Single recombinant cytokine bone repair injection and preparation method thereof

Abstract

The present application relates to a single recombinant cytokine bone repair injection and a preparation method thereof, and belongs to the technical field of medicine.The single recombinant cytokine bone repair injection is composed of the following components in mass percentage: single recombinant human cytokine 0.001%-0.05%, mannitol 2%-5%, human blood albumin 0.5-2%, phosphate buffer 0.1%-0.3%, and the rest is sterile water for injection.The preparation method of the single recombinant cytokine bone repair injection is also disclosed.The single recombinant cytokine bone repair injection has the advantages of domestic originality, greatly improved safety, highly controllable quality, significant clinical advantages, mature and industrialized process, strong compliance, and outstanding clinical value.

Description

Technical Field

[0001] This invention relates to a single recombinant cytokine bone repair injection and its preparation method, belonging to the field of pharmaceutical technology. Background Technology

[0002] Currently, clinical treatment options for bone defects, poor fracture healing, nonunion, and spinal fusion mainly include autologous bone grafting, allogeneic bone grafting, synthetic bone materials, compound growth factor gels, and compound growth factor dressings. Existing technologies have the following limitations:

[0003] First, compound growth factor products have complex components, low purity, many impurities, and uncontrollable residual DNA / host proteins, which can easily trigger immunogenic reactions and pose a high risk of ectopic ossification.

[0004] Secondly, most existing products are in the form of gels, solids, granules, or dressings, which cannot achieve minimally invasive and precise injection, thus limiting their clinical use.

[0005] Third, the active ingredients cannot be accurately quantified, resulting in large batch-to-batch differences, unstable efficacy, and uncontrollable quality.

[0006] Fourth, there are currently no single high-purity cytokine injection bone repair products on the market in China, which presents a huge clinical gap.

[0007] Fifth, traditional bone graft materials are highly invasive, have limited availability, and require a long repair cycle, failing to meet the clinical needs for rapid, minimally invasive, and bioactive repair.

[0008] Therefore, developing a domestically produced, safe, controllable, precisely quantifiable, and minimally invasive single-cytokine bone repair injection has significant clinical value and market implications. Summary of the Invention

[0009] One of the objectives of this invention is to provide a single recombinant cytokine bone repair injection.

[0010] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A single recombinant cytokine bone repair injection, which is composed of the following components in mass percentage: 0.001%-0.05% single recombinant cytokine, 2%-5% mannitol, 0.5%-2% human serum albumin, 0.1%-0.3% phosphate buffer and the balance being sterile water for injection.

[0011] The principle of the single recombinant cytokine bone repair injection of the present invention is as follows:

[0012] In the prior art, there are no reports of preparing bone repair injections using a single recombinant human cytokine, mannitol, human serum albumin, phosphate buffer, and sterile water for injection. In this invention, the roles of each component are as follows:

[0013] Single recombinant human cytokine: recognizes and binds to corresponding receptors on the surface of target cells and initiates intracellular signaling pathways.

[0014] Mannitol: Protects protein activity.

[0015] Human serum albumin: protects recombinant proteins, prevents inactivation, regulates osmotic pressure, improves safety, assists in antifreeze, and resists freeze-drying damage.

[0016] Phosphate buffer: maintains a stable pH environment, reduces local irritation, and synergistically stabilizes protein structure.

[0017] Sterile water for injection: serves as a solvent carrier to ensure the purity and sterility of the injection solution.

[0018] Through numerous experiments, this invention unexpectedly and surprisingly discovered that using the above five raw materials in appropriate proportions yields a bone repair injection with excellent performance, exhibiting the technical advantages of being a single-component, high-purity, low-impurity, low-immunogenicity, and low-risk ectopic ossification.

[0019] The key technical indicators of the single recombinant cytokine bone repair injection of the present invention are as follows:

[0020] Appearance: Colorless / pale yellow clear liquid, free of visible foreign matter; pH: 6.5-8.0; Osmolarity: 280mOsm / kg-320mOsm / kg; Purity: ≥95.0% (SEC-HPLC); Biological activity: 80%-120% of labeled amount; Protein content: 90%-110% of labeled amount; Sterility: Meets the requirements of the Chinese Pharmacopoeia; Bacterial endotoxin: <0.5EU / mL; Mycoplasma: Negative; Residual DNA: ≤10ng / mL; Residual host protein: ≤100ppm; Pyrogen: Qualified.

[0021] The dosage form and administration method of the single recombinant cytokine bone repair injection of the present invention:

[0022] Dosage form: sterile injection; specifications: 0.5mL, 1.0mL, 2.0mL; administration method: local injection at bone defect sites / fracture ends / nonunion sites / segmental fusion; storage conditions: store at -20℃ protected from light.

