method for extracting venom protein from a lepidopteran larva according to claim 1, further comprising step S4: S4, preparing freeze-dried powder, namely freeze-drying the obtained supernatant to obtain freeze-dried powdery crude toxin.
  3. 3. The method for extracting venom protein from lepidoptera larvae according to claim 1, wherein in the step S1, the venom sticks are sheared by adopting ophthalmic scissors, and the sheared venom sticks are placed in a low-temperature container with a temperature of 2-6 ℃.
  4. 4. The method for extracting venom protein from lepidopteran larvae according to claim 1, wherein: In the step S2, the PBS buffer solution is used for soaking by using 1-3 mL (preferably 1.5-2.5 mL) of PBS buffer solution according to the dosage of all the toxins of every 1-5 lepidopteran larvae, wherein the PBS buffer solution is 0.5-5 xPBS buffer solution (preferably 2 xPBS buffer solution); In the step S3, the low-temperature high-speed centrifugation means centrifugation at 7000 to 12000 g for 2 to 8 min (preferably 3 to 5 min) at 2 to 6 ℃.
  5. 5. The method for extracting venom protein from lepidopteran larvae according to claim 1, wherein: In the step S3, adding precooled PBS buffer solution again to the residue after the supernatant is sucked in the centrifuge tube, repeating the step S2 and the step S3 to repeatedly extract toxins, and repeatedly extracting for 1-3 times; The lepidoptera larva is middle-final-stage larva of Araliaceae, spodopteraceae, fagaceae or Podopteraceae, and has thorn or pilus on the surface.
  6. 6. The method for extracting venom protein from lepidopteran larvae according to claim 2, wherein the freeze-drying comprises the steps of quick freezing the supernatant in liquid nitrogen for 1-2 h and freeze-drying.
  7. 7. The method for extracting venom protein from a lepidopteran larva according to claim 2, further comprising the step of S5: S5, LC-MS/MS separation and purification S5.1, sample preparation: (1) Adding ddH 2 O into the lepidopteran larva toxin freeze-dried powder prepared in the step S4 to dissolve the lepidopteran larva toxin freeze-dried powder, and preparing a venom with the concentration of 8-12 mg/mL; (2) Adding the prepared venom into a centrifuge tube, adding ultrapure water to dilute the solution to 8-10 times of the volume, adding 0.8-1.2M ammonium carbonate solution to obtain diluted venom, wherein the pH=10.5-11.5 of the ammonium carbonate solution and the volume ratio of the ammonium carbonate solution to the venom before the dilution by adding the ultrapure water are 1:0.9-1.1, adding an improved reduction/alkylation mixture which is equal to the volume of the diluted venom, sealing the centrifuge tube, incubating the centrifuge tube at 36-38 ℃ for 30-60 min, and freeze-drying after the incubation is completed to obtain a freeze-dried product, wherein the improved reduction/alkylation mixture comprises 97-98% acetonitrile, 1.7-2.3% iodoethanol and 0.3-0.7% triethylphosphine by volume percent; (3) Adding protease solution into the freeze-dried product obtained in the step (2) for enzymolysis digestion; (4) After enzymolysis, the liquid is sucked into an ultrafiltration tube for filtration, the filtrate is collected and freeze-dried to be used as a sample to be tested, preferably, ammonium bicarbonate solution is added into the filtrate obtained after ultrafiltration, and then the filtrate is centrifuged, collected and freeze-dried to be used as the sample to be tested; S5.2, LC-MS/MS detection: Adding 0.08-1.2% (v/v) formic acid aqueous solution into a freeze-dried sample to be tested for re-dissolution, using LC-MS/MS to obtain mass spectrum of venom protein, wherein the mobile phase A liquid is an aqueous solution containing 0.08-1.2% (v/v) formic acid, and the mobile phase B liquid is an acetonitrile solution containing 0.08-1.2% (v/v) formic acid, and performing gradient elution.
  8. 8. The method for extracting venom protein from a lepidopteran larva according to claim 7, wherein: In the step (3) of the S5.1, the protease solution is trypsin solution with the concentration of 0.04-0.06 wt%, and 45-55 mu L of 0.04-0.06 wt% trypsin solution is added into the freeze-dried product prepared in the step (2) of the S5.1 according to the amount of 50 mu g of freeze-dried powdery crude toxin prepared in the step S4, wherein the trypsin solution is prepared by adopting 45-55 mM ammonium bicarbonate solution; In the step (4) of the step S5.1, after enzymolysis is finished, liquid is sucked into a ultrafilter tube, high-speed centrifugation is carried out, filtrate is collected, 45-55 mM ammonium bicarbonate solution is added, 45-55 mu L of 45-55 mM ammonium bicarbonate solution is added to 50 mu g of the freeze-dried powdery crude toxin prepared in the step S4 serving as a starting material, and the mixture is centrifuged, collected and freeze-dried to obtain a sample to be tested.
  9. 9. The method for extracting venom protein from a lepidopteran larva according to claim 7, wherein: the improved reduction/alkylation mixture comprises, by volume, 97.5% acetonitrile, 2% iodoethanol and 0.5% triethylphosphine, wherein the content of ethanol in the 2% iodoethanol is 40% -50% by volume, and the effective iodine content is 18-22 g/L.
