A ptilomyces porphyria polypeptide toxin HC-G02 and a synthesis method and application thereof

Abstract

The present application provides a kind of purple spot sea anemone polypeptide toxin HC-G02 and its synthesis method, application, its amino acid sequence is APCSGCYYQVGNECVYDKLKC-NH2. Linear peptide HC-G02 is synthesized by adopting polypeptide solid-phase synthesis method, and the purity of linear peptide after purification reaches more than 95%. Purple spot sea anemone oxidized peptide HC-G02 (the connection mode of disulfide bond is C1-C3, C2-C4) is obtained by site-directed oxidation. The present application is found through hot plate experiment test that purple spot sea anemone oxidized peptide HC-G02 has good analgesic effect on physical pain of mouse, and the pain threshold of mouse intraperitoneal injection oxidized peptide HC-G02 is gradually increased in 30min, and the pain threshold reaches the highest in 120min after administration. The analgesic effect of oxidized peptide HC-G02 at 50mg / kg is equivalent to the analgesic effect of tramadol at the same concentration, and the analgesic time is prolonged.

Description

Technical Field

[0001] This invention belongs to the field of active polypeptide technology, and relates to a purple-spotted sea anemone polypeptide toxin HC-G02, its synthesis method, and its application. Background Technology

[0002] Sea anemones belong to the phylum Cnidaria, class Anthozoa, and order Anemoneales. They are marine animals found in both deep and shallow waters worldwide. Sea anemones are among the oldest extant orders of venomous animals, with molecular and fossil data indicating their origins predating the Ediacaran period, 750 million years ago. There is a great diversity of sea anemones, with over 1,100 species recorded globally, belonging to approximately 400 genera and 50 families. China accounts for about one-tenth of the world's sea anemone species, with the highest species diversity found in the South China Sea. Sea anemones possess stinging cells on their tentacles. Like other Cnidaria, they concentrate venom in sac-like organelles called nematocysts. Upon contact with prey, the nematocysts penetrate the target organism, releasing venom to paralyze or kill it, thus enabling predation, defense, and deterrence of competitors.

[0003] According to the "Chinese Materia Medica" and "Chinese Medicinal Animal Records," sea anemones have astringent and consolidating effects, and are mainly used in folk medicine to treat hemorrhoids, rectal prolapse, pinworm infection, and tinea corporis. Modern pharmacological studies have shown that sea anemone toxins have analgesic effects. Summary of the Invention

[0004] This invention screened active polypeptide sequences from purple-spotted sea anemones and synthesized functional polypeptides from these anemones through oxidative folding. Analgesic experiments demonstrated that these anemone polypeptides possess certain analgesic activity. This invention lays the foundation for the development of novel marine drugs and provides strong support for human health and marine drug research.

[0005] The solution of this invention is as follows:

[0006] A purple-spotted sea anemone polypeptide toxin HC-G02 has the amino acid sequence APCSGCYYQVGNECVYDKLKC-NH2, which has four cysteine ​​residues forming two disulfide bonds, with the disulfide bonds linked in a C1-C3, C2-C4 configuration.

[0007] This invention also provides a method for synthesizing the purple-spotted sea anemone polypeptide toxin HC-G02, comprising the following steps:

[0008] (1) Weigh the resin, soak it in dichloromethane first, then wash it with dimethylformamide and dichloromethane in sequence, add Fmoc-Ala-OH, dichloromethane and N,N-diisopropylethylamine to react, then add methanol and dichloromethane to react, wash it with dimethylformamide, and then add a dimethylformamide solution containing pyridine to wash it to obtain the first resin;

[0009] (2) Add the second amino acid Fmoc-Pro-OH and a condensing agent to the first resin, then add dimethylformamide and N,N-diisopropylcarbodiimide to react, then add a dimethylformamide solution containing piperidine to wash, and obtain the second resin;

[0010] (3) Based on the aforementioned amino acid sequence of HC-G02, repeat step (2) until the peptide chain ends to obtain linear peptide resin.

[0011] (4) Add methanol to the linear peptide resin for rinsing and then dry it. Add cutting fluid for cutting, filter, add ice-cold ether, centrifuge to remove the upper layer of ice-cold ether, and obtain the crude precipitated peptide.

