Earthworm protein with anticoagulant activity as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of anticoagulant drugs, and particularly relates to lumbricus protein with anticoagulant activity as well as a preparation method and application of the lumbricus protein. The lumbricus protein provided by the invention contains at least one of a c71616 protein with an amino acid sequence as shown in SEQ ID No. 1, a c128686 protein with an amino acid sequence as shown in SEQ ID No. 2, a c58213 protein with an amino acid sequence as shown in SEQ ID No. 3 and a c118923 protein with an amino acid sequence as shown in SEQ ID No. 4. The protein can effectively inhibit thrombin activity and interfere formation of fibrin so as to act on a key link of a blood coagulation process. The protein can be obtained through recombinant expression and purification, and has high expression efficiency and stability, so that the protein can be used as an active component to be applied to development of anticoagulant drugs, surface anticoagulant coatings and other products.

Description

Technical Field

[0001] This invention belongs to the field of anticoagulant drug technology, and particularly relates to a ground worm protein with anticoagulant activity, its preparation method and application. Background Technology

[0002] Anticoagulants are among the most commonly used antithrombotic drugs. Their mechanism of action involves activating plasminogen to convert into plasmin, which directly degrades fibrin in the thrombus, thereby preventing thrombus formation or expansion. They are suitable for the prevention of deep vein thrombosis after atrial fibrillation (stroke prevention), post-artificial valve replacement surgery, or orthopedic surgery, as well as for the maintenance treatment of deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation (DIC). Currently, commonly used anticoagulants in clinical practice include vitamin K antagonists (such as warfarin), heparin derivatives (such as unfractionated heparin and low molecular weight heparin), Xa inhibitors (such as rivaroxaban), thrombin inhibitors (such as dabigatran etexilate), and antiplatelet drugs (such as aspirin and clopidogrel). While the aforementioned anticoagulants are effective, they also have several drawbacks: warfarin has a narrow therapeutic window and significant adverse reactions; heparin can destroy platelets through immune-mediated metabolism, increasing the risk of thrombosis; rivaroxaban can increase the burden on hepatic enzymes, leading to elevated transaminase levels; dabigatran etexilate is expensive; aspirin carries a risk of gastrointestinal bleeding; and clopidogrel has genetic polymorphisms that can lead to drug resistance. Therefore, it is necessary to continue exploring safer anticoagulants. Summary of the Invention

[0003] To address the above-mentioned technical problems, this invention provides a *Pheretima aspergillum* protein with anticoagulant activity, its preparation method, and its applications. The *Pheretima aspergillum* protein provided by this invention is derived from the natural medicinal herb *Pheretima aspergillum*, offering higher safety. It effectively inhibits thrombin activity and interferes with fibrin formation, thereby acting on a key aspect of the coagulation process and exhibiting excellent anticoagulant activity, making it suitable for the formulation of anticoagulant drugs. The preparation method provided by this invention produces this *Pheretima aspergillum* protein through recombinant expression and purification, achieving high expression efficiency and suitability for industrial-scale implementation.

[0004] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a *Pheretima asiatica* protein, wherein the *Pheretima asiatica* protein contains at least one of c71616 protein, c128686 protein, c58213 protein, and c118923 protein; the amino acid sequence of the c71616 protein is shown in SEQ ID No. 1, the amino acid sequence of the c128686 protein is shown in SEQ ID No. 2, the amino acid sequence of the c58213 protein is shown in SEQ ID No. 3, and the amino acid sequence of the c118923 protein is shown in SEQ ID No. 4.

[0005] Earthworm (Pheretima asiatica) Pheretima aspergillum(E. perrier) commonly known as "Guangdilong" is the earliest earthworm medicinal material used. The Guangdilong protein provided in this invention is obtained through extraction and screening from different genetic subtypes of Guangdilong. It can effectively inhibit key coagulation factors such as thrombin and fibrin in vitro. Its binding free energy with thrombin is less than -10 kcal / mol, exhibiting good binding stability. Furthermore, as an animal-derived protein, this Guangdilong protein is safer and particularly suitable for patients with high bleeding risk who require long-term antithrombotic prophylaxis but cannot tolerate the severe side effects of traditional anticoagulant / antiplatelet drugs.

[0006] This protein can be obtained through recombinant expression and purification, and it has high expression efficiency and stability, thus enabling it to be used as an active ingredient in the development of anticoagulant drugs, biomedical materials (such as surface anticoagulant coatings), and other products.

[0007] Preferably, the earthworm protein contains at least one of c71616 protein and c58213 protein. c71616 protein and c58213 protein have superior antithrombin activity and fibrinolytic activity.

[0008] The aforementioned earthworm protein can be chemically synthesized using conventional solid-phase synthesis based on its amino acid sequence, or it can be prepared through recombinant expression and purification.

