A purple-bearded red-bellied sea anemone polypeptide toxin MD-384 and its preparation method and application

Abstract

The present invention provides a purple-bearded red-bellied sea anemone polypeptide toxin MD-384 and a preparation method and application thereof, wherein the amino acid sequence is shown in SEQ ID NO: 1. The present invention synthesizes a linear peptide MD-384 by using polypeptide solid phase synthesis (SPPS) and purifies the linear peptide, and then obtains an oxidized peptide MD-384 containing three disulfide bonds by site-directed oxidation, wherein the disulfide bonds are connected in a manner of C1-C6, C2-C4, and C3-C5, and three protecting groups, Trt, Acm and tBu, are used to protect cysteine ​​to ensure the correct connection of the disulfide bonds, and the product after oxidation and folding is subjected to liquid phase purification, high-performance liquid phase analysis and mass spectrometry identification to obtain the purple-bearded red-bellied sea anemone polypeptide toxin MD-384. The present invention tests the analgesic effect of the synthesized oxidized peptide MD-384 on zebrafish through behavioral trajectory experiments and the analgesic effect on mice through hot plate and writhing experiments. The present invention tests the blocking strength of the synthesized oxidized peptide MD-384 on Kv1.1-1.6 and Kv3.1 through cell patch clamp experiments. The purple-bearded red-bellied sea anemone polypeptide toxin MD-384 of the present invention can be used to prepare analgesic drugs.

Description

Technical Field

[0001] The invention belongs to the technical field related to polypeptides, and particularly relates to a purple-bearded red-bellied sea anemone polypeptide toxin MD-384 and a preparation method and application thereof. Background Art

[0002] As a member of the phylum Cnidaria, sea anemones are one of the oldest venomous animals in existence. They have rich pharmacological and biotechnological value and are known as a marine drug resource library, containing peptide neurotoxins with diverse functions. Marine biotoxins are one of the fastest-growing areas of research on marine active substances and a hot topic in the research of new marine drugs. Some marine biotoxins have been used as tools for neuroscience research or directly developed into new marine drugs. Sea anemones and the peptide toxins they secrete provide broad potential and prospects for the development of marine drugs.

[0003] Modern pharmacological studies have shown that the toxins secreted by sea anemones have a wide range of biological activities, including insecticidal, hypoglycemic, anti-tumor, antibacterial, antihypertensive, analgesic and central nervous system inhibitory effects.

[0004] Patent CN202311445953.4 discloses a purple-spotted sea anemone polypeptide toxin HC-K and its synthesis method and application. The DPPH free radical scavenging experiment shows that the sea anemone polypeptide toxin HC-K has good antioxidant activity.

[0005] Patent CN202310309321.9 discloses a mantle anemone polypeptide toxin Ed79-WR and its preparation method and application. The mantle anemone polypeptide toxin Ed79-WR disclosed therein can be used to prepare insecticides.

[0006] Based on the previous analysis of the transcriptome data of the purple-bearded red-bellied sea anemone, the target polypeptide sequence was screened from the widely studied ShK family, and the anemone functional polypeptide with three disulfide bonds was successfully synthesized by site-directed oxidation. Through targeted analgesic experiments, it was clarified that the anemone polypeptide has a certain basis for analgesic activity. This lays the foundation for the development of new analgesic drugs and new marine drugs, and provides valuable support for human health and marine drug research. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a new polypeptide toxin MD-384 of purple-bearded red-bellied sea anemone, and provide a synthesis method and application thereof.

[0008] The purple-bearded red-bellied sea anemone polypeptide toxin MD-384 prepared by the present invention has analgesic effect, laying a foundation for the development of new, efficient and safe biological analgesics. The present invention uses high-throughput transcriptome to discover the purple-bearded red-bellied sea anemone polypeptide toxin MD-384 from the purple-bearded red-bellied sea anemone (Macrodactyladoreensis), which has six cysteines and forms three disulfide bonds, and its amino acid sequence is as follows:

[0009] SEQ ID NO: 1: AVCAHFFPGLCPKLARGGYCRKGRRDRKARFAAKYCAWACGGC.

