Extraction method of miniature parasitic wasp venom protein

Abstract

The invention discloses a method for extracting venom protein of miniature parasitic wasps, and relates to the technical field of biology. The extraction method comprises the following steps: carrying out anesthesia treatment on the glassogyne brilliant, putting the treated glassogyne brilliant in a buffer solution containing a protease inhibitor, and separating out venom organs; after the venom organ is subjected to low-temperature grinding and ultrasonication treatment, disulfide bonds in protein are reduced, alkylation modification is carried out, then the protein is aggregated through a solvent to form precipitates, and the miniature parasitic wasp venom protein is obtained through separation. The systematic venom protein collection and extraction process is established for the first time, and the operation bottleneck of micro insect venom research is broken through. The extraction efficiency and integrity of venom protein are remarkably improved through key technologies such as material taking process optimization, buffer solution protection, low-temperature grinding and ultrasonication.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for extracting micro-parasitic wasp venom protein. Background Art

[0002] In the field of micro-insect venom protein research, especially the research on the venom of external parasitic wasps such as Tamarixia radiata, there has long been a bottleneck in operational technology. Due to the tiny size of these insects, their venom glands and venom content are extremely limited, so traditional venom extraction methods are difficult to meet the needs of high-quality and high-efficiency research. Existing technologies usually rely on extensive dissection methods and simple physical crushing methods, lacking a systematic operating process, resulting in venom proteins that are easily degraded, especially the low recovery rate of low-abundance proteins, which seriously affects subsequent functional analysis and applied research.

[0003] Furthermore, insufficient control of temperature and buffer systems during conventional extraction further exacerbates protein loss and limits comprehensive analysis of venom components. Currently, there are no mature technical solutions to effectively address these issues, which has slowed progress in micro-insect venom research and hampered in-depth exploration of related biological regulatory mechanisms. Therefore, improving the extraction efficiency and integrity of venom proteins, particularly low-abundance proteins, has become a critical challenge in this field that urgently needs to be overcome. Summary of the Invention

[0004] The present invention aims to provide a method for extracting venom protein from microparasitic wasps to solve the problems of the prior art. The method significantly improves the extraction efficiency and integrity of venom protein.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for extracting microparasitic wasp venom protein, comprising the following steps:

[0007] After the glaucoma wasps were anesthetized, they were placed in a buffer containing protease inhibitors to separate the venom organs.

[0008] The venom organ is subjected to low-temperature grinding and ultrasonic disruption, the disulfide bonds in the protein are reduced, and alkylation modification is performed. Then, a solvent is used to aggregate the protein to form a precipitate, and the micro-parasitic wasp venom protein is separated.

[0009] Furthermore, the bright-bellied enameled wasp is a bright-bellied enameled wasp female that has emerged within 7 days and has fully mated.

[0010] Furthermore, the anesthesia treatment is to place the Mylotrichum glazei in a 4°C environment and freeze it for 8-10 minutes.

[0011] Furthermore, the buffer solution is a phosphate buffer solution.

[0012] Furthermore, the concentration of the protease inhibitor is 1 mM.

[0013] Furthermore, the cryogenic grinding is to firstly freeze the venom organ with liquid nitrogen and then add protein lysis solution for grinding.

[0014] Furthermore, the protein lysis solution comprises: 100 mM NH4HCO3, 6 M urea and 0.2 wt% sodium dodecyl sulfate, pH=8.

[0015] Furthermore, the ultrasonic disruption treatment time is 5 minutes.

[0016] Furthermore, dithiothreitol is used to reduce the disulfide bonds in the protein.

[0017] Furthermore, iodoacetamide was used for alkylation modification.

[0018] The present invention discloses the following technical effects:

[0019] This study establishes a systematic venom protein collection and extraction process for the microscopic ectoparasitic wasp Tamarixia radiata, breaking through the operational bottleneck of micro-insect venom research. By optimizing key technologies such as the sampling process, buffer protection, cryogenic grinding, and ultrasonic disruption, the extraction efficiency and integrity of venom proteins are significantly improved. Compared with traditional methods, the method of the present invention avoids protein degradation, ensures a high recovery rate of venom proteins (especially low-abundance proteins), and provides high-quality samples for subsequent functional studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a flow chart of the three-level nutritional relationship between Murraya osmanthus, Citrus psyllid and Glechoma glazei;

[0022] Figure 2 This is an SDS-PAGE gel image of the venom protein of the bright-bellied enameled wasp. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1. Collection process of venom protein from the bright-bellied enameled wasp

[0030] (1) Rearing system: Establish a three-level nutritional relationship between Murraya odora, citrus psyllids and bright-bellied enameled wasps indoors ( Figure 1 ) to raise the bright-bellied enameled wasp.

