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CN-121974994-A - Fetin anticoagulant peptide, preparation method and application

CN121974994ACN 121974994 ACN121974994 ACN 121974994ACN-121974994-A

Abstract

The invention provides a feta protein anticoagulant peptide, a preparation method and application thereof, wherein the feta protein anticoagulant peptide is VKETMVPK, VLPVPQK or EMPFPK. The invention solves the problems of low safety and high cost of the existing clinical anticoagulants, and low utilization rate of feta protein resource development and high value, and fills the blank of feta protein anticoagulation peptide preparation technology. The feta protein inhibitory peptide provided by the method has the advantages of high safety, capability of avoiding the defects of the existing drug treatment, capability of efficiently excavating the resource value of the feta, capability of reducing the resource waste, capability of filling the technical blank, capability of establishing a systematic preparation system, clear and efficient anticoagulation activity, comprehensive action mechanism, wide application scene and large market potential.

Inventors

  • WU HAIXIA
  • WAN YU
  • HU XUE

Assignees

  • 内蒙古大学

Dates

Publication Date
20260505
Application Date
20260330

Claims (11)

  1. 1. A feta protein anticoagulant peptide is characterized in that the feta protein anticoagulant peptide is EMPFPK, VKETMVPK or VLPVPQK; The amino acid sequence of the feta protein anticoagulant peptide EMPFPK is SEQ ID NO. 1; The amino acid sequence of the feta protein anticoagulant peptide VKETMVPK is SEQ ID NO. 2; The amino acid sequence of the feta protein anticoagulant peptide VLPVPQK is SEQ ID NO. 3.
  2. 2. The method for preparing feta protein anticoagulant peptide according to claim 1, comprising the steps of: S1, centrifuging raw goat milk to remove upper fat, taking a supernatant liquid of the lower layer, regulating the pH to 4.6, centrifuging, collecting precipitate, adding pre-cooled n-hexane at-20 ℃, stirring at 4 ℃ for degreasing for 24 hours, replacing liquid for multiple times, filtering by a Buchner funnel, and freeze-drying for 24 hours to obtain casein; s2, dissolving casein by using Tris-HCl buffer solution with pH=8.0, preparing casein solution with concentration of 40 mg/mL, and adding alkaline protease for enzymolysis to obtain enzymolysis solution; S3, diluting the enzymolysis liquid to 1-5 mg/mL, acidifying with formic acid to pH <3.0, performing solid-phase extraction and desalination column treatment by using Oasis HLB, collecting effluent, performing vacuum centrifugal concentration, and drying to obtain crude polypeptide containing a plurality of polypeptides; S4, identifying the polypeptide by adopting an ion mobility high-resolution mass spectrometer, and screening feta protein anticoagulant peptides EMPFPK, VKETMVPK and VLPVPQK with higher anticoagulant activity; S5, synthesizing feta protein anticoagulant peptides EMPFPK, VKETMVPK and VLPVPQK by adopting a solid-phase synthesis method.
  3. 3. The method for producing feta protein anticoagulant peptide according to claim 2, wherein the S1 is performed under a first centrifugation condition of 7500 Xg, a centrifugation time of 15min, a second centrifugation condition of 9000 Xg, a centrifugation time of 20min, a solid-to-liquid ratio of precipitate to n-hexane of 1:10, and the S3 is performed under a centrifugation condition of 500 Xg.
  4. 4. The method for preparing feta protein anticoagulant peptide according to claim 2, wherein the enzyme amount in S2 is 2500U/g, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 60 min.
  5. 5. The method for preparing feta protein anticoagulant peptide according to claim 2, wherein the steps of S3, oasis HLB solid phase extraction desalting column treatment and crude polypeptide collection comprise: s31, adding 1mL of methanol into the solid phase extraction desalting column, flowing down by gravity, and discarding effluent; S32, adding 700 mu L of Buffer B, pressurizing and flowing out of the column, repeating twice, and discarding effluent; S33, adding 700 mu L of Buffer A, pressurizing and flowing out of the column, and discarding effluent; S34, diluting the enzymolysis liquid sample to 2 times of volume by using Buffer A, loading the sample onto a column of 700 mu L, and collecting effluent liquid under gravity flow.