[0012] (5) Purify the crude polypeptide to obtain pure linear peptide HC-G02;

[0013] (6) Dissolve the linear peptide from step (5) in an aqueous methanol solution, then dilute with acetic acid, add methanol-iodine solution dropwise, and keep stirring to form the first disulfide bond;

[0014] (7) Add an equal volume of hydrochloric acid-methanol solution, then add methanol-iodine solution, and keep stirring to form a second disulfide bond;

[0015] (8) After purification, the pure polypeptide product is obtained.

[0016] Further, step (1) is as follows: weigh the resin, soak it in dichloromethane first, then wash it with dimethylformamide and dichloromethane in sequence, add 0.6-1.0 mmol Fmoc-Ala-OH, 10-15 mL dichloromethane and 1-3 mL N,N-diisopropylethylamine and react for 80-100 min, then add 3-5 mL methanol and 8-12 mL dichloromethane to block the reaction for 25-35 min, wash it with dimethylformamide, and then wash it with a dimethylformamide solution containing pyridine for 15-25 min to obtain the first resin; wherein, the dimethylformamide solution containing piperidine is a DMF solution with a piperidine mass fraction of 20%.

[0017] Further, step (2) is as follows: 1.5-2.0 mmol of the second amino acid Fmoc-Pro-OH and 1.5-2.0 mmol of condensing agent are added to the first resin, followed by 5-15 mL of dimethylformamide and 1-3 mL of N,N-diisopropylcarbodiimide. The mixture is reacted for 0.5-1.5 h, and then washed with a dimethylformamide solution containing piperidine for 15-25 min to obtain the second resin; wherein the dimethylformamide solution containing piperidine is a DMF solution with a piperidine mass fraction of 20%.

[0018] Furthermore, the cutting fluid contains: 95 wt% trifluoroacetic acid, 1 wt% H2O, 2 wt% 1,2-ethylenedithiol and 2 wt% triisopropylsilane.

[0019] Further, step (6) is as follows: dissolve the linear peptide from step (5) in a 50 v / v% methanol aqueous solution, then dilute it with acetic acid to make the final concentration of the peptide solution 1 mg of peptide per milliliter, add 10 mg / mL methanol-iodine solution dropwise, and keep stirring to form the first disulfide bond.

[0020] Further, step (7) is: add an equal volume of hydrochloric acid-methanol solution, then add 10 mg / mL methanol-iodine solution, keep stirring, and form a second disulfide bond;

[0021] Furthermore, in step (8), high performance liquid chromatography is used for purification. The purification conditions are: mobile phase A is H2O, mobile phase B is acetonitrile, flow rate is 5 mL / min, linear gradient elution is performed for 45 min, and the volume percentage of mobile phase B increases linearly from 5% to 50%.

[0022] The present invention also proposes the application of the purple-spotted sea anemone polypeptide toxin HC-G02 in the preparation of analgesics.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. This invention utilizes high-throughput transcriptomics to discover the anemone polypeptide toxin HC-G02 from Heteractis crispa. Its amino acid sequence is APCSGCYYQVGNECVYDKLKC-NH2, which has four cysteine ​​residues that can form two disulfide bonds.

[0025] 2. In this invention, a linear peptide HC-G02 was synthesized by solid-phase peptide synthesis (SPPS). The linear peptide was purified by HPLC, and then the synthesized linear peptide was oxidatively folded by a stepwise method. After site-directed oxidation by disulfide bonds, an oxidized peptide HC-G02 containing two disulfide bonds was obtained. The disulfide bonds were connected in the form of C1-C3 and C2-C4.

[0026] 3. This invention provides a method for synthesizing Heteractis crispa polypeptide toxin HC-G02 and its analgesic effect. The Heteractis crispa polypeptide toxin HC-G02 synthesized by this method has a highly effective analgesic effect, which can lay the foundation for the development of new, efficient and safe biological analgesics.

[0027] 4. This invention, through hot plate experiments, revealed that the purple-spotted sea anemone oxidized peptide HC-G02 has a good analgesic effect on physical pain in mice. After intraperitoneal injection of HC-G02 into mice, the rate of increase in pain threshold gradually increased over 30 minutes, reaching its highest point at 120 minutes post-administration. The analgesic effect of HC-G02 at 50 mg / kg is comparable to that of tramadol at the same concentration, with a prolonged analgesic duration. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of HPLC analysis of the linear peptide HC-G02.

[0029] Figure 2 This is a schematic diagram of mass spectrometry identification of the linear peptide HC-G02.

[0030] Figure 3 This is a schematic diagram of the oxidized peptides after the first step of HPLC analysis of HC-G02.