[0031] (2) Sample preparation: Prepare female bees of the bright-bellied enameled bee that are within 7 days of eclosion and fully mated.

[0032] (3) Place the bright-bellied enameled wasp in a 4°C refrigerator for 10 minutes to paralyze it.

[0033] (4) Place the anesthetized Myxoma glabrae on a glass slide and drip a solution containing 1 mM protease inhibitor (ProteinSafeTM The samples of the anthelmintic were kept in phosphate buffer (8 mM Na2HPO4, 136 mM NaCl, 2 mM KH2PO4, 2.6 mM KCl, pH 7.4) to prevent protein degradation.

[0034] (5) Place the dissecting slide under a stereo microscope (ZEISS, Stemi508) and separate the venom organs (including venom glands and venom sacs).

[0035] (6) Three biological replicates were established for each treatment, with each replicate containing 300 venom organs.

[0036] (7) The collected venom organs were washed three times with high-temperature sterilized PBS buffer (8 mM Na2HPO4, 136 mM NaCl, 2 mM KH2PO4, 2.6 mM KCl, pH 7.4), transferred to an enzyme-free 1.5 mL centrifuge tube, quickly frozen in liquid nitrogen for 2 minutes, and stored at -80°C for later use.

[0037] 2. Extraction of Venom Proteins

[0038] (1) The sample tissue was taken out from the -80℃ freezer, quickly frozen in liquid nitrogen for 3 minutes, and then quickly transferred to a centrifuge tube pre-cooled with liquid nitrogen. Protein lysis buffer was added and the sample was ground on ice using a handheld grinding rod (Tian Gen, OSE-Y30) for 5 minutes.

[0039] Protein lysis buffer: 100 mM NH 4 HCO 3 , 6 M urea (Urea), 0.2 wt % sodium dodecyl sulfate (SDS), pH=8.

[0040] (2) After grinding to a homogenate, shake and mix thoroughly. Use an ultrasonic cell disruptor (Ningbo Xinzhi, JY96-IIN) to disrupt the cells in an ice-water bath for 5 min to ensure complete lysis.

[0041] (3) Centrifuge at 4°C and 12,000 rpm for 15 min and collect the supernatant.

[0042] (4) Add dithiothreitol (DTTred) to a final concentration of 10 mM and react in a water bath at 56°C for 1 h;

[0043] (5) Add sufficient iodoacetamide (IAM) and react at room temperature in the dark for 1 h.

[0044] (6) Add 4 volumes of -20°C pre-cooled acetone and precipitate at -20°C for at least 2 h;

[0045] (7) Centrifuge at 12000 g for 15 min at 4°C and collect the precipitate.

[0046] (8) Add 1 mL of -20°C pre-cooled acetone to resuspend and wash the precipitate, centrifuge at 4°C and 12,000 g for 15 min, collect the precipitate, air-dry, and add protein dissolving solution to dissolve the protein precipitate.

[0047] Protein dissolution solution: 6 M Urea, 100 mM tetraethylammonium bromide (TEAB), pH = 8.5.

[0048] 3. Quantitative detection by BCA method

[0049] (1) Preparation of protein standards

[0050] a. Add 1.2 mL of protein standard solution to a tube of protein standard (30 mg BSA) and dissolve thoroughly to prepare a 25 mg / mL protein standard solution. This solution can be used immediately or stored at -20°C for extended periods.

[0051] b. Take an appropriate amount of 25 mg / mL protein standard solution and dilute it to a final concentration of 0.5 mg / mL. For example, add 20 μL of 25 mg / mL protein standard solution to 980 μL of diluent to make a 0.5 mg / mL protein standard solution. The standard should be diluted in the same solution as the protein sample. However, for simplicity, the standard can also be diluted in 0.9% NaCl or PBS. The diluted 0.5 mg / mL protein standard solution can be stored at -20°C for long periods of time.

[0052] (2) Preparation of BCA working solution

[0053] Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA Reagent A to 1 volume of BCA Reagent B (50:1) and mix thoroughly. For example, add 5 mL of BCA Reagent A to 100 μL of BCA Reagent B and mix thoroughly to make 5.1 mL of BCA working solution. BCA working solution is stable at room temperature for 24 hours.

[0054] (3) Protein concentration determination

[0055] a. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the standard wells of a 96-well plate. Add standard diluent to make up to 20 μL. These concentrations correspond to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively.

[0056] b. Add an appropriate volume of sample to each well of a 96-well plate. If the sample volume is less than 20 μL, add standard diluent to bring the volume up to 20 μL. Please note the sample volume.

[0057] c. Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 minutes.

[0058] d. Measure the absorbance at A562 or other wavelengths between 540-595 nm using a microplate reader.