  10. 10. The method for extracting venom protein from a lepidopteran larva according to claim 7, wherein the sample is detected by a Dionex UltiMate 3000 liquid chromatography system and a Q Exactive mass spectrometer: The liquid chromatography conditions are C18 chromatographic column with specification of 75 μm inner diameter, length of 15 cm, particle diameter of 1.9 μm, pore diameter of 100A, liquid flow rate of 600 nl/min, mobile phase of 0.1% FA/H 2 O for phase A, acetonitrile solution containing 0.1% (v/v) formic acid for phase B, chromatographic gradient of 0min, 5% B phase, 8min, 8% B phase, 16 min,13% B phase, 39 min,28% B phase, 50min, 40% B phase, 51 min,95% B phase, 55 min,95% B phase, 56 min,6% B phase, 60 min,6% B phase, and sample injection amount of 8 μl; The mass spectrum conditions are spray voltage 2100V, capillary temperature 320 ℃, full Scan resolution 70000, scan mass range 300-1400, AGC value 3E6, IT time 60ms, secondary Scan, topN 20, resolution 17500, AGC value 5E4, IT time 80ms, NCE 27.

Description

Extraction method of venom protein of lepidoptera larva Technical Field The invention relates to the technical field of insect toxin extraction, in particular to a method for extracting venom protein of lepidoptera larvae. Background Most lepidopteran larvae have irritating stings or hairs on their body surfaces that meet the human skin and trigger a series of severe reactions, such as reddening, swelling, pain or itching of the skin (Jang et al 2009; areces-Berazain, 2022). In recent years, with the continuous development of urban greening areas, the daily activities such as street trees, parks and the like are increased, and the contact probability of people with lepidoptera larvae is increased, so that the risk of poisoning by caterpillars is greatly increased, and therefore, the research on toxin components of the lepidoptera larvae is very important for the clinical targeted preventive and therapeutic measures. Meanwhile, the research on lepidopteran larva toxins can provide important supplement for revealing complex mechanisms of insect adaptation environment and long-term evolution. Different toxin proteins have different functions and roles, for example, the literature published by Andrew A, walke et al (2021) (https:// doi. Org/10.1073/pnas. 2023815118) reports that the venom of the larvae of the spiny moth (Doratifera vulnerans) contains 151 polypeptide toxins, belonging to 59 families, most of which have a molecular weight less than 10 kDa, and the core family includes family 1: ACP-like peptides: specifically activating insect G Protein Coupled Receptors (GPCRs), presumably involved in physiological regulation of insects involved in ecological defense. Family 2 Cecropin-LIKE PEPTIDES), a pleiotropic defensive action, including induction of vertebrate pain, insecticidal, antibacterial and nematode development, is a key factor in venom pain and broad-spectrum defenses. Family 3 ICK-like peptides which do not contain painful or insecticidal activity but have potent inhibitory effects on the development of Haemonchus contortus larvae and have no apparent cytotoxicity. Different proteins in venom may have different functions and play a variety of roles. The research on the venom of common toxic species such as scorpions, snakes, spiders and the like is mainly carried out by spraying or secreting the venom through electric, mechanical and chemical stimulation or by planing the venom glands (Garb, 2014; von Reumont et al, 2014; undheim et al, 2015, walker et al, 2018), but the extraction of the insect biotoxin is difficult at present, namely (1) the most of the insects are tiny in body type, the venom glands or the toxic organs are difficult to obtain, the toxic thorns are easy to break in the experimental process to cause the loss of the toxin, the extraction content is low, a large number of insects are required to extract, the insect production is tiny, the operation difficulty of the venom extraction is high, the toxin is easy to degrade or lose in the extraction process to cause undetectable, and (3) the toxic liquid amount of part of the insect species is small and the venom glands are lack. The presence of these problems presents a great challenge to the collection of insect venom, resulting in a serious lag in lepidopteran insect toxin studies. The yellow thorn moth (Monema (Cnidocampa) FLAVESCENSWALKER) is a pest of the genus Tripteridae, which is called Trichinella, anise, dioscorea, etc. The yellow thorn moth larva body is yellow green to green, the whole body is fully filled with toxic thorn hair, the toxic thorn hair contains toxin, and the toxic thorn hair can penetrate into skin to release venom after touching, so that red swelling and stinging are caused, and systemic symptoms can be caused when the toxic thorn moth larva body is severe. There are few reports on yellow thorn moth larva toxins and extraction methods thereof. Disclosure of Invention The invention aims at solving the problems and provides a novel, convenient and efficient method for extracting venom protein from lepidoptera larvae, which can realize high-purity venom extraction based on a very small number of insect samples. In order to achieve the purpose, the invention adopts the following technical scheme: The invention provides a method for extracting venom protein from lepidoptera larvae, which comprises the following steps: S1, cutting, namely taking lepidoptera larvae, and cutting the toxic thorns from the basal part without touching the tip of the toxic thorns; S2, leaching, namely soaking the sheared poison thorns in PBS buffer solution precooled to 2-6 ℃ for 20-60 min, preferably 20-40 min; s3, centrifuging at a low temperature and a high speed to collect supernatant, namely centrifuging at a high speed in a centrifuge tube after soaking is completed, and collecting supernatant to obtain the finished product. Preferably, the method for extracting venom protein from lepidopteran larvae of the present invention fur