Eggshell membrane polypeptide with thrombin inhibitory activity and application thereof
The nonapeptide YDGQHGRVS and octapeptide EVPPSPAL were identified from the eggshell membrane through enzymatic digestion and separation purification, overcoming the limitations of existing anticoagulant drugs and achieving a highly efficient and safe thrombin inhibition effect, which has the potential to be developed into a new generation of anticoagulant drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN RESEARCH INSTITUTE OF NORTHWEST A & F UNIVERSITY
- Filing Date
- 2026-03-25
- Publication Date
- 2026-07-03
AI Technical Summary
Existing anticoagulant drugs have limitations such as significant side effects, high cost, and inconvenience of administration, and no polypeptide sequence with strong thrombin inhibitory activity has been identified in the eggshell membrane.
By enzymatically hydrolyzing the eggshell membrane, the nonapeptide YDGQHGRVS and octapeptide EVPPSPAL were isolated, purified, and identified, exhibiting significant thrombin inhibitory activity. Large-scale production was achieved through genetic engineering and chemical synthesis.
The nonapeptide YDGQHGRVS and the octapeptide EVPPSPAL exhibited thrombin inhibitory activity comparable to that of heparin sodium in vitro, significantly prolonging activated partial thromboplastin time and prothrombin time, and have the potential to be developed into safe and highly effective anticoagulant drugs.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of polypeptide preparation and biopharmaceutical technology, specifically relating to eggshell membrane polypeptides with thrombin inhibitory activity and their applications. Background Technology
[0002] Thrombotic diseases are a class of chronic non-communicable diseases. Excessive activation of thrombin is a key step in thrombus formation. Therefore, thrombin inhibitors have significant clinical value in the prevention and treatment of cardiovascular and cerebrovascular thrombotic diseases (such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and ischemic stroke). Currently, commonly used anticoagulants mainly include heparin, vitamin K antagonists, and thrombin inhibitors. However, existing anticoagulants generally have limitations such as significant side effects, high cost, and inconvenient administration. For example, heparin, rivaroxaban, and argatroban can cause bleeding; clopidogrel can cause adverse reactions such as thrombocytopenia, headache, and allergies; and dabigatran may lead to abnormal liver function. Therefore, researching and developing safe, effective, and low-side-effect natural active anticoagulants is of great significance.
[0003] Antithrombin peptides are short-chain amino acid sequences with thrombin-inhibiting activity, attracting significant attention due to their high safety, low side effects, and potential for preventing and treating thrombosis-related diseases. For example, hirudin, a natural antithrombin peptide extracted from the salivary glands of leeches, is a potent thrombin inhibitor and has been successfully applied clinically. The poultry egg processing industry generates massive amounts of eggshell and shell membrane waste annually, primarily composed of protein and polysaccharides. Currently, much of this waste is discarded or used only as low-value feed additives and fertilizers, resulting in resource waste and environmental impact. Although studies have reported that eggshell membrane hydrolysates may possess antioxidant and osteogenic biological activities, no novel polypeptide sequence with potent and specific thrombin-inhibiting activity has been identified from this specific waste resource to date. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention involves enzymatically hydrolyzing the eggshell membrane and separating and purifying the hydrolysate to identify two polypeptides with thrombin-inhibiting activity: nonapeptide YDGQHGRVS and octapeptide EVPPSPAL. Through thrombin-inhibiting activity assays, it was found that both nonapeptide YDGQHGRVS and octapeptide EVPPSPAL possess thrombin-inhibiting activity comparable to that of the positive control heparin sodium. This provides a new biomolecule for inhibiting thrombin activity and offers a novel technical solution for the prevention and treatment of thrombotic diseases.
[0005] On one hand, the present invention provides eggshell membrane polypeptides with thrombin inhibitory activity, wherein the eggshell membrane polypeptides are nonapeptide YDGQHGRVS and octapeptide EVPPSPAL, the amino acid sequence of the nonapeptide YDGQHGRVS is shown in SEQ ID NO:1, and the amino acid sequence of the octapeptide EVPPSPAL is shown in SEQ ID NO:2.