[0010] The present invention is achieved through the following technical solutions:

[0011] A purple-bearded red-bellied sea anemone polypeptide toxin MD-384 has six cysteines, forming three disulfide bonds. Its amino acid sequence is AVCAHFFPGLCPKLARGGYCRKGRRDRKARFAAKYCAWACGGC. The connection patterns of the disulfide bonds are C1-C6, C2-C4, and C3-C5. At the same time, three protecting groups, Trt, Acm and tBu, are used to protect the cysteine ​​to ensure the correct connection of the disulfide bonds.

[0012] The preparation method of the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone comprises the following steps:

[0013] (1) Weighing a resin, soaking it with DCM, then washing it with DMF and DCM in sequence, adding Fmoc-Ala-OH, DCM and DIEA to react, then adding methanol and DCM to react, washing it with DMF, then adding pyridine solution to react, and then washing it with DMF to obtain a first resin;

[0014] (2) adding a second amino acid Fmoc-Val-OH and a condensing agent to the first resin, then adding DMF and DIC to react, washing with DMF, then adding a pyridine solution to react, and then washing with DMF to obtain a second resin;

[0015] (3) According to the above amino acid sequence, repeat step (2) until the peptide chain is terminated to obtain a linear peptide resin;

[0016] (4) adding methanol to the linear peptide resin to rinse and drain, adding cutting solution to cut, filtering, adding icy ether, centrifuging and removing the icy ether in the upper layer to obtain a crude precipitated polypeptide;

[0017] (5) separating and purifying the crude polypeptide by HPLC to obtain a pure polypeptide, i.e., pure linear peptide MD-384;

[0018] (6) dissolving the linear peptide of step (5) in a methanol-water mixed solution, diluting with acetic acid, adding methanol iodine solution dropwise, and maintaining stirring to form the first disulfide bond;

[0019] (7) Add an equal volume of hydrochloric acid-methanol solution, then add methanol-iodine solution, keep stirring, and form a second dihydrogen iodine solution.

[0020] Sulfur bonds;

[0021] (8) separating and purifying by HPLC and then freeze-drying to obtain freeze-dried powder;

[0022] (9) The freeze-dried powder was dissolved in TFA / DMSO / anisole solution, stirred at room temperature, and then TFA / DMSO / anisole solution was added and stirred in a water bath;

[0023] (10) adding icy ether, removing the supernatant liquid icy ether after centrifugation to obtain a crude precipitated polypeptide;

[0024] (11) HPLC separation and purification and MS identification were used to obtain the pure polypeptide, i.e., the oxidized peptide MD-384;

[0025] Furthermore, in step (1), 0.6-1.0 mmol of amino acid Fmoc-Ala-OH, 10-15 mL of DCM and 1-3 mL of DIEA are added to react for 80-100 min, and then 3-5 mL of methanol and 8-12 mL of DCM are added to close the reaction for 25-35 min.

[0026] Furthermore, in step (1) and step (2), a DMF solution containing 20% ​​piperidine by mass is used to remove Fmoc for 15 to 25 minutes.

[0027] Furthermore, in step (2), 1.5-2.0 mmol of amino acid Fmoc-Val-OH and 1.5-2.0 mmol of condensing agent (HOBT) were weighed, 5-15 mL of DMF was added, and then 1-3 mL of DIC was added to react for 0.5-1.5 h.

[0028] Furthermore, in step (4), the cutting solution is 95% TFA, 1% H20, 2% EDT and 2% TIS, and the centrifugation is 4°C, 12000r / min, and 5min for 3 times.

[0029] Furthermore, in step (5), the crude polypeptide was separated and purified by HPLC, and the elution time of the linear peptide MD-384 was 27.42 min, thereby obtaining a pure polypeptide, namely, pure linear peptide MD-384.

[0030] Furthermore, in step (6), the methanol-water mixed solution is 50% methanol / 50% water, which is then diluted with acetic acid to a final concentration of 1 mg of peptide per ml of a mixed solution of 5% methanol / 5% water / 90% acetic acid, and the methanol-iodine solution is 10 mg / mL, and stirred for 1 min.

[0031] Furthermore, in step (7), the hydrochloric acid-methanol solution is 50mM HCL / 50% methanol, the methanolic iodine solution is 10mg / mL (10-20 times excess), and stirring is performed for 1h.

[0032] Furthermore, in step (8) and step (11), HPLC separation and purification mobile phase A (H, O), mobile phase B (ACN), flow rate 5 mL / min, linear gradient elution, detection wavelength 220 nm.