[0009] The second aspect of this invention provides a method for preparing the above-mentioned earthworm protein, specifically comprising the following steps: S1. Codon optimization is performed on the original coding gene sequences of c71616, c128686, c58213 and c118923 proteins in the prokaryotic expression system to obtain the coding genes of each protein; each coding gene is synthesized and then PCR amplification is performed. S2. Through enzyme digestion and ligation reactions, the PCR amplification products encoding the c71616 and c128686 proteins obtained in S1 were ligated to the pET22b(+) expression vector, and the PCR amplification products encoding the c58213 and c118923 proteins obtained in S1 were ligated to the pET28a(+) expression vector to obtain recombinant plasmids. S3. The recombinant plasmid obtained in S2 was transformed into Escherichia coli BL21(DE3), and positive transformants were screened after culture. S4. The positive transformant is induced to express, the bacterial cells are collected, the bacterial cells are resuspended and lysed, the precipitate is collected, the inclusion body protein is purified and refolded, and the refolded protein is purified to obtain the Guangdilong protein.

[0010] Preferably, the restriction sites for the enzyme digestion reaction in S2 are: NdeI and XhoI for the PCR amplification products encoding the genes of c71616 and c128686 proteins and the pET22b(+) expression vector, and BamHI and XhoI for the PCR amplification products encoding the genes of c58213 and c118923 proteins and the pET28a(+) expression vector.

[0011] Preferably, the method for inducing expression in S4 is as follows: culturing positive transformants in LB medium containing 0.1 mM IPTG.

[0012] Preferably, the purification method for the inclusion bodies in S4 is as follows: resuspend the precipitate in STET buffer solution, add DTT to a final concentration of 1 mM; sonicate to promote the dissolution of impurities, with parameters set as follows: power 200 W, operation for 3 s, pause for 3 s, time 10 min; centrifuge at 10,000 rpm at 4℃ for 10 min, and discard the supernatant; repeat the above three steps until the supernatant is clear; resuspend the precipitate in PBS, sonicate, with parameters set as follows: power 200 W, operation for 3 s, pause for 3 s, time 10 min; centrifuge at 16,000 rpm at 4℃ for 10 min, and discard the supernatant; resuspend the inclusion bodies in 6M guanidine hydrochloride aqueous solution, add DTT to a final concentration of 5 mM; shake at 220 rpm at 37℃ for 4 h until all inclusion bodies are dissolved; centrifuge at 10,000 rpm at 4℃ for 10 min, and collect the supernatant.

[0013] Preferably, the refolding step in S4 is as follows: Step a: Dilute the purified protein solution with 2 times the volume of 6 M guanidine hydrochloride, add it to the refolding solution at pH = 8.0, adjust the speed to 1800 r / min, and stir for at least 24 h; reduce the speed to 1500 r / min and stir for at least 24 h; take the protein solution into a dialysis bag with a molecular weight cutoff of 3500D, and concentrate it with PEG20000 until the volume of the concentrate is 1 / 4 to 1 / 2 of the volume of the refolding solution; Step b: Place the concentrate obtained in step a into a dialysis bag, place it in a container containing NTA buffer, and dialyze at 4°C for at least 48 h. Place the resulting protein-containing NTA buffer into a dialysis bag with a molecular weight cutoff of 3500D, and concentrate it with PEG20000 until the volume is reduced to 1 / 20 to 1 / 10 of the volume of the refolding solution. Step c: Place the concentrate obtained in step b into a dialysis bag and place it in a container containing NTA buffer for dialysis at 4°C for at least 48 h.

[0014] Preferably, the method for purifying the dialyzed protein in S4 is as follows: the dialyzed protein solution is filtered through a 0.22 μm filter, loaded onto a Ni-NTA column at a flow rate of 1 mL / min, and washed with NTA-0 buffer at pH 8.0 until the eluent is free of protein; elution is performed with 20 mM, 60 mM, 200 mM, and 500 mM imidazole solutions, and the eluent is collected in fractions until the eluent is free of protein; the collected eluent is dialyzed and concentrated. The presence of protein in the eluent can be determined by using G250 detection solution; no color change in the G250 detection solution indicates that the eluent is free of protein.

[0015] A third aspect of the present invention provides the use of the above-mentioned earthworm protein or the earthworm protein prepared by the above-mentioned preparation method in the preparation of anticoagulant drugs, wherein the active ingredient of the anticoagulant drug includes the above-mentioned earthworm protein or the earthworm protein prepared by the above-mentioned preparation method.

[0016] The fourth aspect of the present invention provides the application of the above-mentioned earthworm protein or the earthworm protein prepared by the above-mentioned preparation method in the preparation of surface anticoating coating products, wherein the active ingredient of the surface anticoating coating product includes the above-mentioned earthworm protein or the earthworm protein prepared by the above-mentioned preparation method.