[0031] Figure 4 This is a schematic diagram of mass spectrometry identification of oxidized peptides after the first step of oxidation by HC-G02.

[0032] Figure 5 This is a schematic diagram of the oxidized peptides after the second step of HC-G02 oxidation, as analyzed by HPLC.

[0033] Figure 6 This is a schematic diagram of mass spectrometry identification of oxidized peptides after the second step of oxidation by HC-G02.

[0034] Figure 7 This is a schematic diagram of the analgesic effect of HC-G02 oxidative peptide on mice. Note: The experimental group was compared with the negative control group (PBS) and the experimental group was compared with the positive control group (tramadol). * indicates a significant difference (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical content of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0036] This invention utilizes high-throughput transcriptomics to discover the polypeptide toxin HC-G02 from the purple-spotted sea anemone (Heteractis crispa). Its amino acid sequence is APCSGCYYQVGNECVYDKLKC-NH2, and the disulfide bond linkage is C1-C3, C2-C4.

[0037] Experimental Example: Synthesis of Purple Spotted Sea Anemone Polypeptide Toxin HC-G02 and Verification of its Analgesic Effect

[0038] 1. Materials and Methods

[0039] 1.1 Experimental Materials

[0040] Chromatographic grade trifluoroacetic acid (TFA) and chromatographic grade acetonitrile (ACN) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; a water analytical C18 column (5 μm, 4.6 mm × 250 mm) was purchased from Waters Corporation, USA; and an Elite preparative C18 column (10 μm, 10 mm × 250 mm) was purchased from Dalian Elite Analytical Instruments Co., Ltd.

[0041] 1.2 Experimental Apparatus

[0042] CEM fully automated microwave peptide synthesizer (LibertyBlue, USA); reversed-phase high-performance liquid chromatography (ThermoFisher, Germany); triple quadrupole liquid chromatography-mass spectrometry (Shimadzu, Japan); benchtop freeze dryer (Saif, China); intelligent hot plate apparatus (Zhenghua, China).

[0043] 1.3 Experimental Methods

[0044] 1.3.1 The synthesis method of purple-spotted sea anemone polypeptide toxin HC-G02 includes the following steps:

[0045] (1) Weigh the resin (2-cl resin with a Sd of 0.8 mmol / g), soak it in DCM (dichloromethane) first, then wash it with DMF (dimethylformamide) and DCM in sequence, add 0.8 mmol amino acid (Fmoc-Ala-OH), 12.5 mL DCM and 2 mL DIEA (N,N-diisopropylethylamine) and react for 90 min, then add 4 mL methanol and 10 mL DCM, block the reaction for 30 min, wash with DMF, and then wash with DMF solution with a piperidine mass fraction of 20% for 20 min to obtain the first resin;

[0046] (2) Add 1.8 mmol of amino acid (Fmoc-Pro-OH) and 1.8 mmol of condensing agent (HOBT) to a resin, add 10 mL of DMF, then add 2 mL of DIC (N,N-diisopropylcarbodiimide) and react for 1 h. Then add DMF solution containing 20% ​​piperidine by mass and wash for 10 min to obtain a second resin.

[0047] (3) Based on the amino acid sequence of HC-G02, repeat step (2) until the peptide chain ends to obtain linear peptide resin;

[0048] (4) Add methanol to the linear peptide resin for rinsing and then dry it. Add cutting fluid (95wt% TFA, 1wt% H2O, 2wt% EDT and 2wt% TIS) for cutting. After filtration, add ice ether and centrifuge three times at 4℃, 12000r / min and 5min to remove the upper layer of ice ether and obtain the crude precipitated peptide.

[0049] (5) The crude peptide was separated and purified by HPLC to obtain the pure peptide, namely the pure linear peptide HC-G02 (purity of 95% or more); the HPLC conditions were the same as in step (8).

[0050] (6) Dissolve the linear peptide from step (5) in a 50 v / v% methanol aqueous solution, then dilute with acetic acid to make the final peptide concentration so that each milliliter of the mixed solution (5 v / v% methanol + 5 v / v% water + 90 v / v% acetic acid) contains 1 mg of peptide. Add 10 mg / mL methanol-iodine solution until the solution turns pale yellow, and keep stirring for 1 min to form the first disulfide bond.

