Search

CN-116478263-B - Sea anemone polypeptide toxin HC-G02 and synthetic method and application thereof

CN116478263BCN 116478263 BCN116478263 BCN 116478263BCN-116478263-B

Abstract

The invention provides a sea anemone polypeptide toxin HC-G02, a synthesis method and application thereof, and an amino acid sequence thereof is APCSGCYYQVGNECVYDKLKC-NH 2 . The linear peptide HC-G02 is synthesized by adopting a polypeptide solid-phase synthesis method, and the purity of the purified linear peptide reaches more than 95 percent. The anemone purpurea oxidized peptide HC-G02 (disulfide bond connection mode is C1-C3, C2-C4) is obtained after fixed-point oxidation. According to the invention, through a hot plate experiment test, the anemone purpurea oxidized peptide HC-G02 has a good analgesic effect on physical pain of a mouse, and after the mouse is injected with the oxidized peptide HC-G02 intraperitoneally, the pain threshold improvement rate is gradually increased within 30min, and the pain threshold improvement rate reaches the highest within 120min after administration. The analgesic effect of the oxidized peptide HC-G02 at 50mg/kg is equivalent to that of tramadol with the same concentration, and the analgesic time is prolonged.

Inventors

  • Gao Bingmiao
  • GUO QIQI
  • LI MING
  • LIN CHENGZHANG
  • HUA ZIQIANG
  • ZHUO YAN
  • LIAO YANLING

Assignees

  • 海南医学院

Dates

Publication Date
20260508
Application Date
20230412

Claims (9)

  1. 1. The echinacea purpurea polypeptide toxin HC-G02 is characterized by having an amino acid sequence of APCSGCYYQVGNECVYDKLKC-NH 2 , wherein the echinacea purpurea polypeptide toxin HC-G02 has four cysteines to form two disulfide bonds, and the disulfide bonds are connected in a mode of C1-C3 and C2-C4.
  2. 2. The method for synthesizing the echinacea purpurea polypeptide toxin HC-G02 according to claim 1, which comprises the following steps: (1) Weighing resin, soaking the resin in dichloromethane, sequentially washing the resin with dimethylformamide and dichloromethane, adding Fmoc-Ala-OH, dichloromethane and N, N-diisopropylethylamine for reaction, adding methanol and dichloromethane for reaction, washing the resin with dimethylformamide, and adding a pyridine-containing dimethylformamide solution for washing to obtain a first resin; (2) Adding a second amino acid Fmoc-Pro-OH and a condensing agent into the first resin, adding dimethylformamide and N, N-diisopropylcarbodiimide for reaction, and adding a piperidine-containing dimethylformamide solution for washing to obtain a second resin; (3) The amino acid sequence according to claim 1, wherein step (2) is repeated until the peptide chain ends, resulting in a linear peptide resin; (4) Adding methanol into linear peptide resin for washing and pumping, adding cutting fluid for cutting, filtering, adding glacial ethyl ether, centrifuging to remove supernatant liquid glacial ethyl ether, and obtaining a precipitated polypeptide crude product; (5) Purifying the crude polypeptide to obtain pure linear peptide HC-G02; (6) Dissolving the linear peptide in the step (5) in methanol water solution, diluting with acetic acid, dropwise adding methanol-iodine solution, and keeping stirring to form a first disulfide bond; (7) Adding an equal volume of hydrochloric acid-methanol solution, then adding a methanol-iodine solution, and keeping stirring to form a second disulfide bond; (8) Purifying to obtain the polypeptide pure product.
  3. 3. The synthetic method according to claim 2, wherein the step (1) is characterized in that resin is weighed, soaked with dichloromethane, washed with dimethylformamide and dichloromethane sequentially, added with 0.6~1.0 mmol Fmoc-Ala-OH, 10-15 mL of dichloromethane and 1-3 mL of N, N-diisopropylethylamine for reaction for 80-100 min, further added with 3-5 mL of methanol and 8-12 mL of dichloromethane for blocking reaction for 25-35 min, washed with dimethylformamide, and further added with a pyridine-containing dimethylformamide solution for 15-25 min to obtain the first resin, wherein the piperidine-containing dimethylformamide solution is DMF solution with 20% of piperidine mass fraction.
  4. 4. The synthesis method according to claim 2, wherein the step (2) is characterized in that 1.5-2.0 mmol of second amino acid Fmoc-Pro-OH and 1.5-2.0 mmol of condensing agent are added into the first resin, 5-15 mL of dimethylformamide and 1-3 mL of N, N-diisopropylcarbodiimide are added to react for 0.5-1.5 h, and a piperidine-containing dimethylformamide solution is added to wash for 15-25 min to obtain the second resin, wherein the piperidine-containing dimethylformamide solution is a DMF solution with 20% of piperidine mass fraction.
  5. 5. The method according to claim 2, wherein the cutting fluid comprises 95wt% trifluoroacetic acid, 1wt% H 2 O, 2wt% 1, 2-ethanedithiol and 2wt% triisopropylsilane.
  6. 6. The method according to claim 2, wherein the step (6) is to dissolve the linear peptide of the step (5) in 50v/v% aqueous methanol solution, dilute the solution with acetic acid to a final concentration of 1mg peptide per mL of the mixed solution, drop 10mg/mL iodine methanol solution, and keep stirring to form the first disulfide bond.
  7. 7. The method of claim 2, wherein the step (7) is to add hydrochloric acid-methanol solution in the same total volume as the step 6, then add 10 mg/mL of methanol iodine solution, and keep stirring to form the second disulfide bond.
  8. 8. The method according to claim 2, wherein in the step (8) and the step (5), the purification is performed by high performance liquid chromatography under the conditions that the mobile phase A is H 2 O, the mobile phase B is acetonitrile, the flow rate is 5 mL/min,45 min is linearly gradient eluted, and the volume ratio of the mobile phase B is increased from 5% to 50%.
  9. 9. Use of the echinacea purpurea polypeptide toxin HC-G02 as claimed in claim 1 for the preparation of an analgesic.