[0017] The beneficial effects of this invention are as follows: 1. The Guangdilong protein provided by this invention is derived from the natural medicinal herb Guangdilong, and has higher safety compared with chemically synthesized anticoagulants.

[0018] 2. The c71616, c128686, c58213, or c118923 proteins in the earthworm protein provided by this invention can effectively inhibit key coagulation factors such as thrombin and fibrin in vitro. The binding free energy with thrombin is less than -10 kcal / mol. Therefore, this earthworm protein can be used to prepare anticoagulant drugs, surface anticoagulant coatings, and other anticoagulant products, providing new molecular basis and application value for the basic research of anticoagulant active substances of earthworm and the in-depth development of earthworm. Attached Figure Description

[0019] Figure 1 This is a small-scale expression and lysis SDS-PAGE electrophoresis image of the positive clone expressing c71616 protein in Example 1 of this invention; in the figure, Lane M: protein marker, Lane 1: control (without IPTG), Lane 2: whole bacterial protein after induced expression and lysis, Lane 3: supernatant protein, and Lane 4: insoluble protein; Figure 2This is a small-scale expression and lysis SDS-PAGE electrophoresis image of the positive clone expressing c128686 protein in Example 1 of this invention; in the figure, Lane M: protein marker, Lane 1: control (without IPTG), Lane 2: whole bacterial protein after induced expression and lysis, Lane 3: supernatant protein, and Lane 4: insoluble protein; Figure 3 This is a small-scale expression and lysis SDS-PAGE electrophoresis image of the positive clone expressing c58213 protein in Example 1 of this invention; in the figure, Lane M: protein marker, Lane 1: control (without IPTG), Lane 2: whole bacterial protein after induced expression and lysis, Lane 3: supernatant protein, and Lane 4: insoluble protein; Figure 4 This is a small-scale expression and lysis SDS-PAGE electrophoresis image of the positive clone expressing c118923 protein in Example 1 of this invention; in the figure, Lane M: protein marker, Lane 1: control (without IPTG), Lane 2: whole bacterial protein after induced expression and lysis, Lane 3: supernatant protein, and Lane 4: insoluble protein; Figure 5 This is an SDS-PAGE electrophoresis image of the purified inclusion body c71616 protein in Example 1 of this invention; in the image, Lane M: protein marker, Lane 1: purified inclusion body protein; Figure 6 This is an SDS-PAGE electrophoresis image of the purified inclusion body c128686 protein in Example 1 of this invention; in the image, Lane M: protein marker, Lane 1: purified inclusion body protein; Figure 7 This is an SDS-PAGE electrophoresis image of the purified inclusion body c58213 protein in Example 1 of this invention; in the image, Lane M: protein marker, Lane 1: purified inclusion body protein; Figure 8 This is an SDS-PAGE electrophoresis image of the purified inclusion body c118923 protein in Example 1 of this invention; in the figure, Lane M: protein marker, Lane 1: purified inclusion body protein; Figure 9 This is an SDS-PAGE electrophoresis image of the refolded and purified c71616 protein in Example 1 of the present invention; in the figure, Lane M: protein marker, Lane 1: refolded and purified protein; Figure 10 This is an SDS-PAGE electrophoresis image of the refolded and purified c128686 protein in Example 1 of this invention; in the figure, Lane M: protein marker, Lane 1: refolded and purified protein; Figure 11 This is an SDS-PAGE electrophoresis image of the refolded and purified c58213 protein in Example 1 of this invention; in the image, Lane M: protein marker, Lane 1: refolded and purified protein; Figure 12 This is an SDS-PAGE electrophoresis image of the refolded and purified c118923 protein in Example 1 of this invention; in the figure, Lane M: protein marker, Lane 1: refolded and purified protein; Figure 13 This is the experimental result of the thrombin titration method in Example 2 of this invention; Figure 14 This is the experimental result of the fiber plate method in Example 3 of this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0021] Currently, most commonly used anticoagulants in clinical practice are chemically synthesized, which have various problems such as significant adverse reactions and high prices. It is necessary to find safer anticoagulants. Naturally derived proteins hold promise as safer anticoagulant active ingredients. Modern pharmacological studies have shown that earthworms possess various effects, including relieving asthma, promoting diuresis, promoting lactation, anti-fibrosis, anti-tumor activity, promoting nerve repair, thrombolysis, and anticoagulation. Guangdilong (Guangdong earthworm) is considered to be of the highest quality due to its characteristics of "white neck and purple body, large size is preferred for medicinal use," and it was the earliest earthworm medicinal material used. The main active components of Guangdilong include small molecule amino acids, nucleosides, bases, and large molecule proteins (approximately 60% to 70% of dry weight), making it a potential source of anticoagulant proteins. However, among the 16,474 earthworm-derived proteins currently included in the Uniprot database, only 53 proteins have anticoagulant and thrombolytic activities, accounting for only 0.32%. Approximately 3,198 proteins can be found in the Lumbriaceae family, but Guangdilong-derived proteins contain only 4 of them. It is evident that there is currently limited research on proteins with anticoagulant activity derived from *Pheretima aspergillum*.