[0023] The beneficial effects of the single recombinant cytokine bone repair injection of the present invention are:

[0024] 1. First in China: The first single-cytokine bone repair injection in China, filling a market gap;

[0025] 2. Significantly improved safety: Single ingredient, high purity (≥95%), low impurities, low immunogenicity, and low risk of ectopic ossification;

[0026] 3. Highly controllable quality: precise quantification of activity, good batch-to-batch consistency, and stable and predictable therapeutic effects;

[0027] 4. Significant clinical advantages: minimally invasive injection, precise positioning, simple operation, minimal trauma, and rapid healing;

[0028] 5. Mature and industrially scalable technology: The entire process can be scaled up industrially, with controllable costs and stable quality;

[0029] 6. Extremely high compliance: Fully compliant with the requirements for Class III medical devices and innovative medical devices;

[0030] 7. Outstanding clinical value: Significantly improves bone healing rate, shortens healing time, and reduces patient suffering.

[0031] Based on the above technical solution, the present invention can be further improved as follows.

[0032] Furthermore, it is composed of the following components by mass percentage: 0.03% single recombinant human cytokine, 2.2% mannitol, 1% human serum albumin, 0.2% phosphate buffer, and the balance being sterile water for injection.

[0033] The further beneficial effect of adopting the above parameters is that the above parameters are the optimal parameters, and the resulting technical effect is also the best.

[0034] Furthermore, it is composed of the following components by mass percentage: 0.001% single recombinant human cytokine, 5% mannitol, 0.5% human serum albumin, 0.3% phosphate buffer, and the balance being sterile water for injection.

[0035] The further beneficial effect of adopting the above parameters is that the above parameters are the optimal parameters, and the resulting technical effect is also the best.

[0036] Furthermore, it consists of the following components by mass percentage: 0.05% single recombinant human cytokine, 2% mannitol, 2% human serum albumin, 0.1% phosphate buffer, and the balance being sterile water for injection.

[0037] The further beneficial effect of adopting the above parameters is that the above parameters are the optimal parameters, and the resulting technical effect is also the best.

[0038] Furthermore, the single cytokine is selected from any one of BMP-2, PDGF-BB, FGF-2, and VEGF.

[0039] The further beneficial effect of the above-mentioned method is that all of the above-mentioned cytokines can be used to prepare the single recombinant cytokine bone repair injection of the present invention. Different cytokines have different effects and correspond to different disease symptoms, specifically:

[0040] BMP-2, or bone morphogenetic protein-2, is an important member of the TGF-β superfamily and plays a central role in bone formation and repair. In this invention, the single recombinant cytokine used is BMP-2, which has anti-inflammatory and repairing effects.

[0041] PDGF-BB, or platelet-derived growth factor BB, is a cytokine that plays an important role in physiological processes such as cell growth and tissue repair. In this invention, the single recombinant cytokine used is PDGF-BB, which has antiviral activity.

[0042] FGF-2, or fibroblast growth factor 2, also known as basic fibroblast growth factor (bFGF), is a signaling protein that plays a crucial role in cell growth, angiogenesis, and tissue repair. In this invention, the single recombinant cytokine used is FGF-2, which has the function of regulating angiogenesis, cell proliferation, and tissue regeneration.

[0043] VEGF, or vascular endothelial growth factor, also known as vascular permeability factor, is a factor that promotes the growth of vascular endothelium. In this invention, the single recombinant cytokine used is VEGF, which has the function of tissue repair and wound healing.

[0044] The second objective of this invention is to provide a method for preparing the above-mentioned single recombinant cytokine bone repair injection.

[0045] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for preparing a single recombinant cytokine bone repair injection, comprising the following steps:

[0046] Take 0.001%-0.05% of a single recombinant cytokine, 2%-5% of mannitol, 0.5%-2% of human serum albumin, 0.1%-0.3% of phosphate buffer, and the balance as sterile water for injection according to the following mass percentages. Mix them evenly, adjust the pH and osmotic pressure, filter sterilely, and fill aseptically to obtain the single recombinant cytokine bone repair injection.

[0047] The beneficial effects of the preparation method of the single recombinant cytokine bone repair injection of the present invention are:

[0048] 1. This invention prepares high-purity single osteogenic cytokines through gene recombination, precisely activates bone regeneration signaling pathways, and directionally induces bone formation to achieve bone defect repair and bone reconstruction.

[0049] 2. The preparation method of the present invention is simple, easy to operate, low in cost, has broad market prospects, and is suitable for large-scale promotion and application.

[0050] Based on the above technical solution, the present invention can be further improved as follows.

[0051] Furthermore, in step 1.1, the single cytokine is selected from any one of BMP-2, PDGF-BB, FGF-2, and VEGF.

[0052] The further beneficial effect of using the above methods is that all of the aforementioned cytokines can be used to prepare the single recombinant cytokine bone repair injection of the present invention. Different cytokines have different effects and correspond to different disease symptoms.