[0059] e. Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0060] Table 1 shows the quality inspection data for the venom protein samples of the glaucoma glaucoma. As can be seen from Table 1, the venom protein samples extracted by the present invention have high concentrations and total amounts. The evaluation grade is A, indicating that the quality meets the experimental requirements, the electrophoresis bands are clear, and the total amount is sufficient for two or more experiments.

[0061] Table 1 Quality inspection data of the venom protein samples of the bright-bellied enameled wasp

[0062]

[0063] 4.SDS-PAGE detection

[0064] (1) Gel preparation: Prepare gel using PAGE Gel Quick Preparation Kit (10%):

[0065] a. Prepare a 1mm gel block and mix equal volumes of separating gel buffer and separating gel solution, i.e., 2.7 mL of each solution.

[0066] b. Add 55 μL of modified ammonium persulfate solution to the mixed solution in step a (if solidification is too fast, the amount of ammonium persulfate can be halved) and mix thoroughly.

[0067] c. Pour the solution from step b into the gel-forming glass plate. Be sure to pour the stacking gel into the gel mold within 2 minutes after adding the separating gel. Pour the stacking gel slowly to prevent mixing of the stacking gel and the separating gel. If you find the stacking gel difficult, you can also block the plate with ethanol before adding the stacking gel.

[0068] d. Preparation of stacking gel: Take equal volumes of stacking gel buffer and stacking gel solution and mix them evenly, i.e., take 0.75 mL of each solution, then add 15 μL of modified ammonium persulfate solution and mix thoroughly.

[0069] e. Inject into the glass plate and insert the comb teeth (do not use excessive force when inserting the comb, insert it gently).

[0070] f. After the stacking gel has solidified for 15 minutes, remove the comb teeth and use it for electrophoresis.

[0071] (2) Protein sample preparation

[0072] The extracted protein sample was taken out from -20℃, thawed on ice, and 40μL of protein solution was taken. 10μL of protein loading buffer was added. After gentle mixing, the sample was centrifuged briefly, boiled in boiling water for 10 minutes, and placed on ice.

[0073] (3) SDS-PAGE electrophoresis

[0074] After the stacking gel has solidified for 15 minutes, place it in the electrophoresis tank, add electrophoresis buffer, and gently remove the comb. Add protein marker and sample, and run at 150V for 1 hour.

[0075] (4) Dyeing and bleaching

[0076] a. After electrophoresis, remove the gel and place it in a staining container. Rinse with water to remove the buffer on the surface of the gel to enhance the staining effect. Discard the water and add an appropriate amount of staining solution (Sanggong, Coomassie Brilliant Blue G-250, C526024) to cover the gel.

[0077] b. Place on a shaker and stain for 30-60 minutes. Remove the gel and add destaining solution (need to be prepared by yourself, 10% ethanol + 10% acetic acid) and place on a shaker to destain for 2 hours or overnight.

[0078] c. After decolorization, the bands can be observed.

[0079] The SDS-PAGE gel of the venom protein of the bright-bellied enameled wasp is shown in Figure 2 .from Figure 2 It can be seen that the protein electrophoresis bands of the venom of the bright-bellied enameled wasp are clear, with the maximum molecular weight at 72KD. The proteins with molecular weights between 8KD-17KD are the most abundant, followed by 25KD-33KD.

[0080] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for extracting protein from microparasitic wasp venom, characterized in that: The following steps are involved: After the glaucoma wasps were anesthetized, they were placed in a buffer containing protease inhibitors to separate the venom organs. The venom organ is subjected to low-temperature grinding and ultrasonic disruption, the disulfide bonds in the protein are reduced, and alkylation modification is performed. Then, a solvent is used to aggregate the protein to form a precipitate, and the micro-parasitic wasp venom protein is separated.

2. The extraction method according to claim 1, wherein The bright-bellied enameled wasp is a bright-bellied enameled wasp female that is within 7 days of emergence and has fully mated.

3. The extraction method according to claim 1, wherein The anesthesia treatment is to place the glaucoma wasp in a 4° C. environment and freeze it for 8-10 minutes.

4. The extraction method according to claim 1, wherein The buffer is phosphate buffer.

5. The extraction method according to claim 4, characterized in that The concentration of the protease inhibitor was 1 mM.

6. The extraction method according to claim 1, characterized in that The cryogenic grinding is to firstly freeze the venom organ with liquid nitrogen and then add protein lysis solution for grinding.

7. The extraction method according to claim 6, characterized in that The protein lysis solution comprises: 100 mM NH 4 HCO 3 , 6 M urea and 0.2 wt % sodium dodecyl sulfate, pH=8.

8. The extraction method according to claim 1, characterized in that The ultrasonic disruption treatment time is 5 min.

9. The extraction method according to claim 1, characterized in that Dithiothreitol is used to reduce disulfide bonds in proteins.

10. The extraction method according to claim 1, characterized in that Alkylation modification was performed using iodoacetamide.

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