[0022] This invention, through experimental research, obtained a *Pheretima aspergillum* protein with anticoagulant activity. This *Pheretima aspergillum* protein contains at least one of the following proteins: c71616, c128686, c58213, and c118923. The amino acid sequence of c71616 protein is shown in SEQ ID No. 1, the amino acid sequence of c128686 protein is shown in SEQ ID No. 2, the amino acid sequence of c58213 protein is shown in SEQ ID No. 3, and the amino acid sequence of c118923 protein is shown in SEQ ID No. 4. This protein can effectively inhibit key coagulation factors such as thrombin and fibrin in vitro. Its binding free energy with thrombin is less than -10 kcal / mol, exhibiting good binding stability.

[0023] This invention also provides a method for preparing the above-mentioned earthworm protein, specifically including the following steps: S1. Codon optimization is performed on the original coding gene sequences of c71616, c128686, c58213 and c118923 proteins in the prokaryotic expression system to obtain the coding genes of each protein; each coding gene is synthesized and then PCR amplification is performed. S2. Through enzyme digestion and ligation reactions, the PCR amplification products encoding the c71616 and c128686 proteins obtained in S1 were ligated to the pET22b(+) expression vector, and the PCR amplification products encoding the c58213 and c118923 proteins obtained in S1 were ligated to the pET28a(+) expression vector to obtain recombinant plasmids. S3. The recombinant plasmid obtained in S2 was transformed into Escherichia coli BL21(DE3), and positive transformants were screened after culture. S4. The positive transformant is induced to express, the bacterial cells are collected, the bacterial cells are resuspended and lysed, the precipitate is collected, the inclusion body protein is purified and refolded, and the refolded protein is purified to obtain the Guangdilong protein.

[0024] The present invention will be described below through specific embodiments.

[0025] The main experimental reagents used in the following examples are: Pfu DNA polymerase (Thermo Fisher Scientific, USA), Xho I, BamHI, and NdeI (NEB Biotechnology, USA), and T4 DNA ligase (Thermo Fisher Scientific, USA).

[0026] Unless otherwise specified, all other reagents, raw materials, pharmaceuticals, and instruments used in the following examples are commercially available products. The methods used in the following examples are conventional methods in the art.

[0027] Example 1 This embodiment provides a method for preparing a ground worm protein with antithrombin activity.

[0028] 1. Vector plasmid construction 1.1 Gene Synthesis Codon optimization was performed on the coding gene sequences of c71616, c128686, c58213, and c118923 proteins in a prokaryotic expression system. The gene synthesis method was as follows: the gene sequence was divided into multiple short oligonucleotide fragments, and corresponding mixed primers were synthesized. Each pair of adjacent primer fragments had an overlap of approximately 17 bp. These were then amplified in a PCR system, where the disordered primers, under the action of DNA polymerase, formed ordered DNA double strands through base pairing. This DNA double strand served as the template for subsequent PCR reactions.

[0029] 1.2 Gene Amplification The PCR amplification reaction system is shown in Table 1: Table 1 PCR reaction system

[0030] The PCR amplification reaction conditions are shown in Table 2. For specific steps of electrophoresis detection and recovery, please refer to the kit instructions.

[0031] Table 2 PCR amplification reaction conditions

[0032] 1.3 Enzyme digestion of vector and target gene The gene fragments encoding c71616 and c128686 proteins were ligated into the pET22b(+) expression vector; the gene fragments encoding c58213 and c118923 proteins were ligated into the pET28a(+) expression vector. The enzyme digestion conditions of the vector and the target gene are shown in Table 3.

[0033] Table 3 Enzyme digestion conditions between vector and target gene

[0034] Note: Enzyme cleavage sites: c71616, c128686: NdeI XhoI; c58213, c118923: BamHI XhoI.

[0035] React at 37℃ for 1-2 hours; perform electrophoresis detection and recovery of the enzyme digestion products, following the instructions in the kit manual.

[0036] 1.4 Ligation of vector and target gene The ligation conditions between the vector and the target gene are shown in Table 4. The constructed recombinant plasmid was used to transform Escherichia coli BL21(DE3).

[0037] Table 4. Ligation reaction conditions between vector and target gene

[0038] React at 16℃ for 0.5-1h.