[0053] Furthermore, the method for preparing the single cytokine includes the following steps:

[0054] Step 1: Construction of engineered cells

[0055] Step 1.1: Target gene codon optimization and fragment acquisition

[0056] Based on the natural gene sequence of a single recombinant human cytokine, codon bias optimization was performed on the E. coli expression system to remove rare codons and optimize the mRNA secondary structure. The optimized sequence was then used for whole-gene synthesis, with restriction endonuclease sites NcoI and XhoI and protective bases introduced upstream and downstream, respectively, followed by PCR amplification. The PCR product was detected by 1% agarose gel electrophoresis and purified using a gel extraction kit to obtain the target gene fragment.

[0057] Step 1.2: Double digestion of the target gene and vector

[0058] The purified target gene fragment from step 1.1 was combined with pET-28a(+) plasmid and verified by double digestion with NcoI / XhoI. The digestion products were subjected to 1% agarose gel electrophoresis, and the linearized vector and the target fragment were recovered separately.

[0059] Step 1.3: Ligation and transformation of Escherichia coli DH5α

[0060] The linearized vector was mixed with the target fragment, T4 DNA ligase was added, and ligation was carried out overnight; the ligation product was transformed into DH5α competent cells, spread on LB solid medium containing 50 μg / mL kanamycin, and cultured upside down.

[0061] Step 1.4: Screening of positive clones, double enzyme digestion verification, and bidirectional sequencing

[0062] Single colonies were extracted for colony PCR identification; plasmids were extracted after the positive strains were expanded and cultured, and verified by double digestion with NcoI / XhoI; bidirectional sequencing was performed on the correctly digested plasmids, and the sequencing results were 100% consistent with the optimized sequence, which is the positive recombinant plasmid.

[0063] Step 1.5: Transformation and expression of Escherichia coli BL21 DE3

[0064] The positive recombinant plasmid was transformed into BL21 DE3 competent cells and cultured on LB-Kan plates. Single colonies were picked and verified to be correct by colony PCR and double enzyme digestion of plasmid, thus obtaining the original engineered bacteria.

[0065] Step 1.6: Preservation of microbial strains

[0066] The original engineered bacteria were inoculated into LB-Kan liquid medium, shaken overnight, and then frozen for later use.

[0067] Step 2: Inducing Expression

[0068] Step 2.1: Seed liquid preparation

[0069] Step 1: Activate the frozen engineered bacteria by streaking onto LB-Kan plates; pick a single colony and inoculate it with 3-5 mL of LB-Kan liquid medium, incubate overnight to obtain the seed culture;

[0070] Step 2.2: Expanded Culture and Induction

[0071] Seed culture was inoculated into the expansion medium and cultured until OD 600 =0.6–0.8, add IPTG to a final concentration of 0.5 mM, induce at 25°C for 12 h; collect bacterial cells by centrifugation, wash twice with PBS, and store at -20°C;

[0072] Step 3: Protein sample processing, purification, and injection solution preparation

[0073] Step 3.1: Cell disruption

[0074] The bacterial cells were resuspended in lysis buffer, sonicated, centrifuged, and the supernatant and precipitate were collected separately.

[0075] Step 3.2: SDS-PAGE and Western blot identification

[0076] The supernatant and precipitate samples were subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining; Western blot was performed using the corresponding cytokine-specific primary antibody to verify the correct expression of the target protein; grayscale analysis was used to calculate the expression level and soluble percentage.

[0077] Step 3.3: Western Blot protein purification

[0078] Expression was confirmed by Western blotting using Anti-His antibody or target protein-specific antibody. The supernatant was purified by Ni affinity chromatography, concentrated by ultrafiltration, and filtered to obtain a single recombinant cytokine.

[0079] The further beneficial effect of using the above method is that the single recombinant cytokine required by the present invention can be prepared by using the above method.

[0080] Furthermore, in step 1.1, the PCR amplification system consists of 5 μL of 10×PCR Buffer; 4 μL of 2.5 mmol / L dNTP Mix; 1 μL of 10 μmol / L upstream primer; 1 μL of 10 μmol / L downstream primer; 1 μL of template DNA; 0.5 μL of Taq DNA polymerase; and 37.5 μL of ddH2O, for a total volume of 50 μL. The PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 55℃-60℃ annealing for 30 s; 72℃ extension for 30 cycles; 72℃ final extension for 10 min; and storage at 4℃.

[0081] The further beneficial effects of the above approach are as follows: Using the online codon optimization tool ExpOptimizer (https: / / www.novopro.cn / tools / codon-optimization.html), reverse gene sequence design is performed. This design focuses on the expression characteristics of the host *E. coli*, screening and selecting optimal codons required for *E. coli* expression. This ensures a high degree of match between codon usage frequency and *E. coli* codon preference, laying the foundation for efficient gene translation. During the design process, NcoI and XhoI restriction sites are precisely identified and removed from the sequence, effectively avoiding restriction site interference during subsequent gene cloning and vector construction, ensuring the smooth progress of subsequent gene manipulation.