[0051] (7) Add an equal volume (i.e., the same as the total volume in step 6) of hydrochloric acid-methanol solution (i.e., methanol containing 50 mM HCl), then add a 10 mg / mL methanol-iodine solution (the amount added is 10 times the amount of methanol-iodine solution added in step 6), and keep stirring for 1 hour to form a second disulfide bond.

[0052] (8) The peptide was separated and purified by HPLC. Mobile phase A was H2O and mobile phase B was ACN (acetonitrile). The flow rate was 5 mL / min and the linear gradient elution was carried out for 45 min at 5%-50% (volume of phase B). The detection wavelength was 220 nm. The peptide was then identified by MS and freeze-dried to obtain the pure peptide, namely HC-GO2.

[0053] Furthermore, in other embodiments of the present invention, the synthesis method of the purple-spotted sea anemone polypeptide toxin HC-G02 can be implemented within the following ranges, all of which yield similar results. The synthesis method includes the following steps:

[0054] (1) Weigh the resin (2-cl resin with a Sd value of 0.2-1.4 mmol / g), soak it in DCM (dichloromethane) first, then wash it with DMF (dimethylformamide) and DCM in sequence, add 0.6-1.0 mmol of amino acid (Fmoc-Ala-OH), 10-15 mL of DCM and 1-3 mL of DIEA (N,N-diisopropylethylamine) and react for 80-100 min, then add 3-5 mL of methanol and 8-12 mL of DCM, block the reaction for 25-35 min, wash with DMF, and then wash with DMF solution with a piperidine mass fraction of 20% for 15-25 min to obtain the first resin;

[0055] (2) Add 1.5-2.0 mmol of amino acid (Fmoc-Pro-OH) and 1.5-2.0 mmol of condensing agent (HOBT) to the first resin, add 5-15 mL of DMF, then add 1-3 mL of DIC (N,N-diisopropylcarbodiimide) and react for 0.5-1.5 h. Then add DMF solution containing 20% ​​piperidine by mass and wash for 15-25 min to obtain the second resin.

[0056] (3) Based on the amino acid sequence of HC-G02, repeat step (2) until the peptide chain ends to obtain linear peptide resin;

[0057] (4) Add methanol to the linear peptide resin for rinsing and then dry it. Add cutting fluid (95wt% TFA, 1wt% H2O, 2wt% EDT and 2wt% TIS) for cutting. After filtration, add ice ether and centrifuge three times at 4℃, 12000r / min and 5min to remove the upper layer of ice ether and obtain the crude precipitated peptide.

[0058] (5) The crude peptide was separated and purified by HPLC to obtain the pure peptide, namely the pure linear peptide HC-G02 (purity of 95% or more); the HPLC conditions were the same as in step (8).

[0059] (6) Dissolve the linear peptide from step (5) in a 50 v / v% methanol aqueous solution, then dilute with acetic acid to make the final peptide concentration so that each milliliter of the mixed solution (5 v / v% methanol + 5 v / v% water + 90 v / v% acetic acid) contains 1 mg of peptide. Add 10 mg / mL methanol-iodine solution dropwise, and keep stirring for 1 min to form the first disulfide bond.

[0060] (7) Add an equal volume (i.e., the same as the total volume in step 6) of hydrochloric acid-methanol solution (i.e., methanol containing 50 mM HCl), then add a methanol-iodine solution with a concentration of 10 mg / mL (the amount added is 10-20 times the amount of methanol-iodine solution added in step 6), and keep stirring for 1 hour to form a second disulfide bond.

[0061] (8) The peptide was separated and purified by HPLC. Mobile phase A was H2O and mobile phase B was ACN (acetonitrile). The flow rate was 5 mL / min and the linear gradient elution was carried out for 45 min at 5%-50% (volume of phase B). The detection wavelength was 220 nm. The peptide was then identified by MS and freeze-dried to obtain pure peptide.

[0062] 1.3.2 Hot Plate Experiment

[0063] Female mice weighing approximately 20±2g were selected for the hot plate test. The method is briefly described below, referring to existing techniques:

[0064] Mice were placed on a smart hot plate (temperature 55±0.5℃) for baseline measurements. Pain thresholds were then determined on the hot plate at 55±0.5℃, using the mouse licking its hind paw as an observation indicator. Measurements were repeated twice, with an interval of more than five minutes, to obtain the normal pain threshold for the mice. Thirty mice that showed a positive reaction within 30 seconds were pre-selected and randomly divided into five groups of six each.