[0039] 2. Construction of recombinant bacteria 2.1 Transformation Add the recombinant plasmid constructed in section 1.4 to a tube containing E. coli BL21(DE3) competent cells (25 ng of recombinant plasmid is needed for 50 μL of competent cells). The volume should not exceed 5% of the competent cells. Gently rotate the tube several times to mix the contents and incubate on ice for 30 min. Place the mixture in a circulating water bath heated to 42°C and heat shock for 90 s. Quickly transfer the tube to an ice bath to cool the cells for 1-2 min. Add 200 μL of SOC liquid medium to each tube and heat the medium to 37°C in a water bath. Then transfer the tube to a shaker set to 37°C and incubate at 220 rpm / min for 45 min to allow the cells to recover and express the resistance marker gene encoded by the plasmid. Transfer an appropriate volume (200 μL per 90 mm plate) of transformed competent cells to LB medium containing the corresponding antibiotic. Invert the plates and incubate at 37°C. Plaques will appear after 12-16 h.

[0040] 2.2 Colony PCR Validation Once colonies have grown on the plate, randomly select several for colony PCR verification to detect transformants.

[0041] 2.3 Sequencing Validation Positive clone samples were selected for sequencing verification.

[0042] 2.4 Competent Transformation and Screening of Positive Clones Rosetta (DE3) competent cells were removed from the ultra-low temperature freezer and thawed on ice. Plasmid (5 μg) was added, and the mixture was gently aspirated and incubated on ice for 30 min. The cells were then heat-shocked at 42°C for 90 s and incubated on ice for 1-2 min. 800 μL of preheated LB liquid medium was added, and the cells were incubated at 37°C at 158 ​​rpm for 50-60 min. The cells were centrifuged at 6,000 rpm for 4 min, and a portion of the supernatant (650 μL) was removed. The remaining bacterial culture (150 μL) was resuspended, mixed, and plated onto kanamycin-resistant LB plates. The plates were inverted and incubated at 37°C for 16 h until single colonies (positive clones) appeared.

[0043] 2.5 Low-level expression and identification of positive clones Single colonies containing the recombinant plasmid were selected and transferred to 5 mL of LB broth (strains expressing c71616 and c128686 were ampicillin-resistant; strains expressing c58213 and c118923 were kanamycin-resistant), incubated overnight at 37°C, and preserved at -20°C; then, single colonies containing the recombinant plasmid were transferred to 5 mL of LB broth (ampicillin / kanamycin-resistant), and cultured at 37°C with shaking until OD... 600 Approximately 0.6; 800 μL of bacterial culture was used as a control group, and the remaining bacterial culture was added with IPTG inducer (final concentration 0.1 mM) and cultured at 37℃ with shaking for 4 h; 0.15 mL of bacterial culture from each group was taken, centrifuged at 12,000 rpm for 2 min, and the bacterial pellet was resuspended in 40 μL of 1× loading buffer for lysis. 10 μL was taken for SDS-PAGE analysis. The results are as follows. Figures 1-4 As shown, differential bands (indicated by red rectangles) were visible in the SDS-PAGE assay, indicating that the recombinant protein gene can be expressed in the E. coli expression system after IPTG induction. 3. Protein expression and purification 3.1 High-level protein expression and bacterial rupture detection 100 μL of the bacterial culture stored at -20℃ was inoculated into 100 mL of LB liquid medium (ampicillin / kanamycin resistant) and cultured with shaking for 16 h; 100 mL of the bacterial culture was then inoculated into 2,000 mL of LB liquid medium and cultured at 37℃ until OD reached. 600 Approximately 0.6, lower the culture temperature to 30℃; add IPTG to induce a final concentration of 0.1 mM, and continue incubation at 30℃ with shaking for 8 h; collect the bacterial culture by centrifugation at 8,000 rpm for 3 min, resuspend in 50 mL of pre-cooled NTA-0 buffer solution, add lysozyme (final concentration 0.1 mg / mL), and incubate on ice for 30 min; sonicate the bacterial cells with parameters set as follows: power 200 W, working time 3 s, pause time 4 s, time 25-30 min; centrifuge at 16,000 rpm at 4℃ for 50 min, separate and collect the supernatant and precipitate; take 10 μL of supernatant and precipitate for SDS-PAGE detection, and store the remaining supernatant and precipitate at 4℃ for later use. If the SDS-PAGE result shows that the protein is expressed in the supernatant, purify the supernatant (proteins that do not form inclusion bodies only need to be purified); if the result shows that the protein is expressed in the precipitate, purify the precipitate. SDS-PAGE analysis showed that the molecular weight of recombinant protein c71616 was approximately 29.6 kDa, that of recombinant protein c128686 was approximately 29.4 kDa, that of recombinant protein c58213 was approximately 28.5 kDa, and that of recombinant protein c118923 was approximately 28.7 kDa, all of which were in line with the expected molecular weight of the target protein.