[0082] Compared with the original gene sequence, the GC content decreased from 86.11% to 64.43%, effectively reducing the complexity of the secondary structure of the gene sequence, which is more in line with the transcription and translation mechanism of E. coli. This significantly improved the expression of the cytokine in E. coli. The reasonable reduction in GC content improved the hydrophilicity of the gene sequence, enhanced transcription efficiency, and thus increased the expression level and protein activity of the cytokine in E. coli. This is more in line with the gene expression preferences of E. coli and effectively improved the high-efficiency expression level and protein solubility of the cytokine in E. coli.

[0083] Using the above amplification system and procedure can ensure that the PCR reaction is carried out efficiently, specifically amplifies the target gene fragment, reduces the generation of non-specific bands, and obtains products with the required concentration and purity, providing high-quality raw materials for subsequent enzyme digestion and ligation steps.

[0084] Furthermore, in step 1.2, the composition of the double enzyme digestion system is as follows: target gene or vector fragment, 20 μL; NcoI enzyme, 1 μL; Xho I enzyme, 1 μL; 10×CutSmart Buffer, 5 μL; ddH2O 23 μL, total system 50 μL; the digestion temperature is 37℃ and the time is 4 h.

[0085] The further beneficial effects of the above-mentioned method are: the components of this dual enzyme digestion system have a reasonable ratio, which can ensure that the two restriction enzymes, Nco I and Xho I, can work at the same time. The digestion efficiency is high and the specificity is strong. It can accurately cut the target gene and the vector, and generate complementary sticky ends, which lays the foundation for the subsequent ligation of the vector and the target gene, while reducing the interference caused by non-specific enzyme digestion.

[0086] Furthermore, in step 1.3, the molar ratio of the linearized vector to the target fragment is 1:5; the overnight ligation temperature is 16°C; the transformation into E. coli DH5α competent cells refers to heat shock at 42°C for 90 seconds, followed by an ice bath for 2 minutes, addition of SOC or LB medium, and recovery at 37°C for 1 hour; the culture temperature is 37°C, and the cells are inverted and cultured overnight.

[0087] Further advantages of using the above methods include: a 1:5 molar ratio of vector to insert improves ligation efficiency and reduces vector self-ligation. Overnight ligation at 16°C ensures a complete ligation reaction and improves the purity of the ligation product. A transformation method combining heat shock at 42°C and an ice bath efficiently introduces the ligation product into DH5α competent cells. Recovery and culture at 37°C promotes the recovery of competent cell activity and proliferation; inverted culture avoids colony spread and facilitates subsequent screening and selection of positive clones.

[0088] Furthermore, in step 1.4, the bidirectional sequencing uses the T7 promoter and the T7 terminator.

[0089] The further beneficial effects of the above method are: double enzyme digestion verification and electrophoresis results showing two bands, with the larger fragment (approximately 5300 bp) representing the pET-28a(+) vector and the smaller fragment representing the corresponding cytokine target fragment. The T7 promoter and T7 terminator are commonly used sequencing primer binding sites, exhibiting high specificity and binding efficiency. Bidirectional sequencing allows for simultaneous sequencing from both ends of the target gene, ensuring the accuracy and completeness of the sequencing results, effectively verifying whether the target gene sequence is correctly inserted into the vector, and avoiding problems such as sequence mutations and deletions. Bidirectional sequencing of correctly digested plasmids yields 100% alignment with the optimized sequence, with no mutations or frameshifts, indicating a positive recombinant plasmid.

[0090] Furthermore, in step 1.5, the transformation into host Escherichia coli BL21 DE3 competent cells refers to heat shock at 42°C for 90 seconds, followed by an ice bath for 2 minutes, addition of SOC medium or LB medium, and recovery at 37°C for 1 hour.

[0091] The further beneficial effects of the above method are: BL21 DE3 competent cells are commonly used host bacteria for protein expression. This transformation method can efficiently introduce recombinant vectors into the host bacteria. 37°C recovery and culture can promote the recovery and proliferation of the host bacteria, providing sufficient positive strains for the subsequent induction of the target protein expression and ensuring the smooth progress of protein expression experiments.

[0092] Furthermore, in step 1.6, the volume of the LB-Kan liquid culture medium is 3mL-5mL; the shaking temperature is 37℃ and the rotation speed is 220rpm; the preservation of the bacterial strain specifically refers to: mixing the bacterial solution after overnight shaking at 37℃ with sterile glycerol at a mass concentration of 50g / L at a volume ratio of 3:1, dispensing it into sterile cryovials, labeling them, and storing them at -80℃ for a long period of time.

[0093] The further beneficial effects of using the above method are: the 3mL-5mL LB-Kan liquid medium is of moderate volume, which can provide sufficient nutrition and growth space for positive strains, ensure rapid proliferation of strains, obtain sufficient cell volume for subsequent strain preservation, avoid waste of culture medium, reduce experimental costs, and facilitate subsequent shaker culture operations, thereby improving experimental efficiency.