[0006] On the other hand, a composition having thrombin inhibitory activity is also provided, the composition comprising the eggshell membrane polypeptide and / or its pharmaceutically acceptable salt as described in this invention, and a pharmaceutically acceptable carrier and / or excipient.
[0007] The use of the eggshell membrane polypeptide in the preparation of formulations for inhibiting thrombin is also provided.
[0008] Furthermore, in the application, the eggshell membrane polypeptide is nonapeptide YDGQHGRVS, and the inhibition of thrombin includes prolonging the activation of partial thromboplastin time and / or prothrombin time.
[0009] The use of the eggshell membrane polypeptide in the preparation of medicaments for the prevention and / or treatment of thrombotic diseases is also provided.
[0010] A method for preparing the eggshell membrane polypeptide of the present invention is also provided, the method comprising the following steps:
[0011] S1. Obtain raw materials containing eggshell membranes;
[0012] S2. The eggshell membrane is sequentially hydrolyzed with alkaline protease and papain to obtain the hydrolysis product;
[0013] S3. Separate and purify the eggshell membrane polypeptide with the amino acid sequence YDGQHGRVS or EVPPSPAL from the enzymatic hydrolysis product.
[0014] Further, in the method, step S3 includes: ultrafiltration of the enzymatic hydrolysis product to collect components with a molecular weight less than 3 kDa; separation of the components by reversed-phase high-performance liquid chromatography to obtain eggshell membrane polypeptides with amino acid sequences YDGQHGRVS or EVPPSPAL.
[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:
[0016] (1) Two novel polypeptides were first identified from the waste resource of eggshell membrane, namely nonapeptide YDGQHGRVS and octapeptide EVPPSPAL. Functional studies revealed that nonapeptide YDGQHGRVS and octapeptide EVPPSPAL have significant inhibitory activity on thrombin. This invention provides core compounds for the development of novel anticoagulant drugs.
[0017] (2) In vitro thrombin inhibition assay results showed that the nonapeptide YDGQHGRVS and octapeptide EVPPSPAL provided in this invention exhibited thrombin inhibition rates of 96.7% and 72.4%, respectively, at a concentration of 2 mg / mL. Their inhibitory efficacy was comparable to that of heparin sodium, a commonly used anticoagulant in clinical practice, demonstrating extremely strong thrombin inhibition ability. The experiment confirmed that the nonapeptide YDGQHGRVS significantly prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT), indicating that it can exert an anticoagulant effect by intervening in intrinsic and common coagulation pathways, and its mechanism of action is clear.
[0018] (3) This invention not only provides a method for preparing polypeptides by enzymatic hydrolysis from natural raw materials, but also provides a feasible technical solution for large-scale, low-cost production of polypeptides by obtaining nonapeptide YDGQHGRVS and octapeptide EVPPSPAL through genetic engineering and chemical synthesis, which is beneficial to industrial production and clinical application.
[0019] (4) Based on the high thrombin inhibitory activity and clear anticoagulant effect of nonapeptide YDGQHGRVS and octapeptide EVPPSPAL, the nonapeptide YDGQHGRVS and octapeptide EVPPSPAL provided by the present invention have great potential for application in the preparation of drugs for the prevention and treatment of cardiovascular and cerebrovascular thrombotic diseases, and are expected to be developed into a next-generation anticoagulant drug with fewer side effects and greater safety. Attached Figure Description
[0020] Figure 1 The results show the assay results of the inhibitory activity of eggshell membrane polypeptides against thrombin in Example 1;
[0021] Figure 2 The mass spectrometry results are for the eggshell membrane nonapeptide YDGQHGRVS.
[0022] Figure 3 The mass spectrometry results are for the determination of eggshell membrane octapeptide EVPPSPAL.
[0023] Figure 4 The results of thrombin inhibition rate determination for different concentrations of nonapeptide YDGQHGRVS and octapeptide EVPPSPAL are shown in the figure.
[0024] Figure 5 The figure shows the effect of nonapeptide YDGQHGRVS on APTT.
[0025] Figure 6 The graph shows the effect of the nonapeptide YDGQHGRVS on PT.
[0026] Figure 7 The graph shows the thrombin inhibition rate of recombinant nonapeptide YDGQHGRVS and recombinant octapeptide EVPPSPAL. Detailed Implementation
[0027] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.
