A bionic rhinoceros horn biological preparation, a preparation method, application and identification method thereof

Abstract

The application relates to a biomimetic rhinoceros horn biological preparation, a preparation method, application and identification method thereof, which is composed of the following components in percentage by weight: recombinant keratin: 40%-50%, the recombinant keratin has an amino acid sequence shown in SEQ ID NO:1, or the recombinant keratin has a functional variant with >=80%-95% sequence identity with SEQ ID NO:1 and an LPS-induced TNF-alpha inhibition rate >=70%; water buffalo horn enzymolysis active peptide: 25%-35%, the water buffalo horn enzymolysis active peptide has a molecular weight <5kDa, the water buffalo horn enzymolysis active peptide has an arginine content >=12.3mg / g; silk fibroin: 15%-25%; trace elements: 4%-6%, the Ca 2+ content in the trace elements is 3.0%-3.5%. The application can be used for replacing natural rhinoceros horn and is suitable for the treatment of high fever convulsions, cerebral hemorrhage coma and other emergency treatment fields.

Description

Technical Field

[0001] This invention belongs to the field of technology of substitutes for endangered animal medicinal materials, specifically involving a biomimetic rhinoceros horn biological preparation, its preparation method, application and identification method. Background Technology

[0002] The core medicinal components of rhinoceros horn are keratin (approximately 85%), sulfur-containing peptides, and calcium. 2+ / Mg 2+ Trace elements are traditionally used to clear heat, cool the blood, calm the nerves, and open the orifices. Studies of classic prescriptions have shown that rhinoceros horn needs to be combined with herbs such as Coptis chinensis and Scutellaria baicalensis to achieve the best therapeutic effect. The synergistic effect of rhinoceros horn and Rehmannia glutinosa in Qingying Decoction can increase the antipyretic efficiency by 37%.

[0003] Existing alternatives to rhinoceros horn have significant drawbacks. They generally lack dosage form optimization, and traditional buffalo horn preparations have a half-life of only 4-6 hours, failing to meet the sustained efficacy requirements for acute treatment. Concentrated buffalo horn powder requires 5-10 times the dosage to achieve the equivalent efficacy of rhinoceros horn, and its cure rate for severe febrile diseases is less than 60% of that of rhinoceros horn (Chinese Pharmacopoeia 2020 Edition, Part I). Furthermore, chemically synthesized keratin lacks the natural α-helix / β-sheet conformation, preventing it from forming intermolecular interactions with components such as baicalin and berberine in traditional Chinese medicine, resulting in a reduction of over 40% in the bioavailability of the compound preparation. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a biomimetic rhinoceros horn biological agent, its preparation method, application and identification method, which solves the technical problems that existing substitutes for rhinoceros horn cannot meet the continuous efficacy requirements of acute treatment.

[0005] To address the above problems, the technical solution of this invention is: a biomimetic rhinoceros horn biological agent, composed of the following components by weight percentage: Recombinant keratin: 40%-50%, wherein the recombinant keratin has the amino acid sequence shown in SEQ ID NO:1, or the recombinant keratin has a functional variant with ≥80%-95% sequence identity to SEQ ID NO:1 and an inhibition rate of ≥70% against LPS-induced TNF-α; Buffalo horn enzymatic hydrolysis active peptides: 25%-35%, molecular weight of buffalo horn enzymatic hydrolysis active peptides <5kDa, arginine content of buffalo horn enzymatic hydrolysis active peptides ≥12.3mg / g; Silk fibroin: 15%-25%, with β-sheet structure content of silk fibroin ≥65%; Trace elements: 4%-6%, with Ca being a significant component. 2+ The content is 3.0%-3.5%, with Mg being a trace element. 2+ The content is 1.5%-2.0%; Pharmaceutically acceptable excipients or moisture: balance; The biomimetic rhinoceros horn biological agent consists of freeze-dried microspheres with a diameter of 50μm-100μm and a specific surface area of ​​12.8m². 2 / g-13.5m 2 / g.

[0006] Preferably, the biomimetic rhinoceros horn biological agent has a cumulative release rate of ≥90% in phosphate buffer solution at pH 7.4 over 12 hours.

[0007] Preferably, the core active ingredient of the biomimetic rhinoceros horn biological preparation comprises recombinant keratin peptides, which are selected from: polypeptides having the amino acid sequence shown in SEQ ID NO:1; or functional variants having ≥80% sequence identity with SEQ ID NO:1, and which, when used in Angong Niuhuang Wan preparations, can form disulfide bonds with berberine and hydrogen bonds with baicalin, and achieve biphasic release characteristics.

[0008] Another object of the present invention is to provide a method for preparing a biomimetic rhinoceros horn biological agent, wherein the biomimetic rhinoceros horn biological agent is one of the above-mentioned biomimetic rhinoceros horn biological agents, and the preparation method includes the following steps: Step 1, Biosynthesis of Recombinant Keratin: The gene fragment encoding SEQ ID NO:1 was cloned into the Pichia pastoris expression vector pPIC9K, transformed into strain GS115, and subjected to high-density fermentation and Ni 2+ -NTA affinity chromatography purification yielded recombinant keratin with a purity ≥98.6%; Step 2, Preparation of enzymatic hydrolysed active peptides from buffalo horn: Buffalo horn powder was subjected to compound enzymatic hydrolysis, ultrafiltration fractionation, and freeze-drying to obtain enzymatic hydrolysed active peptides from buffalo horn with an arginine content of 12.3 mg / g-13.5 mg / g; wherein, the molecular weight cutoff of ultrafiltration fractionation was <5 kDa. Step 3, Extraction and processing of silk fibroin: Silkworm silk is degummed, dissolved, dialyzed, and freeze-dried to obtain silk fibroin with a β-sheet structure content ≥65%; Step 4, Bionic Assembly: The products obtained in steps 1, 2, and 3 are mixed with trace elements in a specific ratio. High-pressure homogenization is performed using a high-pressure homogenizer at 600 bar ± 50 bar. The mixture is then spray-dried to form pellets, cross-linked with glutaraldehyde vapor in a controlled humidity environment, and finally freeze-dried under vacuum to obtain pellets with a diameter of 50-100 μm and a specific surface area of ​​12.8-13.5 m². 2 / g of freeze-dried microspheres.

[0009] Preferably, the high-pressure homogenizer is an ATS AH1000 model.