[0094] Labeling includes, but is not limited to, strain name, number, construction date, constructor, resistance, and sequencing batch number. Through the above operations, a complete workflow management system can be established from the original strain → master cell bank (MCB) → working cell bank (WCB).

[0095] Furthermore, in step 2.1, the culture temperature is 37°C and the rotation speed is 220 rpm.

[0096] The further beneficial effects of adopting the above are: shortening the culture cycle; the shaking speed of 220 rpm can ensure sufficient dissolved oxygen in the bacterial solution, while ensuring full contact between the bacterial solution and the culture medium, avoiding differences in strain growth caused by uneven local nutrition or hypoxia, ensuring uniform concentration and stable activity of the cultured bacterial solution, and providing a high-quality bacterial source for subsequent experimental steps (such as bacterial solution amplification, extraction of target products, etc.).

[0097] Furthermore, in step 2.2, the inoculation refers to inoculating the seed culture at an inoculation rate of 1%-2% into 50mL-200mL of LB-Kan liquid culture medium; the culture temperature is 37℃ and the rotation speed is 220rpm; the induction temperature is 16℃-25℃ and the time is 16h-20h; or the temperature is 37℃ and the time is 3h-4h; the centrifugation temperature is 4℃, the rotation speed is 8000rpm, and the time is 10min.

[0098] The further beneficial effects of using the above methods are: The above inoculum size allows for large-scale culture; the above culture parameters shorten the culture cycle; the 220 rpm shaking speed ensures sufficient dissolved oxygen in the bacterial solution and adequate contact between the bacterial solution and the culture medium, avoiding differences in strain growth caused by uneven local nutrition or hypoxia, ensuring uniform bacterial concentration and stable activity, and providing a high-quality bacterial source for subsequent experimental steps (such as bacterial amplification and extraction of the target product). Low-temperature induction at 16℃-25℃ for 16-20 hours or high-temperature induction at 37℃ for 3-4 hours can be selected. High-temperature induction is often used for inclusion bodies. The above centrifugation parameters allow for the centrifugation separation of the induced culture.

[0099] Furthermore, in step 3.1, the ultrasonic disruption power is 300W–400W, the ultrasonic treatment lasts for 3 seconds, followed by a 5-second pause, for a total time of 20 minutes; the centrifugation temperature is 4℃, the power is 12000rpm, and the time is 20 minutes.

[0100] The further beneficial effect of the above method is that the entire ultrasonic disruption process is performed in an ice bath to prevent protein denaturation. After centrifugation, a supernatant and a precipitate are obtained. The supernatant contains soluble proteins, while the precipitate contains inclusion bodies.

[0101] SDS-PAGE, or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is a commonly used protein expression analysis technique in polyacrylamide gel electrophoresis.

[0102] Furthermore, in step 3.2, the mass concentration of the separating gel is 12%-15%.

[0103] The further beneficial effect of using the above is that the separating gel mentioned above refers to SDS-PAGE separating gel, which is suitable for the separation of proteins with a molecular weight of 10kD-60kD.

[0104] Furthermore, in step 3.3, the pore size of the filter membrane is 0.22 μm.

[0105] The further beneficial effects of using the above-mentioned filter membrane are: the 0.22μm pore size filter membrane can effectively retain insoluble particles, bacterial fragments and large protein aggregates in the protein solution, significantly improving the clarity and purity of the protein solution without losing the target protein, providing qualified sample loading raw materials for subsequent protein chromatography purification steps, while avoiding large particulate impurities from clogging the chromatography column sieve plate, extending the service life of chromatography consumables and reducing experimental costs.

[0106] Furthermore, the pH value is 7.2-7.4, and the osmotic pressure is 280mOsm / L-320mOsm / L.

[0107] The further beneficial effects of the above are: the pH range is highly matched with the physiological pH of the human body, which can maintain the spatial conformation, biological activity and structural stability of the target protein to the greatest extent, and avoid protein denaturation, aggregation and loss of activity due to pH deviation from the appropriate range; the osmotic pressure of this isotonic range is consistent with the osmotic pressure of human body fluids, which can ensure the biocompatibility of protein preparations, while avoiding protein structure damage due to osmotic pressure differences, and providing stable and qualified protein samples for subsequent protein activity detection, functional experiments, animal experiments or preclinical studies.

[0108] Furthermore, the pore size of the filter membrane for sterile filtration is 0.22 μm.

[0109] The further beneficial effects of using the above method are as follows: 0.22 μm is the standard pore size for sterile-grade filter membranes uniformly stipulated by pharmacopoeias of various countries. This allows for the efficient retention and removal of various live microorganisms such as bacteria and fungi from protein solutions, achieving final sterilization of the sample. This room-temperature filtration sterilization method completely avoids the irreversible damage to protein activity caused by high-temperature and high-pressure sterilization. While ensuring the activity and recovery rate of the target protein, it meets the aseptic quality control requirements of bioproducts, avoids sample failure due to microbial contamination, and fully guarantees the safety, compliance, and sample stability of subsequent experiments or applications. Attached Figure Description

[0110] Figure 1 This is an electrophoresis diagram of the target gene fragment in Example 1 of the present invention.