[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings, but the embodiments described are not intended to limit the present invention.
[0029] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.
[0030] Example 1
[0031] This example illustrates the isolation and identification of a novel anticoagulant polypeptide.
[0032] 1.1 Obtaining eggshell membrane peptides of different molecular weights
[0033] Fresh eggshells were collected, and the inner membrane was manually peeled off. The inner membrane was then washed, freeze-dried, and pulverized to obtain pulverized eggshell membrane. The pulverized eggshell membrane was dissolved in 30 mmol / L Na2SO3 to prepare an eggshell membrane solution with a weight-to-volume ratio of 5%. 9 U / mg of alkaline protease was added to the prepared eggshell membrane solution, and hydrolysis was carried out at pH 9.0 and 55℃ for 4 hours to obtain an alkaline protease-hydrolysate. After adjusting the pH of the alkaline protease-hydrolysate to 6.5, 9 U / mg of papain was added at 55℃, and hydrolysis was carried out for 4 hours to obtain an alkaline protease and papain complex-hydrolysate. This alkaline protease and papain complex-hydrolysate was heated at 80℃ for 20 minutes to inactivate the enzymes, and then centrifuged to collect the supernatant. The supernatant was fractionated by ultrafiltration into <3kDa, 3-10kDa and >10kDa molecules, and then freeze-dried to obtain low molecular weight eggshell membrane peptides (<3kDa), medium molecular weight eggshell membrane peptides (3-10kDa) and high molecular weight eggshell membrane peptides (>10kDa), which were then stored at -20℃ for later use.
[0034] 1.2 Detection of thrombin inhibitory activity of eggshell membrane peptides of different molecular weights
[0035] Thrombin inhibitory activity is based on the turbidity change caused by the conversion of fibrinogen to fibrin, which leads to a linear increase in the absorbance of the solution at 405 nm. Based on this principle, the thrombin inhibitory activity of eggshell membrane polypeptides of different molecular weights prepared in 1.1 was determined. The specific procedure is as follows:
[0036] 1.2.1 Preparation of substrate
[0037] Fibrinogen was dissolved in Tris-HCl buffer (0.05 mol / L, pH 7.2, containing 0.12 mmol / L NaCl) to prepare a 0.1% (w / v) substrate of fibrinogen.
[0038] 1.2.2 Sample Preparation
[0039] The pH of the low molecular weight eggshell membrane peptides, medium molecular weight eggshell membrane peptides, and high molecular weight eggshell membrane peptides prepared in step 1.1 was adjusted to 7.2 using 1 mol / L NaOH to prepare the test group samples of the low molecular weight eggshell membrane peptides, medium molecular weight eggshell membrane peptides, and high molecular weight eggshell membrane peptides.
[0040] Heparin sodium powder was dissolved in Tris-HCl buffer to prepare a 0.5 mmol / L heparin sodium solution, which was used as the positive control sample. The 0.05 mol / L Tris-HCl buffer solution described in section 1.2.1 was used as the blank control sample.
[0041] 1.2.3 Determination of thrombin inhibitory activity in each sample
[0042] 140 μL of the fibrinogen substrate prepared in 1.2.1 was mixed with the test samples and positive control samples prepared in 1.2.2. The mixture was incubated at 37°C for 10 minutes, and the absorbance was read at 405 nm (AC). Then, 10 μL of thrombin was added. The mixture was incubated at 37°C for 10 minutes, and the absorbance was read at 405 nm (AD). For the blank control group, 140 μL of fibrinogen substrate was mixed with 40 μL of Tris-HCl buffer. The mixture was incubated at 37°C for 10 minutes, and the absorbance was read at 405 nm (AA). Then, 10 μL of thrombin was added, and the mixture was incubated at 37°C for 10 minutes, and the absorbance was read at 405 nm (AB). The inhibition rate of each treatment was calculated based on the absorbance. The thrombin was prepared using Tris-HCl buffer at a concentration of 12 U / mL.