[0065] The purple-spotted sea anemone polypeptide toxin HC-G02, i.e., oxidized peptide HC-G02, obtained in this invention, was dissolved in phosphate-buffered saline (PBS) to concentrations of 5 mg / ml, 2.5 mg / ml, and 0.5 mg / ml. The corresponding doses were injected into the abdomen of mice at 10 μl / g according to their body weight, resulting in final doses of 50 mg / kg, 25 mg / kg, and 5 mg / kg, respectively. Mice injected intraperitoneally with 10 μl / g PBS solution served as a negative control group, while mice injected with 50 mg / kg tramadol served as a positive control group. The endpoint of each experiment occurred when the animals exhibited characteristic physical responses to harmful thermal stimuli, such as licking their paws or jumping. The maximum cutoff time was set to 60 seconds to prevent tissue damage. Pain thresholds (s) were measured at 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, and 300 min after intraperitoneal injection, and the percentage increase in pain threshold was calculated.

[0066] 1.3.3 Data Processing

[0067] All data were statistically analyzed and processed using GraphPad Prism8 software. The data between the control group and the experimental group were analyzed using t-tests. * indicates a significant difference (p<0.05), ** indicates an extremely significant difference (p<0.01), *** indicates an extremely significant difference (p<0.001), and **** indicates an extremely significant difference (p<0.0001).

[0068] 2 Results

[0069] 2.1 Synthesis and Oxidative Folding of Peptides

[0070] The linear peptide HC-G02 of the purple-spotted sea anemone was synthesized using solid-phase peptide synthesis (SPPS). The linear peptide was purified by HPLC, and the mass spectrum is shown in the figure. Figure 2 The synthesized linear peptide was subjected to a one-step oxidative folding method using site-directed oxidation, and the final oxidative folding product was purified by HPLC and identified by mass spectrometry. The molecular weight of the oxidized peptide HC-G02 after the first step of oxidation is shown in (see...). Figure 4 Compared to the linear peptide HC-GO2, the difference is approximately 2 Da, confirming the correct formation of the first disulfide bond. The molecular weight of the oxidized peptide HC-GO2 after the second oxidation step (see...) Figure 6 The difference between the oxidized peptide and the oxidized peptide after the first step of oxidation is about 144 Da, which proves that the second disulfide bond was correctly formed.

[0071] 2.2 Isolation and purification of oxidized peptides

[0072] The linear peptide HC-G02 was analyzed by analytical HPLC, and the results are as follows: Figure 1 As shown, the elution time for the linear peptide HC-G02 was 21.920 min. Preparative HPLC was used to separate and purify the HC-G02 after three-step oxidation, and then analytical HPLC was used for analysis. The results are shown below. Figure 3 and Figure 5 As shown, the elution times of the oxidized peptide HC-G02 after the first oxidation step and the oxidized peptide HC-G02 after the second oxidation step were 20.972 min and 19.863 min, respectively.

[0073] This invention utilizes a site-directed oxidation method to perform a two-step oxidative folding of the synthesized linear peptide HC-G02 sample, and then purifies the oxidative folding final product by HPLC and identifies it by mass spectrometry to obtain the purple-spotted sea anemone polypeptide toxin HC-G02, namely the oxidized peptide HC-G02, with the sequence APCSGCYYQVGNECVYDKLKC-NH2. It has 4 cysteine ​​residues forming 2 disulfide bonds, with the disulfide bond mode being C1-C3 and C2-C4.

[0074] 2.3 Hot Plate Experiment

[0075] Experimental results are as follows Figure 7 As shown, the pain threshold elevation rate after intraperitoneal injection of oxidized peptide HC-G02 at low, medium, and high doses showed an increasing trend, with the high dose (50 mg / kg) having the greatest impact on the pain threshold. The pain threshold elevation rate increased with increasing concentration, showing a pattern of high-dose group > medium-dose group > low-dose group > negative control group. Hot plate tests revealed that oxidized peptide HC-G02 from the purple-spotted sea anemone had a good analgesic effect on physical pain in mice. After intraperitoneal injection of HC-G02, the pain threshold elevation rate gradually increased after 30 minutes, reaching its highest point at 120 minutes. The analgesic effect of HC-G02 at 50 mg / kg was comparable to that of tramadol at the same concentration, with a prolonged analgesic duration.