[0033] Furthermore, the gradient elution procedure is as follows:

[0034]

[0035] Furthermore, 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 of peptide was added with TFA / DMSO / anisole (97.9% / 2% / 0.1%) solution, and stirred in a 70°C water bath for 3 h.

[0036] Furthermore, in step (10), the centrifugation is performed three times at 4°C, 12000 r / min, and 5 min.

[0037] Furthermore, in step (11), the oxidized peptide was separated and purified by HPLC, and the elution time of the oxidized peptide MD-384 was 26.49 min, thereby obtaining a pure polypeptide, namely, pure oxidized peptide MD-384.

[0038] The present invention also proposes the use of the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone in the preparation of analgesic drugs. Further, the present invention also proposes the use of the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone in the preparation of analgesics for zebrafish and mice.

[0039] The Chinese names of the reagents or raw materials used in the present invention are as follows in Table 1:

[0040] Reagents Chinese name DCM Dichloromethane DIEA N,N-Diisopropylethylamine DIC N,N'-Diisopropylcarbodiimide TFA Trifluoroacetic acid EDT 1,2-Ethanedithiol TIS Triisopropylsilane ACN Acetonitrile DMSO Dimethyl sulfoxide HOB T(condensation agent) 1-Hydroxybenzotriazole Fmoc-Ala(tbu)-OH N-(9-Fluorenylmethoxycarbonyl)-O-tert-butyl-L-alanine Fmoc-Cys-OH N-(9-Fluorenylmethoxycarbonyl)-L-cysteine

[0041] Beneficial effects of the present invention:

[0042] 1. The present invention uses high-throughput transcriptome to discover the purple-bearded red-bellied sea anemone polypeptide toxin MD-384 from the purple-bearded red-bellied sea anemone (Macrodactyla doreensis), which has six cysteines that can form three disulfide bonds, and its amino acid sequence is: AVCAHFFPGLCPKLARGGYCRKGRRDRKARFAAKYCAWACGGC.

[0043] 2. The present invention synthesizes a linear peptide MD-384 by adopting a polypeptide solid phase synthesis method (SPPS), purifies the linear peptide by HPLC, and then uses a step-by-step method to oxidatively fold the synthesized linear peptide, and obtains an oxidized peptide MD-384 containing three disulfide bonds by site-specific oxidation of disulfide bonds. The connection mode of the disulfide bonds is C1-C6, C2-C4, and C3-C5. At the same time, three protecting groups, Trt, Acm and tBu, are used to protect cysteine ​​to ensure the correct connection of the disulfide bonds, and the product after oxidative folding is purified by HPLC and identified by mass spectrometry to obtain the purple-bearded red-bellied sea anemone polypeptide toxin MD-384, that is, the oxidized peptide MD-384.

[0044] 3. The polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone prepared by the method of the present invention has analgesic effect, laying a foundation for the research and development of new, efficient and safe biological analgesics. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of HPLC analysis of linear peptide MD-384.

[0046] Figure 2 Schematic diagram of mass spectrometry identification of linear peptide MD-384.

[0047] Figure 3 Schematic diagram of HPLC analysis of oxidized peptide MD-384.

[0048] Figure 4 Schematic diagram of mass spectrometry identification of oxidized peptide MD-384.

[0049] Figure 5 Schematic diagram of the analgesic effect of oxidized peptide MD-384 on zebrafish. Note: the experimental group was compared with the blank control group (Blank control), and the experimental group was compared with the negative control group (Negative control). * indicates significant difference (*p < 0.05; **p < 0.01; ***p < 0.001).

[0050] Figure 6Schematic diagram of the analgesic effect of the oxidized peptide MD-384 on the hot plate test in mice. Note: the experimental group was compared with the blank control group (Blank control), and the experimental group was compared with the negative control group (Negative control). * indicates significant differences (*p < 0.05; **p < 0.01; ***p < 0.001).

[0051] Figure 7 Schematic diagram of the analgesic effect of the oxidized peptide MD-384 on the writhing experiment in mice. Note: the experimental group was compared with the blank control group (Blank control), and the experimental group was compared with the negative control group (Negative control). * indicates significant differences (*p<0.05; **p<0.01; ***p<0.001).