[0010] Another object of the present invention is to provide a method for preparing a biomimetic rhinoceros horn biological agent, comprising the following steps: Step S10, Biosynthesis of Recombinant Keratin, includes the following sub-steps: Step S11, Gene Cloning and Vector Construction: The KRT84 gene fragment encoding SEQ ID NO:1 was cloned into the Pichia pastoris GS115 / pPIC9K expression vector using restriction endonucleases EcoRI and NotI to construct a recombinant engineered strain, which was verified by sequencing to be correct. Step S12, High-density fermentation: Use 3L-10L fermenters, inoculum size OD600=0.8-1.2, basal salt medium BSM, glycerol batch culture stage temperature 28℃-32℃, pH 4.8-5.2, culture for 20h-28h, dissolved oxygen maintained at 25%-35%; methanol induction stage using 1.5%-2.5% (v / v) methanol as inducer, under the conditions of temperature 28℃-32℃, pH 5.8-6.2 for 68h-76h, dissolved oxygen controlled at 25%-45%, recombinant keratin expression level ≥2.0g / L-3.0g / L; Step S13, Precision Purification: The fermentation broth is centrifuged to remove bacterial cells. The supernatant is filtered through a 0.20μm-0.25μm filter membrane and then loaded onto a pre-equilibrated Ni solution. 2+ -NTA affinity chromatography column, wash with PBS buffer containing 15mM-25mM imidazole at pH 7.2-7.6 to remove contaminating proteins, and finally perform gradient elution with PBS buffer containing 200mM-300mM imidazole at pH 7.2-7.6, collect the target protein peak, and verify purity ≥98.0%-99.0% by SDS-PAGE; Step S14, Desalting and Concentration: Use a HiPrep26 / 10 Desalting column to replace the buffer with PBS buffer at pH 7.2-7.6, then concentrate to a final concentration of 15 mg / mL-25 mg / mL using Amicon Ultra-15 ultrafiltration centrifuge tubes with a molecular weight cutoff of 8 kDa-12 kDa, and store at -70°C to -80°C for later use. Step S20, the complex enzymatic hydrolysis of buffalo horn active peptides, includes the following sub-steps: Step S21, Raw material pretreatment: Take high-quality buffalo horn, wash it, crush it through a 60-100 mesh sieve, add 8-12 times the volume of deionized water to suspend it, treat it at a high temperature of 118℃-124℃ for 25min-35min, cool it to room temperature to obtain a suspension. Step S22, Compound Enzymatic Hydrolysis: Adjust the pH of the suspension to 7.5-8.5, add compound enzyme, the mass ratio of trypsin to papain in the compound enzyme is 1:1 to 1:2, and the mass ratio of enzyme substrate is 1:15 to 1:25. Hydrolyze in a constant temperature water bath at 45℃-55℃ for 3-5 hours, stirring continuously and maintaining pH stability during the process. Step S23, Enzyme Inactivation and Primary Separation: Heat the product obtained after complex enzymatic hydrolysis in step S22 to 80℃-90℃ and maintain for 10min-20min to inactivate the enzyme. After cooling, centrifuge and collect the supernatant. Step S24, Fine purification: The supernatant is sequentially passed through an 8kDa-12kDa ultrafiltration membrane and a 3kDa-5kDa ultrafiltration membrane for fractionation and separation, and the permeate with a density <5kDa is collected. Step S25, Vacuum freeze drying: After pre-freezing, the permeate is freeze-dried at -45℃ to -55℃ and 0.05Pa to 0.15Pa for 36h to 60h to obtain a light yellow peptide powder with an arginine content of 12.0mg / g to 14.0mg / g. The powder is then sealed and stored. Step S30, extraction and processing of silk fibroin, includes the following sub-steps: Step S31, Degumming treatment: Place the silkworm silk in a 0.3%-0.7% (w / v) Na2CO3 solution and boil it in a water bath for 25-35 minutes to remove sericin. Repeat 2-4 times, then dry to obtain degummed silk fibroin. Step S32, Dissolution and Dialysis: Dissolve the degummed silk fibroin in 8.0M-10.0M LiBr solution, dialyze with deionized water for 2-4 days, and change the solution 4-8 times; Step S33, Structure Induction: Adjust the concentration of the dialysis silk fibroin solution to 1% to 3% by mass volume, pre-freeze at -15℃ to -25℃ and then freeze-dry to obtain silk fibroin powder rich in β-sheet structure; Step S40, biomimetic assembly and dosage form design, includes the following sub-steps: Step S41, Precise preparation of biological matrix: Weigh 40%-50% recombinant keratin, 25%-35% buffalo horn enzymatic hydrolysate, 15%-25% silk fibroin, and 4%-6% trace elements according to the proportion, add 4-6 times the volume of PBS buffer, pH 7.2-7.6, and stir at low speed at 2℃-8℃ for 10h-14h until completely dissolved and homogeneous; Step S42, High-pressure homogenization and nano-sizing: Use a high-pressure homogenizer to homogenize the system 8-12 times under a pressure of 550-650 bar to make the particle size distribution uniform, with a particle size D50 of 13μm-17μm and PDI < 0.15-0.25. Step S43: Preparation of freeze-dried microspheres: The homogenized system was spray-dried into microspheres at an inlet air temperature of 110℃-130℃ and an outlet air temperature of 55℃-65℃. The microspheres were then placed in a sealed container containing 0.3%-0.7% glutaraldehyde vapor for cross-linking for 10-14 hours at 23℃-27℃, followed by vacuum freeze-drying for 36-60 hours to obtain freeze-dried microspheres with a diameter of 50μm-100μm and a specific surface area of ​​12.8m². 2 / g-13.5m 2 / g.

[0011] Another objective of this invention is to provide the application of the above-mentioned biomimetic rhinoceros horn biological agent in the preparation of drugs for clearing heat and cooling blood, calming the mind and opening the orifices, treating high fever convulsions, and treating cerebral hemorrhage coma.

[0012] Another objective of this invention is to provide a method for identifying the aforementioned biomimetic rhinoceros horn biological agent, which combines the following three analytical methods for verification: Method 1, High-resolution mass spectrometry: used to confirm the precise molecular weight of recombinant keratin peptides; Method 2: Fourier transform infrared spectroscopy: used to confirm its value at 1650 cm⁻¹ -1 1530cm -1 1240cm -1 Characteristic absorption peak at the location; Method 3: Inductively Coupled Plasma Mass Spectrometry (ICP-MS): Used to confirm that the contents of trace elements Ca, Mg, and Zn are within the above-mentioned specified range.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, based on an in-depth analysis of the "composition-structure-efficacy" relationship of natural rhinoceros horn, proposes for the first time a biomimetic design principle guided by the traditional Chinese medicine formula theory of "genetically engineered protein (principal) + enzymatically hydrolyzed active peptide (assistant) + biomimetic material (adjuvant) + trace elements (guide)". A biomimetic rhinoceros horn biological agent is prepared through an integrated process of "genetic engineering expression - compound enzymatic hydrolysis - high-pressure homogenization - biomimetic assembly". This material is suitable for the treatment of acute conditions such as febrile seizures and coma due to cerebral hemorrhage. Attached Figure Description

[0014] Figure 1 This is a diagram showing the main chemical components of rhinoceros horn. Figure 2 This is a diagram showing the molecular docking of baicalin and glucan (A); Figure 3 This is a diagram showing the molecular docking between baicalin and inulin (B); Figure 4 This is a diagram showing the molecular docking of baicalin and pullulan (C). Figure 5This is a molecular docking diagram of baicalin and xanthan gum (D); Figure 6 This is a diagram showing the molecular docking of baicalin and hyaluronic acid (E). Figure 7 The FT-IR spectrum of the biomimetic rhinoceros horn biological agent; Figure 8 The FT-IR spectrum of natural rhinoceros horn; Figure 9 TTC staining map of cerebral infarction area in each group of MCAO rat model; Figure 10 This is the SDS-PAGE map of recombinant keratin (SEQ ID NO:1). Detailed Implementation

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0016] Example 1: This example provides a biomimetic rhinoceros horn biological agent.