[0076] The above description is only a part of the embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A purple-spotted sea anemone polypeptide toxin HC-G02, characterized in that, Its amino acid sequence is APCSGCYYQVGNECVYDKLKC-NH2; the purple anemone polypeptide toxin HC-G02 has four cysteine ​​residues forming two disulfide bonds, and the disulfide bonds are linked in the manner of C1-C3 and C2-C4.

2. The method for synthesizing the purple-spotted sea anemone polypeptide toxin HC-G02 according to claim 1, characterized in that, Includes the following steps: (1) Weigh the resin, soak it in dichloromethane first, then wash it with dimethylformamide and dichloromethane in sequence, add Fmoc-Ala-OH, dichloromethane and N,N-diisopropylethylamine to react, then add methanol and dichloromethane to react, wash it with dimethylformamide, then add dimethylformamide solution containing pyridine to wash it to obtain the first resin; (2) Add the second amino acid Fmoc-Pro-OH and a condensing agent to the first resin, then add dimethylformamide and N,N-diisopropylcarbodiimide to react, and then wash with a dimethylformamide solution containing piperidine to obtain the second resin; (3) According to the amino acid sequence of claim 1, repeat step (2) until the peptide chain ends to obtain a linear peptide resin; (4) Add methanol to the linear peptide resin for rinsing and then dry it. Add cutting fluid for cutting, filter, add ice-cold ether, centrifuge to remove the upper layer of ice-cold ether, and obtain the crude precipitated peptide. (5) Purify the crude polypeptide to obtain pure linear peptide HC-G02; (6) Dissolve the linear peptide from step (5) in an aqueous methanol solution, then dilute with acetic acid, add methanol-iodine solution dropwise, and keep stirring to form the first disulfide bond; (7) Add an equal volume of hydrochloric acid-methanol solution, then add methanol-iodine solution, and keep stirring to form a second disulfide bond; (8) After purification, the polypeptide was obtained.

3. The synthesis method according to claim 2, characterized in that, Step (1) is as follows: Weigh the resin, soak it in dichloromethane first, then wash it with dimethylformamide and dichloromethane in sequence, add 0.6-1.0 mmol Fmoc-Ala-OH, 10-15 mL dichloromethane and 1-3 mL N,N-diisopropylethylamine and react for 80-100 min, then add 3-5 mL methanol and 8-12 mL dichloromethane to block the reaction for 25-35 min, wash it with dimethylformamide, and then wash it with a dimethylformamide solution containing pyridine for 15-25 min to obtain the first resin; wherein, the dimethylformamide solution containing piperidine is a DMF solution with a piperidine mass fraction of 20%.

4. The synthesis method according to claim 2, characterized in that, Step (2) is as follows: 1.5-2.0 mmol of the second amino acid Fmoc-Pro-OH and 1.5-2.0 mmol of condensing agent are added to the first resin, followed by 5-15 mL of dimethylformamide and 1-3 mL of N,N-diisopropylcarbodiimide. The mixture is reacted for 0.5-1.5 h, and then washed with a dimethylformamide solution containing piperidine for 15-25 min to obtain the second resin. The dimethylformamide solution containing piperidine is a DMF solution with a piperidine mass fraction of 20%.

5. The synthesis method according to claim 2, characterized in that, The cutting fluid contains: 95 wt% trifluoroacetic acid, 1 wt% H2O, 2 wt% 1,2-ethylenedithiol and 2 wt% triisopropylsilane.

6. The synthesis method according to claim 2, characterized in that, Step (6) is as follows: Dissolve the linear peptide from step (5) in a 50 v / v% methanol aqueous solution, then dilute with acetic acid to make the final concentration of the peptide solution 1 mg of peptide per milliliter of mixed solution, add 10 mg / mL methanol-iodine solution, and keep stirring to form the first disulfide bond.

7. The synthesis method according to claim 2, characterized in that, Step (7) is to add hydrochloric acid-methanol solution of the same volume as in step 6, then add 10 mg / mL methanol-iodine solution, and keep stirring to form a second disulfide bond.

8. The synthesis method according to claim 2, characterized in that, In steps (8) and (5), high performance liquid chromatography is used for purification. The purification conditions are: mobile phase A is H2O, mobile phase B is acetonitrile, flow rate is 5 mL / min, linear gradient elution is performed for 45 min, and the volume percentage of mobile phase B is increased from 5% to 50%.

9. The use of the purple-spotted sea anemone polypeptide toxin HC-G02 according to claim 1 in the preparation of analgesics.

Images