[0044] 3.2 Inclusion body protein purification The precipitate was resuspended in 50 mL of STET buffer, and DTT was added to a final concentration of 1 mM. The precipitate was sonicated to promote the dissolution of contaminating proteins, with parameters set as follows: power 200 W, 3 s on, 3 s off, time 10 min. The precipitate was centrifuged at 10,000 rpm at 4°C for 10 min, and the supernatant was discarded. The above three steps were repeated until the supernatant became clear. The precipitate was resuspended in PBS, and sonicated with parameters set as follows: power 200 W, 3 s on, 3 s off, time 10 min. The precipitate was centrifuged at 16,000 rpm at 4°C for 10 min, and the supernatant was discarded. The inclusion bodies were resuspended in 3 mL of 6M guanidine hydrochloride aqueous solution, and DTT was added to a final concentration of 5 mM. The precipitate was shaken at 220 rpm at 37°C for 4 h until all inclusion bodies were dissolved. The precipitate was centrifuged at 10,000 rpm at 4°C for 10 min, and 10 μL of the supernatant was collected for SDS-PAGE analysis. The electrophoresis pattern is shown below. Figures 5-8 As shown, the fusion protein is of normal size and has high purity, and can be further refolded and purified.

[0045] 3.3 Inclusion body protein refolding (1) Dilute the protein solution obtained from 3.2 with 2 times the volume of 6 M guanidine hydrochloride, and add it dropwise to 200 mL of refolding solution (pH = 8.0) with a syringe at 4°C. Adjust the speed to 1800 r / min and stir for 24 h. Reduce the speed to 1500 r / min and stir for 24 h to obtain a mixed solution. Take 200 mL of the mixed solution and put it into a dialysis bag with a molecular weight cutoff of 3500D. Place it on PEG20000 powder to concentrate the liquid volume in the dialysis bag to 50-100 mL. (2) Put the concentrated solution obtained in step (1) into a dialysis bag, tie both ends tightly, put it into a beaker containing a large amount of NTA buffer, place it in a 4°C refrigerator, stir gently with a stirrer to ensure the external fluid flows, and dialyze for 48 h (during which the dialysate is changed 3 to 4 times). The protein has been basically replaced into the NTA buffer. Place the obtained NTA buffer containing protein into a dialysis bag with a molecular weight cutoff of 3500D, place it on PEG20000 powder, and concentrate the liquid volume in the dialysis bag to 10-20 mL. (3) Put the concentrated solution obtained in step (2) into a dialysis bag, tie both ends tightly, put it into a beaker containing a large amount of NTA buffer, place it in a 4°C refrigerator, stir gently with a stirrer to ensure the external fluid flows, dialyze for 48 h, and store the resulting protein solution at 4°C for later use.

[0046] 3.4 Protein purification Prepare the Ni-NTA column: Pack the Ni-NTA column material and rinse with 3 column volumes of deionized water; --- flush the column with 3 column volumes of 0.1M EDTA; --- wash with 3 column volumes of deionized water; --- rinse with 3 column volumes of NTA-0 buffer (pH 8.0); --- bind with 5 column volumes of 0.1M NiSO4 solution; --- wash with 3 column volumes of equilibration buffer (pH 4.0); --- wash with 3 column volumes of NTA-0 buffer (pH 8.0) until the effluent pH is 8.0.

[0047] The protein solution obtained in step 3.3 was filtered through a 0.22 μm filter and set aside. The prepared Ni-NTA column was loaded with the protein solution at a flow rate of 1 mL / min. The column was washed with NTA-0 buffer (pH 8.0) until the eluent was free of protein (G250 detection solution remained colorless). Elution was performed with 20 mM, 60 mM, 200 mM, and 500 mM imidazole solutions (NTA-0 buffer as solvent), and the eluent was collected in fractions until the G250 detection solution remained colorless. The column material was washed with 3 times its volume of deionized water, and the column was sealed with 20% ethanol. The collected eluent was dialyzed through a dialysis bag with a molecular weight cutoff of 8 kDa. The cutoff solution in the dialysis bag was concentrated to a recombinant protein concentration of 90%. 10 μL of the recombinant protein concentrate was analyzed by SDS-PAGE electrophoresis. The electrophoresis result is shown below. Figures 9-12 As shown.

[0048] Example 2 This embodiment provides a c71616 protein with antithrombin activity. The c71616 protein was synthesized using a conventional solid-phase synthesis method, following the amino acid sequence shown in SEQ ID No. 1.

[0049] Example 3 This embodiment provides a c128686 protein with antithrombin activity. The c128686 protein was synthesized using a conventional solid-phase synthesis method, following the amino acid sequence shown in SEQ ID No. 2.

[0050] Example 4 This embodiment provides a c58213 protein with antithrombin activity. The c58213 protein was synthesized using a conventional solid-phase synthesis method, following the amino acid sequence shown in SEQ ID No. 3.

[0051] Example 5 This embodiment provides a c118923 protein with antithrombin activity. The c118923 protein was synthesized using a conventional solid-phase synthesis method, following the amino acid sequence shown in SEQ ID No. 4.