[0052] Figure 8 This is a diagram showing the blocking effect of the oxidized peptide MD-384 on Kv1.1-1.6 and Kv3.1 ion channels at a concentration of 10 μM. DETAILED DESCRIPTION

[0053] The technical solution of the present invention is further described below in conjunction with the accompanying drawings, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be included in the protection scope of the present invention.

[0054] Example 1

[0055] In this example, a new purple-bearded red-bellied sea anemone polypeptide toxin MD-384 was discovered from the purple-bearded red-bellied sea anemone (Macrodactyla doreensis) using high-throughput transcriptomics. The toxin has six cysteines that can form three disulfide bonds. Its amino acid sequence is AVCAHFFPGLCPKLARGGYCRKGRRDRKARFAAKYCAWACGGC, and the disulfide bond patterns are C1-C6, C2-C4, and C3-C5. At the same time, three protecting groups, Trt, Acm, and tBu, are used to protect the cysteine ​​to ensure the correct connection of the disulfide bonds.

[0056] Example 2

[0057] This embodiment provides a method for synthesizing the above-mentioned purple-bearded red-bellied sea anemone polypeptide toxin MD-384, and the method comprises the following steps:

[0058] 1. Materials and methods

[0059] 1.1 Experimental Materials

[0060] Chromatographic grade trifluoroacetic acid (TFA) and chromatographic grade acetonitrile (ACN) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; water analytical C18 column (5 μm, 4.6 mm×250 mm) was purchased from Waters Company, USA; Elite preparative C18 column (10 μm, 10 mm×250 mm) was purchased from Dalian Elite Analytical Instrument Co., Ltd.

[0061] 1.2 Experimental instruments

[0062] CEM fully automatic microwave peptide synthesizer (LibertyBlue, USA); reversed-phase high performance liquid chromatography (ThermoFisher, Germany); triple quadrupole liquid chromatography-mass spectrometry (Shimadzu, Japan); desktop freeze dryer (Saifei, China)

[0063] 1.3 Experimental methods

[0064] 1.3.1 The preparation method of the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone comprises the following steps:

[0065] (1) Weighing a 2-cl resin with a degree of substitution Sd = 0.2-1.4 mmol / g, soaking it with DCM, then washing it with DMF and DCM in sequence, adding 0.6-1.0 mmol Fmoc-Val-OH, 10-15 mL DCM and 1-3 mL DIEA to react for 80-100 min, then adding 3-5 mL methanol and 8-12 mL DCM to block the reaction for 25-35 min, washing it with DMF, and then adding a DMF solution with a piperidine mass fraction of 20% to wash it for 15-25 min to obtain a first resin;

[0066] Specifically, in this embodiment, a 2-cl resin with a substitution degree Sd=0.4mmol / g is used, first soaked with DCM, then washed with DMF and DCM in sequence, 0.8mmolFmoc-Ala-OH, 12mL DCM and 2mL DIEA are added to react for 90min, then 4mL methanol and 10mL DCM are added, the reaction is closed for 30min, washed with DMF, and then a DMF solution with a piperidine mass fraction of 20% is added to wash for 20min to obtain the first resin; in other embodiments, adjustments within the scope of the present invention can achieve the purpose of the present invention;

[0067] (2) adding 1.5-2.0 mmol of amino acid (Fmoc-Ala-OH) and 1.5-2.0 mmol of condensing agent (HOBT) to the first resin, adding 5-15 mL of DMF, and then adding 1-3 mL of DIC to react for 0.5-1.5 h, and then adding a DMF solution containing 20% ​​by mass of piperidine to wash for 15-25 min to obtain a second resin;

[0068] Specifically, in this embodiment, 1.8 mmol of amino acid (Fmoc-Val-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, and then a DMF solution containing 20% ​​of piperidine was added to wash for 20 min to obtain a second resin;

[0069] In other embodiments, adjustments within the scope of the present invention can achieve the purpose of the present invention;

[0070] (3) According to the above amino acid sequence, repeat step (2) until the peptide chain is terminated to obtain a linear peptide resin;

[0071] (4) Add methanol to the linear peptide resin for washing and draining, and add cutting solution (95% TFA, 1% H 2 O, 2% EDT and 2% TIS), filtered, added with icy ether, centrifuged at 4°C and 12000r / min for 5min, repeated centrifugation 3 times, and then removed the icy ether in the upper layer to obtain the crude precipitated polypeptide;