[0111] Figure 2 This is a double enzyme digestion diagram of the target gene and vector in Example 1 of the present invention.

[0112] Figure 3 This is a diagram of the ligation product transformed into DH5α competent cells in Example 1 of the present invention.

[0113] Figure 4 This is a double enzyme digestion verification diagram from Example 1 of the present invention.

[0114] Figure 5 This is a double enzyme digestion verification diagram from Example 1 of the present invention.

[0115] Figure 6 This is an SDS-PAGE diagram from Embodiment 1 of the present invention.

[0116] Figure 7 This is a Western blotting image from Embodiment 1 of the present invention.

[0117] Figure 8 This is a CT scan of Case 1 of the present invention, taken before treatment.

[0118] Figure 9 This is a CT image of Case 1 of the present invention after treatment.

[0119] Figure 10 The following is a flowchart of the treatment plan in Case 1 of the present invention. Detailed Implementation

[0120] The principles and features of the present invention are described below with reference to embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0121] I. Strain and Plasmid Information

[0122] Host bacterium: Escherichia coli BL21 DE3

[0123] Promoter: T7 promoter

[0124] Tags: N-terminal His-Tag, resectable thrombin / Xa site

[0125] Resistance: Kanamycin resistance (Kanᵣ)

[0126] Full name of engineered bacteria: Escherichia coli BL21 DE3 / pET-28a-recombinant cytokine

[0127] II. Reagents and Consumables

[0128] Sterile centrifuge tubes: Corning, model 430052 (1.5mL), 430290 (50mL); Axygen, model AXY-2001 (1.5mL).

[0129] Petri dishes: Cotton's Choice (high borosilicate glass); Corning, model 430167 (60mm, disposable sterile plastic), model 430599 (100mm, disposable sterile plastic).

[0130] Shaker: Shanghai Zhicheng, model ZHWY-100B (constant temperature shaking shaker, suitable for culture requirements of 37℃ and 220rpm).

[0131] Ultrasonic cell disruptor: Ningbo Xinzhi, model JY92-IIN (ultrasonic cell disruptor, adjustable power, suitable for small-batch protein extraction in the laboratory), SW-CJ-2FD (vertical laminar flow, ultrasonic frequency ≥25KHz).

[0132] Protein electrophoresis apparatus: Bio-Rad, model Mini-PROTEAN Tetra (small vertical electrophoresis apparatus, suitable for routine protein separation experiments).

[0133] Mannitol: Jiahuqi, model WLBM08737683 (analytical grade AR, 100g / bottle, CAS code 69-65-8); National Institute of Metrology, China, model GBW(E)100681 (D-mannitol purity standard material, 200mg / bottle).

[0134] Human serum albumin: Hualan Biological, 20% 5g / bottle (pharmaceutical grade, suitable for experimental protein protection needs); Shanghai Raas, 20% 10g / bottle.

[0135] Phosphate buffer: Fire Blue Warrior (0.1M, pH 7.4, sterile solution, 500mL / bottle); Corning, model 21-040-CV (10×PBS buffer, pH 7.4).

[0136] III. Solution Formulation

[0137] LB-Kan solid medium (1L): 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar, bring to a final volume of 1L, sterilize at 121℃ for 20min, cool to about 50℃ and add Kan to a final concentration of 50μg / mL.

[0138] LB-Kan liquid medium (1L): 10g tryptone, 5g yeast extract, 10g NaCl, bring to a final volume of 1L, sterilize at 121℃ for 20min, cool to about 50℃ and add Kan to a final concentration of 50μg / mL.

[0139] Kan stock solution: 50 mg / mL, sterile filtered, stored at -20℃, working concentration 50 μg / mL. Kan, or Kanamycin, is a medicinal ingredient. Kan must be added and used immediately to avoid plasmid loss due to inactivation.

[0140] IPTG stock solution: 1 mol / L, sterile filtered, stored at -20℃.

[0141] Cell lysis buffer (50 mL): 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM PMSF, 50 μL protease inhibitor.

[0142] Binding / Washing / Eluting Buffer

[0143] Binding: 20mM Tris, pH 8.0, 500mM NaCl, 20mM imidazole.

[0144] Washing: 20mM Tris, pH 8.0, 500mM NaCl, 50mM imidazole.

[0145] Elution: 20 mM Tris, pH 8.0, 500 mM NaCl, 250 mM–500 mM imidazole.

[0146] Example 1

[0147] The single recombinant cytokine bone repair injection of this embodiment is composed of the following components by mass percentage: 0.03% single recombinant human cytokine, 2.2% mannitol, 1% human serum albumin, 0.2% phosphate buffer, and the balance being sterile water for injection.

[0148] The single cytokine is selected from any one of BMP-2, PDGF-BB, FGF-2, and VEGF.