[0052] Example 6 This embodiment provides a ground worm protein with antithrombin activity, which is composed of at least two of the c71616 protein, c128686 protein, c58213 protein and c118923 protein from Examples 2 to 5.

[0053] Test Example 1 Using GRAMM molecular docking software, c71616, c128686, c58213, and c118923 proteins were molecularly docked with receptor proteins 1FZC (fibrin) and 1DWC (thrombin), respectively, and the binding free energy (ΔG) between these proteins and their receptors was investigated. The results are shown in Table 5.

[0054] Table 5 Molecular docking results

[0055] Test Example 2 The anticoagulant activity of C71616, C128686, C58213, and C118923 proteins was verified and compared using thrombin titration.

[0056] The procedure for the thrombin titration method: Take 100 µL each of the following protein solutions (c71616, c128686, c58213, and c118923, each at a concentration of 0.5 mg / mL) and transfer them to 2 mL centrifuge tubes containing 200 µL of fibrinogen (5 mg / mL, PBS). Incubate the mixture in a 37°C water bath for 5 min. Then, add 2 µL of thrombin (10 U / mL, physiological saline) to the mixture every 4 min until fibrin precipitation is observed. For the blank control, administer an equal volume of distilled water. Perform the experiment three times. Substitute the consumed thrombin solution into the following equation to calculate the anticoagulant activity of the above-mentioned earthworm proteins.

[0057]

[0058] In the formula, U represents the thrombin activity units per 1 g (U / g); C1 is the thrombin solution concentration (µ / mL); C2 is the test sample solution concentration (g / mL); V1 is the volume of thrombin solution consumed (µL); and V2 is the amount of test sample solution added (µL). If the amount of thrombin consumed is less than the blank control, it is considered as a lack of antithrombin activity.

[0059] The experimental results of the thrombin titration method are shown in Figure 13 Compared with the blank control group, the thrombin consumption volume was significantly increased in the c71616 protein, c128686 protein, c58213 protein, and c118923 protein groups. P<0.001); among them, the c71616 protein group consumed a significantly larger volume of thrombin compared to the c128686 protein group ( P (<0.001), there was no difference in thrombin consumption volume between the c58213 protein group and the c118923 protein group. These results indicate that c71616, c128686, c58213, and c118923 proteins all possess antithrombin activity. Among them, c71616 protein exhibits significantly better antithrombin activity than c128686 protein, and c118923 protein exhibits better antithrombin activity than c58213 protein, but the difference is not statistically significant.

[0060] Test Example 3 The thrombolytic activity of C71616, C128686, C58213, and C118923 proteins was evaluated and compared using the fiber plate method.

[0061] Experimental procedure for the fibrin plate method: Place 8 mL of 2 mg / mL fibrinogen solution in a beaker, add 10 mL of 1.5% agarose solution at 55-60℃, stir quickly and evenly, then add 0.75 mL of 1 U / mL thrombin solution. After mixing the above solutions evenly, pour them into a plastic petri dish with a radius of 5 cm, and place horizontally at room temperature for 1-2 h. Punch holes with a 2 mm radius punch and set aside.

[0062] 10 μL of c71616 protein, c128686 protein, c58213 protein and c118923 protein solution with a concentration of 0.5 mg / ml were added to the fiber plate prepared above, and the plate was placed in a constant temperature and humidity incubator and incubated at 37℃ for 18 h before the diameter of the fibrinolytic ring was measured.

[0063] Using physiological saline as a blank control and urokinase (UK) and lumbrokinase (LK) standards as positive controls, the direct fibrinolytic activity was investigated. Experimental results are shown below. Figure 14 Compared with the blank control group, the fibrinolytic ring area increased in the c71616, c128686, c58213, and c118923 protein groups, indicating that these proteins all have the function of directly dissolving fibrin. Furthermore, the fibrinolytic ring area increased in the c71616 protein group compared with the c128686 protein group, and the fibrinolytic ring area increased in the c58213 protein group compared with the c118923 protein group.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of earthworm protein, characterized in that, The *Geopelta purpureus* protein contains at least one of c71616 protein, c128686 protein, c58213 protein, and c118923 protein; the amino acid sequence of the c71616 protein is shown in SEQ ID No. 1, the amino acid sequence of the c128686 protein is shown in SEQ ID No. 2, the amino acid sequence of the c58213 protein is shown in SEQ ID No. 3, and the amino acid sequence of the c118923 protein is shown in SEQ ID No.

4.

2. The earthworm protein according to claim 1, characterized in that, The Guangdilong protein contains at least one of c71616 protein and c58213 protein.