[0072] (5) Separate and purify the crude polypeptide by HPLC, using mobile phase A (H 2 O), mobile phase B (ACN), flow rate 5 mL / min, linear gradient elution, elution program as shown in Table 2, detection wavelength 220 nm, to obtain pure polypeptide, i.e. pure linear peptide MD-384;

[0073] (6) Dissolve the linear peptide from step (5) in a mixed solution of methanol and water in a volume ratio of 1:1, and then dilute with acetic acid to make the final concentration of the peptide solution 1 mg of peptide per ml of the mixed solution (5% methanol + 5% water + 90% acetic acid), and add 10 mg / ml methanol iodine solution (drop the methanol iodine solution drop by drop, and stop dropping once a light yellow color appears and the light yellow color does not fade; about 10 μL of methanol iodine solution is needed for each milligram of peptide). Keep stirring for 1 min to form the first disulfide bond;

[0074] (7) Add an equal volume (i.e., the total volume equal to that in step 6) of 50 mM HCl / 50% methanol hydrochloric acid-methanol solution, and then add 10 mg / mL methanol iodine solution (approximately 0.2 mL of methanol iodine solution is required for each mg of peptide), and keep stirring for 1 h to form a second disulfide bond;

[0075] (8) Separation and purification by HPLC, mobile phase A (H, O), mobile phase B (ACN), flow rate 5 mL / min, linear gradient elution, elution program as shown in Table 2, detection wavelength 220 nm, and then freeze-dried;

[0076] (9) The lyophilized powder was dissolved in 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, TFA / DMSO / anisole (v / v / v = 97.9% / 2% / 0.1%) solution was added at 40 μL / mg of peptide and stirred in a 70°C water bath for 3 h.

[0077] (10) Add icy ether, centrifuge at 4°C and 12000 r / min for 5 min, repeat the centrifugation three times, and then remove the icy ether in the supernatant to obtain the crude precipitated polypeptide;

[0078] (11) HPLC was used for separation and purification with mobile phase A (H, O) and mobile phase B (ACN) at a flow rate of 5 mL / min and linear gradient elution. The elution program was as shown in Table 2. The detection wavelength was 220 nm, and then MS was used for identification to obtain a pure polypeptide, namely, oxidized peptide MD-384.

[0079] Table 2 Gradient elution program

[0080]

[0081] 2 Results

[0082] 2.1 Synthesis and oxidative folding of peptides

[0083] The linear peptide MD-384 from the purple-bearded red-bellied sea anemone was synthesized by solid phase peptide synthesis (SPPS). The linear peptide was purified by HPLC and its molecular weight was 4977.80Da by mass spectrometry (see the results). Figure 2 The synthesized linear peptide was subjected to one-step oxidation folding by site-directed oxidation method, and the final oxidation folding product was purified by HPLC and identified by mass spectrometry. The actual molecular weight of the linear peptide MD-384 was 4978.08.01Da, and the molecular weight of the oxidized peptide MD-384 was 4715.04Da (see the results). Figure 4 ), the difference between the two is about 260Da, proving that three disulfide bonds are formed correctly.

[0084] 2.2 Isolation and purification of oxidized peptides

[0085] The linear peptide MD-384 was analyzed by analytical HPLC. Figure 1As shown in FIG. 1 , the elution time of the linear peptide MD-384 is 27.42 min. The MD-384 after three-step oxidation was separated and purified by preparative HPLC, and then analyzed by analytical HPLC. The results are shown in FIG. Figure 3 As shown, the elution time of the oxidized peptide MD-384 was 26.49 min.

[0086] In this example, a three-step oxidation folding method was performed on the synthesized linear peptide MD-384 sample by a site-directed oxidation method, and the final oxidation folding product was purified by HPLC and identified by mass spectrometry to obtain the linear peptide MD-384 of the purple-bearded red-bellied sea anemone, i.e., the oxidized peptide MD-384, whose sequence is AVCAHFFPGLCPKLARGGYCRKGRRDRKARFAAKYCAWACGGC, with 6 cysteines forming 3 disulfide bonds, and the disulfide bond patterns are C1-C6, C2-C4, and C3-C5. At the same time, three protecting groups, Trt, Acm, and tBu, were used to protect the cysteine ​​to ensure the correct connection of the disulfide bonds.