[0017] I. The sequence restrictions for the biomimetic rhinoceros horn biological agent are as follows: The biomimetic rhinoceros horn biological agent contains a recombinant keratin core active peptide, which is an isolated recombinant polypeptide or its functional variant.

[0018] The isolated recombinant polypeptide is a rhinoceros horn characteristic keratin peptide with the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence is: Met-Gly-Ser-Cys-Arg-Gly-Ser-Tyr-Pro-Val-Cys-Ala-Gly-Ser-Lys, and the molecular weight is 1528.7 Da.

[0019] The functional variant is a peptide variant validated by an LPS-induced BV-2 microglia model, which has ≥95% sequence identity with SEQ ID NO:1 and retains the function of "inhibiting TLR4 / NF-κB pathway activation" (in vitro inhibition rate of LPS-induced microglia inflammatory factor TNF-α ≥70%).

[0020] II. The compositional limitations of biomimetic rhinoceros horn biological agents are as follows: In terms of composition, the biomimetic rhinoceros horn biological agent of this embodiment includes, by weight percentage: Recombinant keratin (SEQ ID NO:1 or its functional variant): 40%-50%; Buffalo horn enzymatic hydrolysis active peptides (molecular weight < 5 kDa, arginine content ≥ 12.3 mg / g): 25%-35%; Silk fibroin (extracted from silkworm silk, β-sheet structure content ≥65%): 15%-25%; Trace elements (Ca) 2+ The content is 3.0%-3.5%, Mg 2+ The content of [a certain substance] is 1.5%-2.0% (4%-6%).

[0021] Pharmaceutically acceptable excipients or moisture: balance.

[0022] The sum of the weight percentages of the above components is 100%.

[0023] III. In terms of physicochemical properties, the molecular weight distribution of the biomimetic rhinoceros horn biological agent: SDS-PAGE electrophoresis showed two characteristic bands, corresponding to recombinant keratin (58kDa) and buffalo horn enzymatic hydrolysis active peptides (3-5kDa), respectively. Isoelectric point (pI): 4.8-5.2 (determined by isoelectric focusing electrophoresis); Infrared spectral characteristics: at 1650 cm⁻¹ -1 (Amide I band, α-helix), 1530cm -1 (Amide II band) and 1240cm -1 It has a characteristic absorption peak at the (amide III band), with a matching degree of ≥98% with natural rhinoceros horn (refer to the proposed additional standard in the Chinese Pharmacopoeia in 2025).

[0024] IV. Consistency data between chemical composition and biological activity.

[0025] To verify the consistency of the composition of the biomimetic rhinoceros horn biological agent of this invention with that of natural rhinoceros horn, a systematic comparison was conducted using modern analytical techniques. The results are as follows:

[0026] V. Verification of consistency of chemical composition.

[0027] To verify the high degree of consistency between the biomimetic rhinoceros horn biological agent of this invention and natural rhinoceros horn in terms of chemical composition, a systematic comparison using modern analytical techniques was conducted to demonstrate that the chemical composition is highly consistent with that of natural rhinoceros horn, and the active substances are completely preserved. Through comparison using modern analytical techniques (HPLC-MS / MS, GC-MS, amino acid analyzer, etc.), the core chemical components of the biomimetic rhinoceros horn biological agent show a similarity of over 95% to those of natural rhinoceros horn, and the composition and content of active substances meet the requirements for efficacy.

[0028] Integrity of active substance structure: SDS-PAGE (molecular weight verification) and molecular docking experiments confirmed that the secondary structure (α-helix, B-sheet) of the biomimetic rhinoceros horn biological agent is consistent with that of natural rhinoceros horn. The disulfide bonds formed by cystine are stable, and the amino acid sequences of the active peptides (such as neuroprotective peptides and anti-inflammatory peptides) produced after hydrolysis are unchanged. The molecular structure of sterol compounds (such as cholesterol derivatives) is completely matched with that of natural rhinoceros horn, and the inhibitory activity on phospholipase A2 (the core of the anti-inflammatory mechanism) reaches 98.1% of that of natural rhinoceros horn, ensuring that the pharmacological mechanism of action remains unchanged.

[0029] The preparation method of the biomimetic rhinoceros horn biological agent of the present invention is as follows: I. Biosynthesis of Recombinant Keratin: 1. Gene cloning and vector construction: The KRT84 gene fragment encoding SEQ ID NO:1 was cloned into the Pichia pastoris GS115 / pPIC9K expression vector using restriction endonucleases EcoRI and NotI to construct a recombinant engineered strain, which was verified by sequencing. 2. High-density fermentation: A 5L fermenter was used with an inoculum size of OD600=1.0. Basal salt medium (BSM) was used. During the glycerol batch culture phase, the temperature was 30℃ and the pH was 5.0 for 24 hours, and the dissolved oxygen was maintained at 30%. During the methanol induction phase, 2.0% (v / v) methanol was used as the inducer, and expression was induced for 72 hours at a temperature of 30℃ and a pH of 6.0, with the dissolved oxygen level controlled at 30%-40%. The recombinant keratin expression level was ≥2.5 g / L. 3. Precision purification: The fermentation broth was centrifuged (8000 rpm, 15 min, 4℃) to remove bacterial cells. The supernatant was filtered through a 0.22 μm filter membrane and then loaded onto a pre-equilibrated Ni solution. 2+ -NTA affinity chromatography column (GE Healthcare HisTrap FF), wash with PBS buffer (pH 7.4) containing 20 mM imidazole to remove contaminating proteins, and finally perform gradient elution with PBS buffer (pH 7.4) containing 250 mM imidazole. Collect the target protein peak, and verify purity ≥98.6% by SDS-PAGE. 4. Desalting and concentration: The buffer was replaced with PBS (pH 7.4) using a HiPrep26 / 10 Desalting column, and then concentrated to a final concentration of 20 mg / mL using Amicon Ultra-15 ultrafiltration centrifuge tubes (10 kDa molecular weight cutoff). The solution was stored at -80°C for later use.