[0043] Thrombin inhibition rate =
[0044] The measurement results are as follows Figure 1 As shown, low molecular weight eggshell membrane peptides (<3kDa), medium molecular weight eggshell membrane peptides (3-10kDa), and high molecular weight eggshell membrane peptides (>10kDa) all exhibited varying degrees of thrombin inhibitory activity. Among them, the low molecular weight eggshell membrane peptides (<3kDa) had a thrombin inhibition rate of approximately 85%, and their thrombin inhibitory activity was close to that of the positive control group. This indicates that there may be active peptide components with thrombin inhibitory activity in eggshell membrane peptides, and the <3kDa component has the strongest activity.
[0045] 1.3 Isolation and Identification of Novel Anticoagulant Peptides
[0046] The fraction with the highest thrombin inhibitory activity (low molecular weight eggshell membrane polypeptide) obtained from screening in section 1.2 was sequentially purified by reversed-phase high-performance liquid chromatography (RP-HPLC, C18 column). The HPLC conditions were as follows: phase A: 0.1% formic acid; phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 300 nL / min.
[0047] Chromatographic peaks were collected, and thrombin inhibitory activity was tested according to steps 1.2.3. Two main peaks were isolated and purified from the low molecular weight eggshell membrane polypeptide, exhibiting significant thrombin inhibitory activity. Further sequence identification of the two chromatographic peaks with significant thrombin inhibitory activity was performed using Ultra-High Resolution LC-MS in positive ion mode. The primary mass spectrometry resolution was 12000 nm, with a scan range of 350-1800 nm; the secondary mass spectrometry resolution was 3000 nm, in automatic scan mode, with an HCD collision energy of 30%. The target protein was identified by searching the gallusNCBI20240514 database using Proteome Discovered 2.2 software. Figure 2 and Figure 3 The two polypeptides shown are, respectively, a nonapeptide with the amino acid sequence YDGQHGRVS (as shown in SEQ ID NO:1) and an octapeptide with the amino acid sequence EVPPSPAL (as shown in SEQ ID NO:2). Comparison with the BIOPEP database revealed that neither the octapeptide nor the nonapeptide has any known homologous functional peptides, making them novel anticoagulant polypeptides discovered for the first time from eggshell membranes.
[0048] Example 2
[0049] This embodiment is to verify the in vitro thrombin inhibitory activity of the nonapeptide YDGQHGRVS and the octapeptide EVPPSPAL.
[0050] A commissioned biotechnology company synthesized the nonapeptide YDGQHGRVS and octapeptide EVPPSPAL using conventional solid-phase synthesis methods, obtaining nonapeptide YDGQHGRVS and octapeptide EVPPSPAL with a purity >95%. Using a chromogenic substrate method, different concentrations of nonapeptide YDGQHGRVS and octapeptide EVPPSPAL, thrombin standard, and buffer were added to 96-well plates. After incubation, the chromogenic substrate was added, and the absorbance change at 405 nm was measured. Simultaneously, 2 mg / mL heparin sodium (a commonly used clinical anticoagulant) was used as a positive control, and the buffer was used as a negative control. The thrombin inhibition rate of samples at concentrations of 0.5–4 mg / mL was measured. Figure 4As shown, the inhibition of thrombin activity by the nonapeptide YDGQHGRVS and the octapeptide EVPPSPAL exhibits a dose-dependent relationship, and their inhibitory effect increases with increasing peptide concentration. When the concentration of nonapeptide YDGQHGRVS is 2 mg / mL, its inhibition rate reaches 96.7%, while the inhibition rate of the octapeptide EVPPSPAL at the same concentration is 72.4%. Under the same experimental conditions, the inhibition rate of heparin sodium is 98.1%. This indicates that the newly discovered nonapeptide YDGQHGRVS and octapeptide EVPPSPAL possess good thrombin inhibitory activity.
[0051] Example 3
[0052] This example is an analysis of the anticoagulation pathway of the nonapeptide YDGQHGRVS.
[0053] The anticoagulant pathway of nonapeptide YDGQHGRVS was investigated by measuring its regulatory effects on activated partial thromboplastin time (APTT) and prothrombin time (PT).
[0054] Healthy cat sodium citrate anticoagulated plasma was used as the experimental sample. The test was performed using the qLabs coagulation test card and the qLabs fully automated coagulation analyzer.