[0149] The preparation method of the above-mentioned single recombinant cytokine bone repair injection includes the following steps: Take 0.03% single recombinant human cytokine, 2.2% mannitol, 1% human serum albumin, 0.2% phosphate buffer, and the remainder sterile water for injection according to the following mass percentages, mix them evenly, adjust the pH to 7.2-7.4, and the osmotic pressure to 280mOsm / L-320mOsm / L; sterile filter, aseptically fill, and the filter membrane pore size is 0.22μm to obtain the single recombinant cytokine bone repair injection.

[0150] The method for preparing the single cytokine described above includes the following steps:

[0151] Step 1: Construction of engineered cells

[0152] Step 1.1: Target gene codon optimization and fragment acquisition

[0153] Based on the natural gene sequence of a single recombinant human cytokine, codon bias optimization was performed on the *E. coli* expression system, rare codons were removed, and the mRNA secondary structure was optimized. The optimized sequence was then used for whole-genome synthesis, with restriction endonuclease sites NcoI and XhoI, along with protective bases, introduced upstream and downstream, respectively, followed by PCR amplification. The PCR product was detected by 1% agarose gel electrophoresis and purified using a gel extraction kit to obtain the target gene fragment. Figure 1 As shown.

[0154] The PCR amplification system consisted of 5 μL of 10×PCR Buffer; 4 μL of 2.5 mmol / L dNTP Mix; 1 μL of 10 μmol / L upstream primer; 1 μL of 10 μmol / L downstream primer; 1 μL of template DNA; 0.5 μL of Taq DNA polymerase; and 37.5 μL of ddH2O, for a total volume of 50 μL. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 55℃-60℃ annealing for 30 s; 72℃ extension for 30 cycles; 72℃ final extension for 10 min; and storage at 4℃.

[0155] Step 1.2: Double digestion of the target gene and vector

[0156] The purified target gene fragment from step 1.1 was combined with the pET-28a(+) plasmid and verified by double digestion with NcoI / XhoI. The digestion products were then subjected to 1% agarose gel electrophoresis, and the linearized vector and target fragment were recovered separately. Figure 2 As shown.

[0157] The double enzyme digestion system consisted of: 20 μL of the target gene or vector fragment; 1 μL of Nco I enzyme; 1 μL of Xho I enzyme; 5 μL of 10×CutSmart Buffer; and 23 μL of ddH2O, for a total volume of 50 μL. The digestion was performed at 37°C for 4 hours.

[0158] Step 1.3: Ligation and transformation of Escherichia coli DH5α

[0159] The linearized vector was mixed with the target fragment, T4 DNA ligase was added, and ligation was carried out overnight. The ligation product was transformed into DH5α competent cells, plated on LB solid medium containing 50 μg / mL kanamycin, and incubated upside down. Figure 3 As shown.

[0160] The molar ratio of the linearized vector to the target fragment is 1:5; the overnight ligation temperature is 16°C; the transformation into E. coli DH5α competent cells refers to heat shock at 42°C for 90 seconds, followed by an ice bath for 2 minutes, addition of SOC or LB medium, and recovery at 37°C for 1 hour; the culture temperature is 37°C, and the cells are cultured upside down overnight.

[0161] Step 1.4: Screening of positive clones, double enzyme digestion verification, and bidirectional sequencing

[0162] Single colonies were extracted for colony PCR identification; after amplification culture of positive strains, plasmids were extracted and verified by NcoI / XhoI double digestion; the correctly digested plasmids were subjected to bidirectional sequencing, and the sequencing results were 100% consistent with the optimized sequence, indicating a positive recombinant plasmid. Figure 4 As shown.

[0163] The bidirectional sequencing uses the T7 promoter and T7 terminator.

[0164] Step 1.5: Transformation and expression of Escherichia coli BL21 DE3

[0165] The positive recombinant plasmid was transformed into BL21 DE3 competent cells and cultured on LB-Kan plates. Single colonies were picked and verified to be correct by colony PCR and double enzyme digestion of the plasmid, thus obtaining the original engineered bacteria. Figure 5 As shown.

[0166] The transformation into host Escherichia coli BL21 DE3 competent cells refers to a heat shock at 42°C for 90 seconds, followed by an ice bath for 2 minutes, addition of SOC or LB medium, and recovery at 37°C for 1 hour.

[0167] Step 1.6: Preservation of microbial strains

[0168] The original engineered bacteria were inoculated into LB-Kan liquid medium, shaken overnight, and then frozen for later use.

[0169] The volume of the LB-Kan liquid culture medium is 3mL-5mL; the shaking temperature is 37℃ and the rotation speed is 220rpm; the preservation of the bacterial strain specifically refers to: mixing the bacterial solution after overnight shaking at 37℃ with sterile glycerol at a mass concentration of 50g / L at a volume ratio of 3:1, dispensing it into sterile cryovials, labeling them, and storing them at -80℃ for a long time.