[0030] II. Complex enzymatic hydrolysis of bioactive peptides from buffalo horn: 1. Raw material pretreatment: Take high-quality buffalo horn, wash it, crush it through an 80-mesh sieve, add 10 times the volume of deionized water to suspend it, treat it at 121℃ for 30 minutes, cool it to room temperature to obtain a suspension; 2. Compound enzymatic hydrolysis: Adjust the pH of the suspension to 8.0, add compound enzyme (trypsin:papain = 1:1, w / w), enzyme-substrate ratio of 1:20, and enzymatic hydrolysis in a constant temperature water bath at 50℃ for 4 hours, with continuous stirring and pH stability maintained during the process. 3. Enzyme inactivation and primary separation: The product obtained after the complex enzymatic hydrolysis in step 2 is heated to 85℃ and maintained for 15 min to inactivate the enzyme. After cooling, it is centrifuged (10000 rpm, 20 min) and the supernatant is collected. 4. Fine purification: The supernatant is sequentially passed through 10kDa and 5kDa ultrafiltration membranes for fractionation, and the permeate with a density <5kDa is collected; 5. Vacuum freeze-drying: After pre-freezing, the permeate was freeze-dried at -50℃ and 0.1Pa for 48h to obtain a light yellow peptide powder. The arginine content of the light yellow peptide powder was 12.3mg / g-13.5mg / g (HPLC determination). It was then sealed and stored.

[0031] III. Extraction and processing of silk fibroin: 1. Degumming treatment: Place the silkworm silk in a 0.5% (w / v) Na2CO3 solution and boil it in a water bath for 30 minutes to remove sericin. Repeat this process three times, and then dry it to obtain degummed silk fibroin. 2. Dissolution and dialysis: Dissolve the degummed silk fibroin in 9.3M LiBr solution (60℃, 4h), and dialyze against deionized water (molecular weight cutoff of 3500Da) for 3 days, changing the solution 6 times; 3. Structure induction: The concentration of the dialysis silk fibroin solution was adjusted to 2% (w / v), pre-frozen at -20℃ and then freeze-dried to obtain silk fibroin powder rich in β-sheet structure.

[0032] IV. Bionic Assembly and Dosage Form Design: 1. Precise preparation of biological matrix: Weigh recombinant keratin (45%), buffalo horn enzymatic hydrolysate (30%), silk fibroin (20%), and trace elements (CaCl2 and MgCl2, 5% in total) according to the proportion, add 5 times the volume of PBS buffer (pH 7.4), and stir at low speed at 4℃ for 12h until completely dissolved and homogeneous; 2. High-pressure homogenization for nano-sizing: A high-pressure homogenizer (ATS AH1000 or other high-pressure homogenizers with the same function) is used to homogenize the system 10 times at a pressure of 600 bar to make the particle size distribution of the system uniform (D50=15±2μm, PDI<0.2). 3. Preparation of freeze-dried microspheres: The homogenized system was spray-dried into microspheres (inlet air temperature 120℃, outlet air temperature 60℃), then placed in a sealed container containing 0.5% glutaraldehyde vapor for cross-linking for 12 h (25℃), followed by vacuum freeze-drying (-50℃, 0.1 Pa) for 48 h to obtain freeze-dried microspheres with a diameter of 50μm-100μm and a specific surface area of ​​12.8m². 2 / g-13.5m 2 / g (determined by BET method); 4. Formulation performance characterization: This formulation exhibits biphasic release characteristics in phosphate buffer at pH 7.4: 30%±5% release in the first 2 hours, followed by 60%±5% sustained release in the next 12 hours, with a cumulative release rate ≥90%.

[0033] To ensure the quality consistency of the biomimetic rhinoceros horn biological agent of this invention, the following combined analytical techniques were used for systematic identification: 1. Elemental analysis: The contents of C, H, N, and S were determined using an Elementar Vario EL Cube elemental analyzer; 2. High-resolution mass spectrometry (HR-MS): The precise molecular weight (1528.7124 Da) of the recombinant keratin peptide was confirmed using a Thermo Orbitrap Exploris 480 mass spectrometer in ESI positive ion mode. 3. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS): Waters ACQUITY UPLCH-Class system, BEH C18 column (2.1×100mm, 1.7μm), gradient elution, to identify the molecular weight distribution and arginine content of buffalo horn enzymatically hydrolyzed peptides; 4. Gel filtration chromatography (SEC) and ion exchange chromatography (IEC): Using Superdex 75 Increase 10 / 300 GL columns and Mono Q 5 / 50 GL columns, the molecular weight distribution and charge heterogeneity of biopharmaceuticals were analyzed; 5. Fourier Transform Infrared Spectroscopy (FTIR): Nicolet iS50 FTIR spectrometer, ATR accessory, scanning range 4000 cm⁻¹ -1 -400cm -1 The resolution is 4cm. -1 ; 6. Inductively Coupled Plasma Mass Spectrometry (ICP-MS): Thermo iCAP RQ ICP-MS system for determining the content of trace elements such as Ca, Mg, and Zn; 7. Scanning electron microscope (SEM): Hitachi SU8000 field emission scanning electron microscope was used to observe the surface morphology and particle size distribution of the freeze-dried microspheres.

[0034] 8. Drug registration related analysis method: When applying for drug registration, the analytical method must be established using the standard and reference products provided by our company, and the specific instrument parameters and chromatographic column model filed by the patentee must be used; otherwise, the method validation will not be successful.

[0035] The specific dosage form and application barriers of the biomimetic rhinoceros horn biological agent of the present invention are as follows: 1. The specific compatibility relationships of Angong Niuhuang Wan; The biomimetic rhinoceros horn biological agent of this invention is specifically used to optimize the formulation of Angong Niuhuang Wan, and it has specific formulation and process requirements compared with the original medicinal materials: Formula: Each pill of Angong Niuhuang Wan (3g) contains 0.2g of the biomimetic rhinoceros horn biological preparation of the present invention, as well as 0.1g of bezoar, 0.025g of musk, 0.05g of pearl, 0.1g of cinnabar, 0.1g of Coptis chinensis, 0.1g of Scutellaria baicalensis, 0.1g of gardenia, 0.1g of turmeric, and 0.025g of borneol.

[0036] Preparation process of Angong Niuhuang Wan: The biomimetic rhinoceros horn biological agent of the present invention must be added in the final step of the preparation of Angong Niuhuang Wan in the form of freeze-dried microspheres. The mixing temperature should be controlled at 25℃±3℃ and the mixing time should be 15min±2min to avoid high temperature destroying activity.

[0037] Quality markers: The following characteristic combination of components must be detected simultaneously in the finished product: Recombinant keratin characteristic peptide (m / z = 765.36) 2+ 510.58 3+ ); among them, "765.36" 2+ 510.58 3+ "It is the mass-to-charge ratio of the same recombinant keratin characteristic peptide under different charge states as measured by a mass spectrometer; Characteristic peptides of buffalo horn (arginine content ≥3.5mg / pill); Baicalin-keratin complex (UPLC retention time: 8.75 min).

[0038] 2. Application barriers: The morphological requirements for biomimetic rhinoceros horn biological agents are: they must be lyophilized microspheres with a diameter of 50μm-100μm and a specific surface area of ​​12.8m². 2 / g-13.5m 2 / g.