Description

Sea anemone polypeptide toxin HC-G02 and synthetic method and application thereof Technical Field The invention belongs to the technical field of active polypeptides, and relates to a sea anemone polypeptide toxin HC-G02, a synthesis method and application thereof. Background Sea anemone is a kind of marine animal found in deep and shallow sea areas worldwide, and is a kind of organism from the phylum of the division Corallinita, class Corallidae, order Heliothis. Sea anemone is one of the oldest existing orders of toxic animals, and molecular and fossil data indicate that their origin was earlier than the Eddie karst years ago of 7.5 hundred million. Sea anemones are of a wide variety, more than 1100 species of sea anemones have been recorded worldwide, belonging to about 400 genera of 50 families, whereas the chinese sea anemone species account for about 1/10 of the world. The diversity of the Chinese sea anemone species is highest in the south China sea. Sea anemones have spiny cells on their hands, and like other spiny animals, sea anemones concentrate venom in a saccular organelle called a spiny capsule. The stab capsule penetrates the target organism when in contact with the prey, releasing the venom paralysis or killing the other party for predation, defense and deterrence of the competitor. The Chinese herbal medicine and the Chinese medicinal animal mind state that sea anemone has the effects of astringing and inducing astringency and the like, and is mainly used for treating hemorrhoids, rectocele, around worm disease, tinea corporis and the like. Modern pharmacological studies show that the anemone toxin has analgesic effects. Disclosure of Invention The invention screens out active polypeptide sequence from the anemone, and synthesizes the anemone functional polypeptide through oxidative folding. The analgesic experiment shows that the sea anemone polypeptide has certain analgesic activity. The invention lays a foundation for developing novel ocean medicaments and provides powerful support for human health and ocean medicament research. The scheme of the invention is as follows: The sea anemone polypeptide toxin HC-G02 has amino acid sequence APCSGCYYQVGNECVYDKLKC-NH 2, and four cysteines form two disulfide bonds, and the disulfide bonds are connected in a mode of C1-C3 and C2-C4. The invention also provides a synthesis method of the echinacea purpurea polypeptide toxin HC-G02, which comprises the following steps: (1) Weighing resin, soaking the resin in dichloromethane, sequentially washing the resin with dimethylformamide and dichloromethane, adding Fmoc-Ala-OH, dichloromethane and N, N-diisopropylethylamine for reaction, adding methanol and dichloromethane for reaction, washing the resin with dimethylformamide, and adding a pyridine-containing dimethylformamide solution for washing to obtain a first resin; (2) Adding a second amino acid Fmoc-Pro-OH and a condensing agent into the first resin, adding dimethylformamide and N, N-diisopropylcarbodiimide for reaction, and adding a piperidine-containing dimethylformamide solution for washing to obtain a second resin; (3) Repeating the step (2) until the peptide chain is finished according to the amino acid sequence of HC-G02, so as to obtain linear peptide resin; (4) Adding methanol into linear peptide resin for washing and pumping, adding cutting fluid for cutting, filtering, adding glacial ethyl ether, centrifuging to remove supernatant liquid glacial ethyl ether, and obtaining a precipitated polypeptide crude product; (5) Purifying the crude polypeptide to obtain pure linear peptide HC-G02; (6) Dissolving the linear peptide in the step (5) in methanol water solution, diluting with acetic acid, dropwise adding methanol-iodine solution, and keeping stirring to form a first disulfide bond; (7) Adding an equal volume of hydrochloric acid-methanol solution, then adding a methanol-iodine solution, and keeping stirring to form a second disulfide bond; (8) Purifying to obtain the polypeptide pure product. The method comprises the steps of (1) weighing resin, soaking the resin in dichloromethane, washing the resin with dimethylformamide and dichloromethane sequentially, adding 0.6-1.0 mmol of Fmoc-Ala-OH, 10-15 mL of dichloromethane and 1-3 mL of N, N-diisopropylethylamine to react for 80-100 min, adding 3-5 mL of methanol and 8-12 mL of dichloromethane to carry out a blocking reaction for 25-35 min, washing the resin with dimethylformamide, and adding a pyridine-containing dimethylformamide solution to wash the resin for 15-25 min to obtain the first resin, wherein the piperidine-containing dimethylformamide solution is a DMF solution with 20% of piperidine mass fraction. Further, in the step (2), 1.5-2.0 mmol of second amino acid Fmoc-Pro-OH and 1.5-2.0 mmol of condensing agent are added into the first resin, 5-15 mL of dimethylformamide and 1-3 mL of N, N-diisopropylcarbodiimide are added to react for 0.5-1.5 h, and then a piperidine-containing dimeth