[0087] Example 3 This example provides an application experiment of the above-mentioned purple beard red belly sea anemone polypeptide toxin MD-384, specifically an analgesic experiment

[0088] 1. Analgesia Experiment

[0089] The zebrafish larvae were selected for analgesia experiment, referring to the existing method as follows:

[0090] The polypeptide toxin MD-384 obtained in this example, namely the oxidized peptide MD-384, was dissolved in 0.7% NaCl to 19.5nM, 39nM, 78nM, 156nM and 312nM, and zebrafish fry were added to a 24-well plate, with six wells in a row for each concentration, and 1 mL of the drug solution was added to each well. Pure water was used in a row of six wells, with 1 mL in each well as a blank control group.

[0091] 2. Data processing

[0092] The data were statistically analyzed and processed using GraphPad Prism 7 software. The data between the control group and the experimental group were analyzed using t-test. * indicates significant difference (p<0.05), ** indicates extremely significant difference (p<0.01), *** indicates extremely significant difference (p<0.001), and so on.

[0093] 3 Analgesia Experiment Results

[0094] The experimental results are as follows Figure 5As shown in the figure, the total swimming distance of zebrafish larvae in each experimental group compared with the blank control group was significantly reduced, indicating that it is feasible to use the zebrafish behavioral trajectory experiment to evaluate the analgesic effect of the oxidized peptide MD-384. The total swimming distance of zebrafish larvae decreased with the increase of the dose of the oxidized peptide MD-384, and there was a significant difference compared with the control group. The low dose of 19.5nM oxidized peptide MD-384 had a significant analgesic effect on zebrafish larvae, and the total swimming distance was reduced by 26.65%. The high dose of 320nM oxidized peptide MD-384 reduced the total swimming distance of zebrafish larvae by 61.34%. The total swimming distance of zebrafish larvae decreased with the increase of the oxidized peptide dose, and the half effective concentration calculated by the software was 97.59nM.

[0095] Example 4

[0096] 1. Analgesia Experiment

[0097] 42 C57 female mice weighing 20±2g were selected and randomly divided into 7 groups, 6 mice / group for hot plate analgesia experiment, referring to the existing method as follows:

[0098] The polypeptide toxin MD-384 obtained in this example, namely the oxidized peptide MD-384, was dissolved in 0.625mM, 1.25mM, 2.5mM, 5mM and 10mM with 0.7% NaCl and injected into the mouse by intraperitoneal injection, about 0.1ml each time, and the pain threshold was tested every 30 minutes using a 55°C hot plate test device. Analgesic dose of tramadol was used as a positive control group, and saline was used as a negative control group.

[0099] 2. Data processing

[0100] The data were statistically analyzed and processed using GraphPad Prism 7 software. The data between the control group and the experimental group were analyzed using t-test. * indicates significant difference (p<0.05), ** indicates extremely significant difference (p<0.01), *** indicates extremely significant difference (p<0.001), and so on.

[0101] 3 Analgesia Experiment Results

[0102] The experimental results are as follows Figure 6As shown, the pain threshold of mice in each experimental group increased compared with the negative control group, indicating that it is feasible to use the mouse hot plate analgesia experiment to evaluate the analgesic effect of oxidized peptide MD-384. The pain threshold of mice increased with the increase of the dose of oxidized peptide MD-384, and there were significant differences compared with the control group except for the 0.625mM group. The second lowest dose of 1.25mM oxidized peptide MD-384 has a significant effect on the pain threshold of mice, with a pain threshold increase rate of 61.41%. The high dose of 10mM oxidized peptide MD-384 has a pain threshold increase rate of 246.71%. The pain threshold of mice increases with the increase of the dose of oxidized peptide, and the half effective concentration calculated by the software is 2.848mM.

[0103] Example 5

[0104] 1. Analgesia Experiment

[0105] C57 mice weighing 20±2g were selected, and 21 male and female mice were randomly divided into 7 groups, with 6 mice in each group, for writhing analgesia experiments, referring to the existing method as follows:

[0106] The polypeptide toxin MD-384 obtained in this example, namely the oxidized peptide MD-384, was dissolved in 0.625mM, 1.25mM, 2.5mM, 5mM and 10mM with 0.7% NaCl, and injected into the mice by intraperitoneal injection, about 0.1ml each time, and 1% acetic acid solution was injected intraperitoneally 20 minutes later, and the number of twisting reactions within 20 minutes was observed. The analgesic dose of tramadol was used as the positive control group, and the normal saline was used as the negative control group.