[0055] The experiment included two control groups: a blank control group consisting of a 1:1 mixture of test animal plasma and physiological saline; and a nonapeptide YDGQHGRVS treatment group consisting of 0.5 mL of test animal plasma mixed with an equal volume of 1 mg / mL nonapeptide YDGQHGRVS solution and incubated for 3 min. The treated plasma samples were then added to test strips, and the data curve was automatically generated after measurement by a coagulation analyzer. The test results are as follows: Figure 5 and Figure 6 As shown.
[0056] Depend on Figure 5 and Figure 6 Compared with the blank control group, the nonapeptide YDGQHGRVS significantly prolonged APTT (a key indicator of the intrinsic coagulation pathway) and PT (thrombin time, reflecting the common coagulation pathway). This suggests that the nonapeptide YDGQHGRVS may achieve its anticoagulant effect by intervening in the intrinsic coagulation cascade and the activity of coagulation factors in the common pathway.
[0057] Example 4
[0058] This embodiment describes the preparation of recombinant nonapeptide YDGQHGRVS and recombinant octapeptide EVPPSPAL using genetic engineering methods.
[0059] Based on the amino acid sequence of the nonapeptide YDGQHGRVS shown in SEQ ID NO:1, a codon optimization strategy was used to design and synthesize the encoding DNA sequence of the nonapeptide shown in SEQ ID NO:3. The encoding DNA sequence of this nonapeptide is ATGTATGATGGTCAACATGGTCGTGTTTCTTAA. This nonapeptide encoding DNA fragment was double-digested and cloned into the pET expression vector to obtain the recombinant expression plasmid pET-SEQ1. The recombinant expression plasmid pET-SEQ1 was transformed into *E. coli* BL21 competent cells. Positive clones were obtained after antibiotic selection, and sequencing confirmed their correctness, thus obtaining the pET-SEQ1 genetically engineered strain. The pET-SEQ1 genetically engineered strain was cultured, and the target protein was induced to express using IPTG. The bacterial cells were collected, lysed, and purified by nickel affinity chromatography to obtain the recombinant nonapeptide YDGQHGRVS. Using the above-described genetic engineering method, based on the amino acid sequence of the octapeptide EVPPSPAL shown in SEQ ID NO:2, the coding DNA sequence of the octapeptide shown in SEQ ID NO:4 was designed and synthesized through a codon optimization strategy. The coding DNA sequence of the octapeptide is ATGGAAGTTCCTCCTTCTCCTGCTTTATAA. The recombinant octapeptide EVPPSPAL was obtained through the same cloning, expression and purification process.
[0060] The thrombin inhibitory activities of recombinant nonapeptide YDGQHGRVS and recombinant octapeptide EVPPSPAL at a concentration of 2 mg / mL were determined according to the method in Example 2. A buffer-added group was used as a blank control group instead of the peptide-added group. The test results are as follows: Figure 7 The results showed that the blank control group had almost no thrombin inhibitory activity, indicating that thrombin activity was normal without inhibitors. The recombinant nonapeptide YDGQHGRVS group had a thrombin activity inhibition rate of 92%, indicating that thrombin activity was strongly inhibited. The recombinant octapeptide EVPPSPAL group had a thrombin activity inhibition rate of 77%.
[0061] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.
Claims
1. An eggshell membrane polypeptide with thrombin inhibitory activity, characterized in that, The eggshell membrane polypeptide is a nonapeptide YDGQHGRVS or an octapeptide EVPPSPAL. The amino acid sequence of the nonapeptide YDGQHGRVS is shown in SEQ ID NO:1, and the amino acid sequence of the octapeptide EVPPSPAL is shown in SEQ ID NO:
2.
2. A composition having thrombin inhibitory activity, characterized in that, It comprises the eggshell membrane polypeptide of claim 1 and / or its pharmaceutically acceptable salt, as well as a pharmaceutically acceptable excipient.
3. The use of the eggshell membrane polypeptide of claim 1 in the preparation of a medicament for the prevention and / or treatment of thrombotic diseases.
4. Use according to claim 3, characterized in that, The eggshell membrane polypeptide is a nonapeptide YDGQHGRVS, and the drug inhibits thrombin by prolonging the activation of partial thromboplastin time and / or prothrombin time.