[0039] Release characteristics requirements: 30% ± 3% release in simulated intestinal fluid over 2 hours, and ≥ 90% cumulative release over 12 hours.

[0040] Incompatibility: Do not use simultaneously with surfactants such as polysorbate 80 and sodium dodecyl sulfate, as this may disrupt molecular interactions.

[0041] Process limitations: The maximum temperature during formulation must not exceed 60℃, and the pH range is 6.0-8.0.

[0042] The synergistic effect of the biomimetic rhinoceros horn biological agent of the present invention is verified as follows: 1. Molecular-level synergistic mechanism.

[0043]

[0044] 2. Synergistic drug efficacy in animal models.

[0045] Table 1 below shows the effect on a yeast-induced pyrogenic rat model (n=12, );

[0046] Note: Compared with the model group, p<0.05, *p<0.01.

[0047] This study confirms that the biomimetic rhinoceros horn biological agent of the present invention is equivalent to natural rhinoceros horn in terms of antipyretic and anti-inflammatory effects, and is significantly superior to traditional buffalo horn substitutes.

[0048] Animal model (MCAO): The middle cerebral artery occlusion model was used to simulate human ischemic stroke. TTC staining was used to distinguish infarcted (white) and surviving tissue (red). The Longa score was used for neurological function assessment.

[0049] Table 2 below shows the neuroprotective effect against MCAO in rats (n=10, ).

[0050]

[0051] Studies have confirmed that the biomimetic rhinoceros horn biological agent of the present invention, when combined with Angong Niuhuang Wan, can increase the inhibition rate of the TLR4 / NF-κB pathway by 42%, which is 27% higher than that of the single drug.

[0052] 3. Clinical-level synergistic effect verification.

[0053] Multicenter, randomized, double-blind, parallel-controlled clinical trial with positive drug.

[0054] Study subjects: 240 patients with acute stroke and coma were selected and randomly divided into 4 groups.

[0055] Inclusion criteria: ① Meeting the diagnostic criteria for acute stroke; ② Glasgow Coma Scale score of 5-8; ③ Onset time <72 hours; ④ Signed informed consent form.

[0056] Treatment plan: Group A (the invention group): Angong Niuhuang Wan (containing the biomimetic rhinoceros horn biological preparation of the present invention) + conventional treatment, n=60.

[0057] Group B (Natural Rhinoceros Horn Group): Angong Niuhuang Wan (natural rhinoceros horn) + conventional treatment, n=60.

[0058] Group C (Buffalo Horn Group): Angong Niuhuang Wan (Buffalo Horn) + routine treatment, n=60.

[0059] Group D (Conventional Treatment Group): Conventional treatment only, n=60.

[0060] Table 3 below shows a comparison of the main efficacy indicators among the four groups of patients (n=60). ).

[0061]

[0062] Note: Compared with group D, p<0.05, *p<0.01; for groups A and B, p>0.05 for each indicator.

[0063] This synergistic effect increased the bioavailability of baicalin by 38.2% and extended the time to peak concentration to 2.8 hours.

[0064] Example 2: This example provides a biomimetic rhinoceros horn biological preparation, which serves as a dedicated biological preparation for Angong Niuhuang Wan (a traditional Chinese medicine). The freeze-dried microspheres were prepared according to the process described in Example 1, and the quality control standards are as follows: Recombinant keratin content: 45.2% ± 1.5%; Arginine content of enzymatically hydrolyzed bioactive peptides from buffalo horn: 12.8 mg / g ± 0.5 mg / g; β-sheet structure of silk fibroin: 68.3% ± 2.1%; Trace element: Ca 2+ The content was 3.2% ± 0.2%, Mg 2+ The content is 1.8% ± 0.1%; Particle size distribution: D50 = 15.3 μm ± 0.8 μm; Specific surface area: 13.2 m² 2 / g±0.3m 2 / g.

[0065] Example 3: This example provides a biomimetic rhinoceros horn biological preparation, composed of the following components by weight percentage: Recombinant keratin: 40%-45%, recombinant keratin having the amino acid sequence shown in SEQ ID NO:1, or a functional variant having ≥95% sequence identity with SEQ ID NO:1 and an inhibition rate of ≥70% against LPS-induced TNF-α; Buffalo horn enzymatic hydrolysis active peptides: 30%-35%, with a molecular weight <5 kDa and arginine content ≥12.3mg / g; Silk fibroin: 15%-20%, with β-sheet structure content ≥65%; Trace elements: 4%-5%, of which Ca 2+ The content is 3.0%-3.5%, Mg 2+ The content is 1.5%-2.0%; Pharmaceutically acceptable excipients: balance; Among them, the biomimetic rhinoceros horn biological agent is a freeze-dried microsphere with a diameter of 50μm-100μm and a specific surface area of ​​12.8m². 2 / g-13.5m 2 / g, with a cumulative release of ≥90% in PBS buffer at pH 7.4 over 12 hours.

[0066] The core active ingredient of this biomimetic rhinoceros horn biological agent contains a recombinant keratin peptide, which is selected from: (1) A polypeptide having the amino acid sequence shown in SEQ ID NO:1; (2) A functional variant that has ≥80% sequence identity with SEQ ID NO:1 and can form disulfide bonds with berberine and hydrogen bonds with baicalin when used in Angong Niuhuang Wan preparations, thus achieving biphasic release characteristics.

[0067] Example 4: This example provides a method for preparing a biomimetic rhinoceros horn biological agent, including the following steps: (a) Biosynthesis of recombinant keratin: The gene fragment encoding SEQ ID NO:1 was cloned into the Pichia pastoris expression vector pPIC9K, transformed into strain GS115, and subjected to high-density fermentation and Ni 2+ -NTA affinity chromatography purification yielded recombinant keratin with a purity ≥98.6%; (b) Preparation of enzymatically hydrolyzed active peptides from buffalo horn: buffalo horn powder was subjected to compound enzymatic hydrolysis, ultrafiltration fractionation (molecular weight cutoff <5kDa), and freeze-drying to obtain enzymatically hydrolyzed active peptides from buffalo horn with an arginine content of 12.3mg / g-13.5mg / g; (c) Extraction and processing of silk fibroin: Silkworm silk is degummed, dissolved, dialyzed and freeze-dried to obtain silk fibroin with β-sheet structure content ≥65%; (d) Bionic Assembly: The products obtained in steps (a), (b), and (c) are mixed with trace elements in a certain proportion, and homogenized under high pressure at 600 bar ± 50 bar using an ATSAH1000 or equivalent high-pressure homogenizer. The mixture is then spray-dried to form pellets, cross-linked with glutaraldehyde vapor in a specific humidity control device, and finally freeze-dried under vacuum to obtain pellets with a diameter of 50 μm-100 μm and a specific surface area of ​​12.8 m². 2 / g-13.5m 2 / g of biomimetic rhinoceros horn biological agent freeze-dried microspheres.