  6. 6. The method for preparing feta protein anticoagulant peptide according to claim 2, wherein the step of synthesizing feta protein anticoagulant peptides VKETMVPK, VLPVPQK and EMPFPK by solid phase synthesis in S5 comprises the steps of: s51, soaking the 2-chlorotrityl chloride resin in dichloromethane to fully swell the 2-chlorotrityl chloride resin, removing dichloromethane on the surface of the 2-chlorotrityl chloride resin, and cleaning the 2-chlorotrityl chloride resin by using N, N-dimethylformamide; s52, taking the 2-chlorotrityl chloride resin treated in the S51 as a solid phase carrier, dissolving Fmoc-amino acid in N, N-dimethylformamide, adding diisopropylethylamine for full reaction, and realizing preliminary connection of the amino acid and the resin; S53, adding a mixed solution of methanol, diisopropylethylamine and dichloromethane, reacting at room temperature for 0.5 h, and sealing unreacted active sites on the resin; S54, adding a mixed solution of piperidine and N, N-dimethylformamide, reacting for 0.5 h, and removing Fmoc protecting groups; S55, dissolving Fmoc-amino acid and benzotriazol-1-yl-oxy-tripyrrolidinyl phosphate hexafluorophosphate in N, N-dimethylformamide, adding diisopropylethylamine, reacting at room temperature for 2-4 h to complete coupling, sequentially repeating S54, and performing the next coupling until all amino acids are connected according to a target sequence; s56, adding a cutting agent into the coupled 2-chlorotrityl chloride resin, reacting at room temperature for 2h, filtering the reaction solution to remove the resin, collecting filtrate, removing impurities through rotary evaporation, adding excessive glacial ethyl ether for precipitation, centrifugally collecting the precipitate, washing the precipitate with the glacial ethyl ether for 2 times, and then drying in vacuum to obtain fetin anticoagulant peptide VKETMVPK, VLPVPQK and EMPFPK crude products; S57, detecting the obtained feta protein anticoagulant peptide crude product by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrum to confirm whether a target peptide segment exists, and if a target molecular weight peak is detected, purifying the feta protein anticoagulant peptide crude product by adopting an aqueous solution containing 0.1% of trifluoroacetic acid and an acetonitrile solution containing 0.1% of trifluoroacetic acid as mobile phases through semi-preparation high performance liquid chromatography; and S58, collecting target peak components, performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection again after rotary evaporation concentration to confirm the components, and measuring the purity by adopting analytical HPLC.
  7. 7. The method for preparing feta protein anticoagulant peptide according to claim 6, wherein: amino acids used in preparing the feta protein anticoagulant peptide EMPFPK are Fmoc-Glu (OtBu) -OH, fmoc-Met-OH, fmoc-Pro-OH, fmoc-Phe-OH and Fmoc-Lys (Boc) -OH; the amino acids used in the preparation of the feta protein anticoagulant peptide VKETMVPK are Fmoc-Val-OH, fmoc-Lys (Boc) -OH, fmoc-Glu (OtBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Met-OH and Fmoc-Pro-OH; the amino acids used in the preparation of the feta protein anticoagulant peptide VLPVPQK are Fmoc-Val-OH, fmoc-Leu-OH, fmoc-Pro-OH, fmoc-Gln (Trt) -OH and Fmoc-Lys (Boc) -OH.
  8. 8. The method for preparing feta protein anticoagulant peptide according to claim 6, wherein the volume ratio of methanol, diisopropylethylamine and dichloromethane in S53 is 2:1:17.
  9. 9. The method for preparing feta protein anticoagulant peptide according to claim 6, wherein the volume ratio of piperidine to N, N-dimethylformamide in S54 is 1:4.
  10. 10. The method of claim 6, wherein in S56, the cutting agent is a mixture of trifluoroacetic acid, triisobutylsilane and deionized water, and the volume ratio of the trifluoroacetic acid, the triisobutylsilane and the deionized water is 95:2.5:2.5.
  11. 11. The use of the feta protein anticoagulant peptide according to claim 1, wherein the feta protein anticoagulant peptide is used for preparing a pharmaceutical preparation having an antithrombotic function.