[0107] 2. Data processing

[0108] The data were statistically analyzed and processed using GraphPad Prism 7 software. The data between the control group and the experimental group were analyzed using t-test. * indicates significant difference (p<0.05), ** indicates extremely significant difference (p<0.01), *** indicates extremely significant difference (p<0.001), and so on.

[0109] 3 Analgesia Experiment Results

[0110] The experimental results are as follows Figure 7As shown, the number of writhing of mice in each experimental group compared with the negative control group was significantly reduced, indicating that it is feasible to use the mouse writhing experiment to evaluate the analgesic effect of the oxidized peptide MD-384. The number of writhing of mice decreased with the increase of the dose of the oxidized peptide MD-384, and there was a significant difference compared with the control group. The low dose of 0.625mM oxidized peptide MD-384 had a significant analgesic effect on mice, and the number of writhing was reduced by 44.78%. The high dose of 10mM oxidized peptide MD-384 reduced the writhing by nearly 100%. The number of writhing of mice decreased with the increase of the dose of oxidized peptide. The half effective concentration calculated by the software was 1.107mM.

[0111] Example 6

[0112] This example provides a potassium channel selectivity screening experiment of the above-mentioned purple beard red belly sea anemone polypeptide toxin MD-384

[0113] 1. Cell patch clamp experiment

[0114] The test concentration was 10 μM, and seven ion channels, Kv1.1-1.6 and Kv3.1, were screened. All experiments were performed at room temperature. Each cell was used as a control. The compounds were perfused using a perfusion system that used their own gravity. At least one cell was tested for each concentration. After the current stabilized, the change in current size before and after the compound was used was compared to calculate the blocking effect of the compound. The cells were transferred to the perfusion tank and perfused with extracellular solution. The intracellular solution (mM) was: Aspartic acid, 140; MgCl2, 2; EGTA11; HEPES, 10; pH7.2 (CsOH titration). The intracellular solution was stored in a small amount in batches at -80 degrees and thawed on the day of the experiment. The electrode was pulled with PC-10 (Narishige, Japan). Whole-cell patch clamp recording, noise was filtered at one-fifth of the sampling frequency. The cells were clamped at -80 mV and then depolarized to -10 mV with a 10 ms square wave to obtain potassium channel currents. This procedure was repeated every 5 seconds. The maximum current induced by the square wave was measured and after it stabilized, the test compound was perfused. When the response stabilized, the intensity of the blockade was calculated.

[0115] 2. Data processing

[0116] The data will be stored in a computer system for analysis. Data collection and analysis will be performed using pCLAMP 10 (Molecular Devices, Union City, CA), and the management will review the analysis results. Current stability refers to the change of current over time within a limited range. The magnitude of the current after stability is used to calculate the effect of the compound at this solubility.

[0117] 3. Results Analysis

[0118] The results are as follows Figure 8 As shown, the blocking effects of MD-384 at a concentration of 10 μM on steady-state expressed human potassium channel cells (Kv1.1-1.6 and Kv3.1) were 87.99%, 68.84%, 69.21%, 20.48%, 8.72%, 91.71% and 1.02%, respectively.

[0119] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A polypeptide toxin MD-384 from Macrodactyla doreensis, characterized in that: The amino acid sequence of the primary structure is shown in SEQ ID NO: 1: AVCAHFFPGLCPKLARGGYCRKGRRDRKARFAAKYCAWACGGC, with six cysteine ​​residues; the disulfide bond linkage is C1-C6, C2-C4, and C3-C5.