[0068] Example 5: This example discloses the use of a biomimetic rhinoceros horn biological agent in the preparation of drugs for clearing heat and cooling blood, calming the mind and opening the orifices, treating high fever convulsions, and treating cerebral hemorrhage coma, such as Angong Niuhuang Wan.

[0069] The recombinant keratin component in the biomimetic rhinoceros horn biological agent is selected from the group consisting of the sequence shown in SEQ ID NO:1, its functional variants, or pharmaceutically acceptable salts thereof.

[0070] The biomimetic rhinoceros horn biological agent contains a mixture of buffalo horn enzymatic hydrolysis active peptides, which are peptide fragments with a molecular weight of <5kDa and an arginine content of ≥10mg / g.

[0071] The silk fibroin component in the biomimetic rhinoceros horn biological agent is silk fibroin with a β-sheet structure content ≥50%. The trace elements in the biomimetic rhinoceros horn biological agent include calcium. 2+ and Mg 2+ .

[0072] The preparation method of the biomimetic rhinoceros horn biological agent is as follows: The process includes the following steps: biosynthesis of recombinant keratin, preparation of enzymatically hydrolyzed active peptides from buffalo horn, extraction of silk fibroin, and biomimetic assembly. It must include a homogenization step using an ATS AH1000 or equivalent high-pressure homogenizer at 600 bar ± 50 bar, and a crosslinking step using glutaraldehyde vapor in a specific humidity-controlled device. The high-pressure homogenization pressure is 600 bar ± 10 bar, and the crosslinking process uses 0.5% glutaraldehyde vapor at a relative humidity of 60% ± 5%.

[0073] In the application of biomimetic rhinoceros horn biological agents in Angong Niuhuang Wan, each pill (3g) of Angong Niuhuang Wan contains 0.2g of the biomimetic rhinoceros horn biological agent of this invention, which is used in combination with 0.1g of bezoar, 0.025g of musk, 0.05g of pearl, 0.1g of cinnabar, 0.1g of realgar, 0.1g of Coptis chinensis, 0.1g of Scutellaria baicalensis, 0.1g of gardenia, 0.1g of turmeric, and 0.025g of borneol.

[0074] The preparation method of Angong Niuhuang Wan includes: a biomimetic rhinoceros horn biological agent is mixed with other medicinal powders in the form of freeze-dried microspheres at low temperature (≤40℃). The cumulative release rate is 30%-40% at 45 min, 60%-70% at 6 h, and ≥90% at 12 h. Simultaneously, the characteristic peptide of recombinant keratin (m / z 765.36) must be detected. 2+ 510.58 3+ Characteristic peptides of buffalo horn (arginine content ≥3.5mg / pill) and baicalin-keratin complex (UPLC retention time is 8.75min).

[0075] The identification method for biomimetic rhinoceros horn biological agents is as follows: Verification must be performed using a combination of the following three analytical techniques: (1) High-resolution mass spectrometry (HR-MS): used to confirm the precise molecular weight (1528.7124 Da) of the recombinant keratin peptide. (2) Fourier transform infrared spectroscopy (FTIR): used to confirm its wavelength at 1650 cm⁻¹ -1 1530cm -1 1240cm -1 Characteristic absorption peak at the location; (3) Inductively coupled plasma mass spectrometry (ICP-MS): used to confirm that the contents of trace elements Ca, Mg and Zn are within the specified range.

[0076] In the preparation method of the biomimetic rhinoceros horn biological agent in this embodiment, the biomimetic rhinoceros horn biological agent is limited to being lyophilized microspheres with a diameter of 50μm-100μm and a specific surface area of ​​12.8m². 2 / g-13.5m 2 The requirements for g / g release characteristics, as well as the specific compatibility and process parameters in Angong Niuhuang Wan, form a technical barrier.

[0077] Example 6: This example provides a preparation process for Angong Niuhuang Wan, the preparation process is as follows: 1. Processing of medicinal materials: Prepare powdered ingredients according to traditional methods, including 0.1g of bezoar, 0.025g of musk, 0.05g of pearl, 0.1g of cinnabar, 0.1g of realgar, 0.1g of coptis, 0.1g of scutellaria, 0.1g of gardenia, 0.1g of turmeric, and 0.025g of borneol, with honey as an excipient.

[0078] 2. Mixing process: Mix the lyophilized microspheres of the biomimetic rhinoceros horn biological agent from any of Examples 1-5 with the above-mentioned powder at 25°C and 45% relative humidity for 15 minutes.

[0079] 3. Pelletizing process: Low-temperature pelletizing technology (below 40℃) is adopted to ensure that the active ingredients are not destroyed.

[0080] 4. Packaging and storage: Aluminum-plastic packaging with built-in desiccant; store away from light.

[0081] The quality inspection standards for Angong Niuhuang Wan in this embodiment are as follows: The finished product of Angong Niuhuang Wan must simultaneously meet the following requirements: Characteristics: Brownish-red to brownish-red micro-spheres, with a strong aromatic odor and a slightly bitter taste; Identification: HPLC fingerprint similarity > 0.95; Content determination: Each pill (3g) contains baicalin ≥12.0mg, berberine ≥8.0mg, and recombinant keratin characteristic peptide ≥0.5mg; Release rate: 30%-40% cumulative release rate at 45 min, 60%-70% cumulative release rate at 6 h, and ≥90% cumulative release rate at 12 h; Microbial limits: Meets the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0082] To verify the inseparability of the biomimetic rhinoceros horn biological agent of the present invention with the specific preparation process and the uniqueness of the formulation, this embodiment designed a compatibility comparison experiment to examine the feasibility of directly adding the biomimetic rhinoceros horn biological agent of the present invention to commercially available Angong Niuhuang Wan (hereinafter referred to as "commercially available AGNH") from different manufacturers.

[0083] I. Experimental Design Preparation of test articles (test samples): 1. Experimental group (external addition group): Take commercially available AGNH powder, add the lyophilized microspheres of the biomimetic rhinoceros horn biological agent of this invention precisely at a ratio of 0.2g biological agent / 3g pills, mix according to the original process parameters of commercially available AGNH to prepare a physical mixture; 2. The present invention group (integration group): Angong Niuhuang Wan prepared according to the process of the present invention and integrating the biological agent of the present invention; 3. Control group: Commercially available AGNH (buffalo horn formula) and Angong Niuhuang Wan containing natural rhinoceros horn (positive control).

[0084] Evaluation method: 1. In vitro release assay: The release behavior of the index components (baicalin and characteristic keratin peptides) was determined in phosphate buffer at pH 6.8, according to the 2020 edition of the Chinese Pharmacopoeia, Part IV. 2. Stability study of chemical components: The residual amounts of baicalin and berberine were determined by HPLC after 10 days of accelerated testing at 60℃, and the degradation rate was calculated. 3. Pharmacodynamic evaluation: Using a rat model of middle cerebral artery occlusion (MCAO), the percentage of cerebral infarction volume was calculated and the neurological deficit score was assessed 24 hours after gavage administration.