Description

Fetin anticoagulant peptide, preparation method and application Technical Field The invention relates to the technical field of biological medicines, in particular to feta protein anticoagulant peptide, a preparation method and application. Background Thrombus is a solid or semi-solid substance formed by abnormal aggregation of blood components in the vascular system, and the core component contains fibrin, platelets, packed red blood cells, white blood cells and the like. The formation of the compound is often related to vascular endothelial injury, hemodynamic changes and hypercoagulability of blood, can obstruct local blood flow and cause tissue ischemia necrosis, and is a key pathogenesis of cardiovascular and cerebrovascular diseases. The thrombus is hidden in onset and rapid in progress, and is easy to cause serious consequences such as myocardial infarction, cerebral infarction, venous thrombosis and the like, thus forming a great threat to public health. The current anticoagulants commonly used in clinic mainly comprise heparin and derivatives thereof, hirudin and analogues thereof, and the like, and the anticoagulants can effectively delay thrombosis, but have the problems of high price, easy bleeding risk, poor long-term use compliance, drug resistance of partial patients, and the like, so that the wide application of the anticoagulants is limited. Therefore, the development of natural anticoagulant active substances with high safety, suitability for oral administration and small potential side effects becomes a research hotspot in the field of antithrombotic medicaments. The milk protein is taken as a natural protein resource with balanced amino acid composition and controllable structure, and is a high-quality raw material for preparing bioactive peptide. Researches prove that the milk-derived polypeptide has good activities in the aspects of antioxidation, depressurization, immunoregulation, antibiosis, anticoagulation and the like, wherein the anticoagulation peptide can play an antithrombotic role through mechanisms of inhibiting thrombin activity, delaying fibrin formation, interfering platelet aggregation and the like, and has wide application prospect. Compared with cow milk, goat milk has the characteristics of higher casein content and tighter structure, and is rich in various potential bioactive peptide precursors. By means of targeted enzymolysis technology, small molecular peptide with anticoagulant activity can be released from goat milk casein to prepare active matter with high selectivity and high activity. After enzymolysis and activity screening are carried out on the goat milk casein hydrolysate, specific anticoagulant active peptide fragments can be identified, and then high-purity and multi-batch preparation can be realized through chemical synthesis, so that a stable material is provided for subsequent activity verification and application research. Disclosure of Invention Based on the above, the invention aims to provide a fetoprotein anticoagulant peptide, a preparation method and an application thereof, so as to solve the multiple problems in the prior art, namely, the existing anticoagulant drugs such as heparin, derivatives thereof, hirudin, analogues thereof and the like which are commonly used in clinic have the defects of high price, easy bleeding risk, poor long-term use compliance, drug resistance of partial patients and the like, the feta is rich in potential anticoagulant active peptide precursors, but the related active value is not effectively excavated, the problem of low utilization rate of resources is solved, and the prior art lacks a systematic preparation scheme aiming at the fetoprotein anticoagulant peptide, and does not form a complete technical chain of raw material extraction-enzymolysis-separation identification-large-scale synthesis, so that the feta protein anticoagulant peptide with high purity and high activity is difficult to obtain stably. In order to solve the technical problems described above, it is an object of the present invention to provide a feta protein anticoagulant peptide, which is EMPFPK, VKETMVPK, VLPVPQK; The amino acid sequence of the feta protein anticoagulant peptide EMPFPK is SEQ ID NO. 1; The amino acid sequence of the feta protein anticoagulant peptide VKETMVPK is SEQ ID NO. 2; The amino acid sequence of the feta protein anticoagulant peptide VLPVPQK is SEQ ID NO. 3. Based on the same inventive concept, the invention also provides a preparation method of feta protein anticoagulant peptide, which comprises the following steps: S1, centrifuging raw goat milk to remove upper fat, taking a supernatant liquid of the lower layer, regulating the pH to 4.6, centrifuging, collecting precipitate, adding pre-cooled n-hexane at-20 ℃, stirring at 4 ℃ for degreasing for 24 hours, replacing liquid for multiple times, filtering by a Buchner funnel, and freeze-drying for 24 hours to obtain casein; s2, dissolving casein by using Tris-HCl buf