2. The method for preparing the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone according to claim 1, characterized in that: The steps include: (1) Weighing a resin, soaking it with DCM, then washing it with DMF and DCM in sequence, adding Fmoc-Ala-OH, DCM and DIEA to react, then adding methanol and DCM to react, washing it with DMF, then adding a pyridine solution to react, and then washing it with a piperidine-DMF solution to obtain a first resin; (2) adding a second amino acid Fmoc-Val-OH and 1-hydroxybenzotriazole to the first resin, then adding DMF and DIC to react, washing with DMF, then adding a pyridine solution to react, and then washing with a piperidine-DMF solution to obtain a second resin; (3) according to the amino acid sequence shown in SEQ ID NO: 1, repeat step (2) until the peptide chain is complete to obtain a linear peptide resin; (4) adding methanol to the linear peptide resin of step (3) to rinse and drain, adding cutting solution to cut, filtering, adding ice ether, centrifuging and removing the upper ether liquid to obtain a crude precipitated polypeptide; (5) separating and purifying the crude polypeptide in step (4) by HPLC to obtain pure linear peptide MD-384; (6) dissolving the linear peptide obtained in step (5) in a methanol-water mixed solution, diluting with acetic acid to obtain an acetic acid mixed solution, adding methanol iodine solution dropwise, and maintaining stirring to form the first disulfide bond; (7) Add hydrochloric acid-methanol solution, then add methanol-iodine solution, keep stirring, to form a second disulfide bond; (8) separating and purifying by HPLC and then freeze-drying to obtain freeze-dried powder; (9) dissolving the freeze-dried powder in a TFA-DMSO-anisole mixture, stirring at room temperature, then adding the TFA-DMSO-anisole mixture, stirring in a water bath to form a third disulfide bond; (10) adding icy ether, removing the supernatant ether after centrifugation to obtain a crude precipitated oxidized polypeptide; (11) The crude polypeptide obtained in step (10) is separated and purified by HPLC to obtain pure oxidized peptide MD-384.

3. The method for preparing the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone according to claim 2, characterized in that: In the reaction system of step (1), the degree of substitution of the resin is Sd=0.2-1.4 mmol / g, 0.6-1.0 mmol of Fmoc-Ala-OH, 10-15 mL of DCM and 1-3 mL of DIEA are added in proportion to react for 80-100 min, and then 3-5 mL of methanol and 8-12 mL of DCM are added to react for 25-35 min.

4. The method for preparing the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone according to claim 2, characterized in that: In step (2), in the reaction system, 1.5-2.0 mmol of amino acid Fmoc-Val-0H and 1.5-2.0 mmol of 1-hydroxybenzotriazole are added proportionally to the first resin obtained in step (1), 5-15 mL of DMF is poured into it, and then 1-3 mL of DIC is added to react for 0.5-1.5 h.

5. The method for preparing the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone according to claim 2, characterized in that: In step (4), the cutting fluid is prepared by 95% TFA, 1% H20, 2% EDT and 2% TIS by mass percentage; in step (4) and step (10), the centrifugation conditions are centrifugation at 3-5°C and 11000-13000 r / min for 4-6 min, and the centrifugation is repeated 2-4 times.

6. The method for preparing the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone according to claim 2, characterized in that: In step (6), the volume ratio of methanol to water in the methanol-water mixed solution is 1:0.8-1.2, the volume ratio of acetic acid to the methanol-water mixed solution is 8.5-9.5:1, and each milliliter of the acetic acid mixed solution contains 0.8-1.2 mg of peptide; the concentration of the methanol iodine solution is 9-11 mg / mL, 9-11 μL of methanol iodine solution is added dropwise for each milligram of peptide, and the stirring time is 0.5-2 min.

7. The method for preparing the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone according to claim 2, characterized in that: In step (7), the hydrochloric acid-methanol solution is prepared from 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 methanol-iodine solution is added per mg of peptide.

8. The method for preparing the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone according to claim 2, characterized in that: In step (5), step (8) and step (11), the HPLC separation and purification mobile phase A is H20, the mobile phase B is ACN, the flow rate is 4-6 mL / min, and the detection wavelength is 220 nm.

9. The method for preparing the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone according to claim 2, characterized in that: 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 TFA-DMSO-anisole solution is added per mg of freeze-dried powder before stirring at room temperature, 35-45 μL of TFA-DMSO-anisole solution is added after stirring at room temperature, and the stirring time at room temperature is 35-45 min; the stirring temperature of the water bath is 65-75 ° C, and the stirring time is 2-4 h.

10. Use of the polypeptide toxin MD-384 of the purple-bearded red-bellied sea anemone according to claim 1 in the preparation of analgesic drugs.

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