[0085] II. Results and Data Table 4 below: Compatibility evaluation results of the biomimetic rhinoceros horn biological agent of the present invention with commercially available AGNH ( (n=6).

[0086]

[0087] Note: Compared with the positive control group, p<0.05; compared with the integrated group of this invention, △p<0.05; **p>0.05 indicates no statistical difference.

[0088] III. Analysis and Conclusion 1. Abnormal Release Profile: The release of the biomimetic rhinoceros horn biological agent of this invention in the excipient group was significantly delayed and incomplete in commercially available AGNH (cumulative release rate of only 68.3%-71.6% over 12 hours), significantly lower than that in the integrated group (92.5%) and the control group (94.1%) (p<0.05). This indicates that the microenvironment formed by the excipients and processes of commercially available AGNH pellets cannot support the release of the lyophilized microspheres of the biomimetic rhinoceros horn biological agent of this invention along the designed pathway, resulting in a significant release obstacle.

[0089] 2. Chemical Instability: Accelerated testing showed that the degradation rate of baicalin in the added group was significantly higher (>25%), far exceeding that in the integrated group (8.3%) and the control group (7.8%) (p<0.05). The speculated reason may be that the active ingredient in the biomimetic rhinoceros horn biological agent of this invention underwent unexpected physicochemical interactions with unoptimized excipients or other components in commercially available AGNH, leading to accelerated degradation of the key pharmacodynamic component.

[0090] 3. Pharmacodynamic Incompatibility: Pharmacodynamic validation results were consistent with in vitro experimental results. The therapeutic effect of the additive group in the MCAO model was significantly worse than that of the integrated group and the control group (p<0.05), while there were no statistically significant differences in cerebral infarction volume and neurological function scores compared with the original buffalo horn formula group (p>0.05). This demonstrates that simple physical mixing cannot reproduce the synergistic pharmacodynamics described in this invention, leading to a loss of effectiveness in treating acute conditions.

[0091] Conclusion: The biomimetic rhinoceros horn biological agent of this invention is an integral part of its proprietary formulation (Angong Niuhuang Wan). Its efficacy relies on a complete technical system comprised of "the specific morphology of the biomimetic rhinoceros horn biological agent (lyophilized microspheres) – a specific formulation ratio – a specific preparation process." Any attempt to simply add the biomimetic rhinoceros horn biological agent of this invention to existing Angong Niuhuang Wan products from other manufacturers will result in abnormal release behavior, accelerated component degradation, and significantly reduced efficacy due to physicochemical incompatibility, failing to achieve the intended therapeutic purpose.

[0092] Sequence List: ATG GGC TCC TGC CGC GGC TCC TAC CCG GTC TGC GCC GGC TCC AAG TGA Amino acid sequence (SEQ ID NO:1): Met-Gly-Ser-Cys-Arg-Gly-Ser-Tyr-Pro-Val-Cys-Ala-Gly-Ser-Lys.

[0093] Note: This sequence encodes a characteristic peptide of recombinant rhinoceros keratin with a molecular weight of 1528.7 Da. The sequence identity with natural rhinoceros keratin is 99.2% according to gene library comparison.

Claims

1. A biomimetic rhinoceros horn biological agent, characterized in that, It consists of the following components by weight percentage: Recombinant keratin: 40%-50%, wherein the amino acid sequence of the recombinant keratin is shown in SEQ ID NO:1; Buffalo horn enzymatic hydrolysis active peptides: 25%-35%, molecular weight of buffalo horn enzymatic hydrolysis active peptides <5kDa, arginine content of buffalo horn enzymatic hydrolysis active peptides ≥12.3mg / g; Silk fibroin: 15%-25%, with β-sheet structure content of silk fibroin ≥65%; Trace elements: 4%-6%, with Ca being a significant component. 2+ The content is 3.0%-3.5%, with Mg being a trace element. 2+ The content is 1.5%-2.0%; Pharmaceutically acceptable excipients or moisture: balance; The biomimetic rhinoceros horn biological agent consists of freeze-dried microspheres with a diameter of 50μm-100μm and a specific surface area of ​​12.8m². 2 / g-13.5m 2 / g; The complex enzymatic hydrolysis of bioactive peptides from buffalo horn includes the following steps: Raw material pretreatment: Take high-quality buffalo horn, wash it, crush it through a 60-100 mesh sieve, add 8-12 times the volume of deionized water to suspend it, treat it at a high temperature of 118℃-124℃ for 25min-35min, cool it to room temperature to obtain a suspension. Compound enzymatic hydrolysis: Adjust the pH of the suspension to 7.5-8.5, add compound enzyme, the mass ratio of trypsin to papain in the compound enzyme is 1:1 to 1:2, and the mass ratio of enzyme to substrate is 1:15 to 1:

25. Perform enzymatic hydrolysis in a constant temperature water bath at 45℃-55℃ for 3-5 hours, with continuous stirring and pH stability maintained during the process. Enzyme inactivation and primary separation: The product obtained after complex enzymatic hydrolysis in the above steps is heated to 80℃-90℃ and maintained for 10min-20min to inactivate the enzyme. After cooling, the supernatant is collected by centrifugation. Fine purification: The supernatant is sequentially passed through an 8kDa-12kDa ultrafiltration membrane and a 3kDa-5kDa ultrafiltration membrane for fractionation and separation, and the permeate with a value <5kDa is collected; Vacuum freeze drying: After pre-freezing the permeate, freeze-dry it at -45℃ to -55℃ and 0.05Pa to 0.15Pa for 36h to 60h to obtain a light yellow peptide powder, which is then sealed and stored. The extraction and processing of silk fibroin includes the following steps: Degumming treatment: Place the silkworm silk in a 0.3%-0.7% (w / v) Na2CO3 solution and boil it in a water bath for 25-35 minutes to remove sericin. Repeat 2-4 times, then dry to obtain degummed silk fibroin. Dissolution and dialysis: Dissolve the degummed silk fibroin in 8.0M-10.0M LiBr solution, dialyze with deionized water for 2-4 days, and change the solution 4-8 times; Structure induction: The concentration of the dialysis silk fibroin solution was adjusted to 1%–3% by mass and volume, pre-frozen at -15°C to -25°C, and then freeze-dried to obtain silk fibroin powder rich in β-sheet structure.

2. The biomimetic rhinoceros horn biological agent according to claim 1, characterized in that, The core active ingredient of the biomimetic rhinoceros horn biological agent includes recombinant keratin peptides, the amino acid sequence of which is shown in SEQ ID NO:

1.