3. The method for preparing the earthworm protein according to claim 1 or 2 specifically includes the following steps: S1. Codon optimization is performed on the original coding gene sequences of c71616, c128686, c58213 and c118923 proteins in the prokaryotic expression system to obtain the coding genes of each protein; each coding gene is synthesized and then PCR amplification is performed. S2. Through enzyme digestion and ligation reactions, the PCR amplification products encoding the c71616 and c128686 proteins obtained in S1 were ligated to the pET22b(+) expression vector, and the PCR amplification products encoding the c58213 and c118923 proteins obtained in S1 were ligated to the pET28a(+) expression vector to obtain recombinant plasmids. S3. The recombinant plasmid obtained in S2 was transformed into Escherichia coli BL21(DE3), and positive transformants were screened after culture. S4. The positive transformant is induced to express, the bacterial cells are collected, the bacterial cells are resuspended and lysed, the precipitate is collected, the inclusion body protein is purified and refolded, and the refolded protein is purified to obtain the Guangdilong protein.

4. The preparation method according to claim 3, characterized in that, The restriction enzyme sites for the enzyme digestion reactions described in S2 are as follows: NdeI and XhoI are used for the PCR amplification products encoding the genes of c71616 and c128686 proteins and for the pET22b(+) expression vector; BamHI and XhoI are used for the PCR amplification products encoding the genes of c58213 and c118923 proteins and for the pET28a(+) expression vector.

5. The preparation method according to claim 3, characterized in that, The method for inducing expression described in S4 is as follows: positive transformants are cultured in LB medium containing 0.1 mM IPTG.

6. The preparation method according to claim 3, characterized in that, The purification method for the inclusion bodies in S4 is as follows: The precipitate is resuspended in STET buffer solution, and DTT is added to a final concentration of 1 mM; sonication is used to promote the dissolution of contaminating proteins, with parameters set as follows: power 200 W, operation for 3 s, pause for 3 s, time 10 min; centrifugation at 10,000 rpm at 4℃ for 10 min, and the supernatant is discarded; the above three steps are repeated until the supernatant is clear; the precipitate is resuspended in PBS, and sonicated, with parameters set as follows: power 200 W, operation for 3 s, pause for 3 s, time 10 min; centrifugation at 16,000 rpm at 4℃ for 10 min, and the supernatant is discarded; the inclusion bodies are resuspended in 6M guanidine hydrochloride aqueous solution, and DTT is added to a final concentration of 5 mM; oscillation is performed at 220 rpm at 37℃ for 4 h until all inclusion bodies are dissolved; centrifugation is performed at 10,000 rpm at 4℃ for 10 min, and the supernatant is collected.

7. The preparation method according to claim 6, characterized in that, The steps for recombination described in S4 are as follows: Step a: Dilute the purified protein solution with 2 times the volume of 6 M guanidine hydrochloride, add it to the refolding solution at pH = 8.0, adjust the speed to 1800 r / min, and stir for at least 24 h; reduce the speed to 1500 r / min and stir for at least 24 h; take the protein solution into a dialysis bag with a molecular weight cutoff of 3500D, and concentrate it with PEG20000 until the volume of the concentrate is 1 / 4 to 1 / 2 of the volume of the refolding solution; Step b: Place the concentrate obtained in step a into a dialysis bag, place it in a container containing NTA buffer, and dialyze at 4°C for at least 48 h. Place the resulting protein-containing NTA buffer into a dialysis bag with a molecular weight cutoff of 3500D, and concentrate it with PEG20000 until the volume is reduced to 1 / 20 to 1 / 10 of the volume of the refolding solution. Step c: Place the concentrate obtained in step b into a dialysis bag and place it in a container containing NTA buffer for dialysis at 4°C for at least 48 h.

8. The preparation method according to claim 7, characterized in that, The method for purifying the dialyzed protein in S4 is as follows: the dialyzed protein solution is filtered through a 0.22 μm filter, loaded onto a Ni-NTA column at a flow rate of 1 mL / min, and washed with NTA-0 buffer at pH 8.0 until the eluent is free of protein; elution is performed with 20 mM, 60 mM, 200 mM and 500 mM imidazole solutions, and the eluent is collected in fractions until the eluent is free of protein; the collected eluent is dialyzed and concentrated.

9. The use of the earthworm protein according to claim 1 or 2, or the earthworm protein prepared by the preparation method according to any one of claims 3 to 8, in the preparation of anticoagulant drugs, characterized in that, The active ingredient of the anticoagulant includes the earthworm protein as described in claim 1 or 2, or the earthworm protein prepared by any one of claims 3 to 8.

10. The application of the *Pheretima aspergillum* protein according to claim 1 or 2, or the *Pheretima aspergillum* protein prepared by the preparation method according to any one of claims 3 to 8, in the preparation of surface anticoating coating products, characterized in that, The active ingredient of the surface anticoating coating product includes the earthworm protein as described in claim 1 or 2, or the earthworm protein prepared by the preparation method described in any one of claims 3 to 8.

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