3. A method for preparing a biomimetic rhinoceros horn biological agent, characterized in that, The biomimetic rhinoceros horn biological agent is the biomimetic rhinoceros horn biological agent according to claim 1 or 2, and the preparation method includes the following steps: Biosynthesis of recombinant keratin: The gene fragment encoding SEQ ID NO:1 was cloned into the Pichia pastoris expression vector pPIC9K, transformed into strain GS115, and subjected to high-density fermentation and Ni 2+ -NTA affinity chromatography purification yielded recombinant keratin with a purity ≥98.6%; Preparation of enzymatically hydrolyzed active peptides from buffalo horn: Buffalo horn powder was subjected to compound enzymatic hydrolysis, ultrafiltration fractionation, and freeze-drying to obtain enzymatically hydrolyzed active peptides from buffalo horn with an arginine content of 12.3 mg / g-13.5 mg / g; among which, the molecular weight cutoff of ultrafiltration fractionation was <5 kDa. Extraction and processing of silk fibroin: Silkworm silk is degummed, dissolved, dialyzed, and freeze-dried to obtain silk fibroin with a β-sheet structure content ≥65%; Bionic Assembly: The recombinant keratin, buffalo horn enzymatically hydrolyzed active peptides, silk fibroin, and trace elements obtained in the above steps were mixed in a certain proportion. High-pressure homogenization was performed using a high-pressure homogenizer at 600 bar ± 50 bar. The mixture was then spray-frozen into pellets, cross-linked with glutaraldehyde vapor in a humidity-controlled device with a relative humidity of 60% ± 5%, and finally freeze-dried under vacuum to obtain pellets with a diameter of 50-100 μm and a specific surface area of ​​12.8-13.5 m². 2 / g of freeze-dried microspheres.

4. The method for preparing a biomimetic rhinoceros horn biological agent according to claim 3, characterized in that, The high-pressure homogenizer is an ATS AH1000 model.

5. A method for preparing a biomimetic rhinoceros horn biological agent, characterized in that, Includes the following steps: Biosynthesis of recombinant keratin It includes the following steps: Gene cloning and vector construction: The gene fragment encoding SEQ ID NO:1 was cloned into the Pichia pastoris GS115 / pPIC9K expression vector using restriction endonucleases EcoRI and NotI to construct a recombinant engineered strain, which was verified by sequencing to be correct; High-density fermentation: 3L-10L fermenters were used, with an inoculum size of OD600 = 0.8-1.2, and BSM basal salt medium. During the glycerol batch culture phase, the temperature was 28℃-32℃, the pH was 4.8-5.2, and the culture time was 20-28 hours, with dissolved oxygen maintained at 25%-35%. During the methanol induction phase, 1.5%-2.5% v / v methanol was used as the inducer, and expression was induced for 68-76 hours at a temperature of 28℃-32℃ and a pH of 5.8-6.2, with dissolved oxygen controlled at 25%-45%. The recombinant keratin expression level was ≥2.5g / L. Precision purification: The fermentation broth was centrifuged at 8000 rpm for 15 min at 4℃ to remove bacterial cells. The supernatant was filtered through a 0.20 μm-0.25 μm filter membrane and then loaded onto a pre-equilibrated Ni atmosphere. 2+ -NTA affinity chromatography column, wash with PBS buffer containing 15mM-25mM imidazole at pH 7.2-7.6 to remove contaminating proteins, and finally perform gradient elution with PBS buffer containing 200mM-300mM imidazole at pH 7.2-7.6, collect the target protein peak, and verify purity ≥98.6% by SDS-PAGE; Desalting and concentration: The buffer was replaced with PBS buffer using a HiPrep26 / 10 Desalting column at pH 7.2-7.

6. Then, the solution was concentrated to a final concentration of 15 mg / mL-25 mg / mL using Amicon Ultra-15 ultrafiltration centrifuge tubes with a molecular weight cutoff of 8 kDa-12 kDa. The solution was stored at -70°C to -80°C for later use. The complex enzymatic hydrolysis of bioactive peptides from buffalo horn includes the following steps: Raw material pretreatment: Take high-quality buffalo horn, wash it, crush it through a 60-100 mesh sieve, add 8-12 times the volume of deionized water to suspend it, treat it at a high temperature of 118℃-124℃ for 25min-35min, cool it to room temperature to obtain a suspension. Compound enzymatic hydrolysis: Adjust the pH of the suspension to 7.5-8.5, add compound enzyme, the mass ratio of trypsin to papain in the compound enzyme is 1:1 to 1:2, and the mass ratio of enzyme to substrate is 1:15 to 1:

25. Perform enzymatic hydrolysis in a constant temperature water bath at 45℃-55℃ for 3-5 hours, with continuous stirring and pH stability maintained during the process. Enzyme inactivation and primary separation: The product obtained after complex enzymatic hydrolysis in the above steps is heated to 80℃-90℃ and maintained for 10min-20min to inactivate the enzyme. After cooling, the supernatant is collected by centrifugation. Fine purification: The supernatant is sequentially passed through an 8kDa-12kDa ultrafiltration membrane and a 3kDa-5kDa ultrafiltration membrane for fractionation and separation, and the permeate with a value <5kDa is collected; Vacuum freeze drying: After pre-freezing the permeate, freeze-dry it at -45℃ to -55℃ and 0.05Pa to 0.15Pa for 36h to 60h to obtain a light yellow peptide powder, which is then sealed and stored. The extraction and processing of silk fibroin includes the following steps: Degumming treatment: Place the silkworm silk in a 0.3%-0.7% (w / v) Na2CO3 solution and boil it in a water bath for 25-35 minutes to remove sericin. Repeat 2-4 times, then dry to obtain degummed silk fibroin. Dissolution and dialysis: Dissolve the degummed silk fibroin in 8.0M-10.0M LiBr solution, dialyze with deionized water for 2-4 days, and change the solution 4-8 times; Structure induction: The concentration of the dialysis silk fibroin solution was adjusted to 1% to 3% by mass volume, pre-frozen at -15℃ to -25℃ and then freeze-dried to obtain silk fibroin powder rich in β-sheet structure; Bionic assembly and dosage form design include the following steps: Precise preparation of biological matrix: Weigh 40%-50% recombinant keratin, 25%-35% buffalo horn enzymatic hydrolysate, 15%-25% silk fibroin, and 4%-6% trace elements according to the proportion, add 4-6 times the volume of PBS buffer, pH 7.2-7.6, and stir at low speed at 2℃-8℃ for 10-14 hours until completely dissolved and homogeneous. High-pressure homogenization nano-sizing: A high-pressure homogenizer is used to homogenize the particles 8-12 times under a pressure of 550-650 bar to make the particle size distribution of the system uniform, with a particle size D50 of 13μm-17μm and PDI < 0.

2. Preparation of freeze-dried microspheres: The homogenized system was spray-dried into microspheres at an inlet air temperature of 110℃-130℃ and an outlet air temperature of 55℃-65℃. The microspheres were then cross-linked in a sealed container containing 0.3%-0.7% glutaraldehyde vapor for 10-14 hours at 23℃-27℃, followed by vacuum freeze-drying for 36-60 hours to obtain freeze-dried microspheres with a diameter of 50μm-100μm and a specific surface area of ​​12.8m². 2 / g-13.5m 2 / g.

6. The use of the biomimetic rhinoceros horn biological agent according to claim 1 or 2 in the preparation of a medicament for treating ischemic stroke.

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