[0170] Step 2: Inducing Expression

[0171] Step 2.1: Seed liquid preparation

[0172] Step 1: Activate the frozen engineered bacteria by streaking onto LB-Kan plates; pick a single colony and inoculate it with 3-5 mL of LB-Kan liquid medium, incubate overnight at 37°C and 220 rpm to obtain the seed culture.

[0173] Step 2.2: Expanded Culture and Induction

[0174] Seed culture was inoculated into the expansion medium and cultured until OD 600 =0.6–0.8, add IPTG to a final concentration of 0.5 mM, induce at 25°C for 12 h; collect the cells by centrifugation, wash twice with PBS, and store at -20°C.

[0175] The inoculation refers to inoculating the seed culture at a rate of 1%-2% into 50mL-200mL of LB-Kan liquid culture medium; the culture temperature is 37℃ and the rotation speed is 220rpm; the induction temperature is 16℃-25℃ and the time is 16h-20h; or the temperature is 37℃ and the time is 3h-4h; the centrifugation temperature is 4℃, the rotation speed is 8000rpm, and the time is 10min.

[0176] Step 3: Protein sample processing, purification, and injection solution preparation

[0177] Step 3.1: Cell disruption

[0178] The bacterial cells were resuspended in lysis buffer, sonicated, and centrifuged. The supernatant and precipitate were collected separately. The sonication power was 300W–400W, with a sonication time of 3 seconds followed by a 5-second pause, for a total of 20 minutes. The centrifugation temperature was 4℃, the power was 12000 rpm, and the time was 20 minutes.

[0179] Step 3.2: SDS-PAGE and Western blot identification

[0180] Supernatant and precipitate samples were subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining; Western blot was performed using specific primary antibodies against corresponding cytokines to verify the correct expression of the target protein; grayscale analysis was used to calculate the expression level and soluble percentage. Figure 6 As shown.

[0181] Step 3.3: Western Blot protein purification

[0182] Expression was confirmed by Western blotting using anti-His antibody or a protein-specific antibody. The supernatant was purified by Ni affinity chromatography, concentrated by ultrafiltration, and filtered to obtain a single recombinant cytokine. Figure 7 As shown.

[0183] Example 2

[0184] The single recombinant cytokine bone repair injection of this embodiment is composed of the following components by mass percentage: 0.001% single recombinant human cytokine, 5% mannitol, 0.5% human serum albumin, 0.3% phosphate buffer, and the balance being sterile water for injection.

[0185] The single cytokine is selected from any one of BMP-2, PDGF-BB, FGF-2, and VEGF.

[0186] The preparation method of the above-mentioned single recombinant cytokine bone repair injection includes the following steps: taking 0.001% single recombinant human cytokine, 5% mannitol, 0.5% human serum albumin, 0.3% phosphate buffer, and the balance as sterile water for injection according to the following mass percentages, mixing them evenly, adjusting the pH and osmotic pressure; sterile filtration, aseptic filling, and thus obtaining the single recombinant cytokine bone repair injection.

[0187] The preparation method of the single cytokine described above is the same as in Example 1.

[0188] Example 3

[0189] The single recombinant cytokine bone repair injection of this embodiment is composed of the following components by mass percentage: 0.05% single recombinant human cytokine, 2% mannitol, 2% human serum albumin, 0.1% phosphate buffer, and the balance being sterile water for injection.

[0190] The single cytokine is selected from any one of BMP-2, PDGF-BB, FGF-2, and VEGF.

[0191] The preparation method of the above-mentioned single recombinant cytokine bone repair injection includes the following steps: taking 0.05% single recombinant human cytokine, 2% mannitol, 2% human serum albumin, 0.1% phosphate buffer, and the remainder as sterile water for injection according to the following mass percentages, mixing them evenly, adjusting the pH and osmotic pressure; sterile filtration, aseptic filling, and thus obtaining the single recombinant cytokine bone repair injection.

[0192] The preparation method of the single cytokine described above is the same as in Example 1.

[0193] Case 1

[0194] Patient information: Mr. Wang, 60 years old, knee joint dysfunction or injury.

[0195] Treatment plan: Inject 2 mL / vial / time of the single recombinant cytokine bone repair injection solution from Example 2 into the knee joint cavity, with a course of 2 mL × 6 vials. Knee joint function significantly improved. CT images before and after treatment are shown below. Figure 8 and Figure 9 As shown, the treatment plan is as follows Figure 10 As shown.

[0196] Comparative Example 1

[0197] This comparative example corresponds to Case 1 above: knee joint function degeneration / injury, a parallel control of first-line clinical drugs.

[0198] Patient Information: Mr. Zhang, 61 years old, male, with knee osteoarthritis and grade II cartilage damage. Baseline data matched Mr. Wang's. He experienced knee pain, limited flexion and extension, and a continuous walking distance of <200 meters. His WOMAC score was 78.2, and his VAS pain score (0-100) was 58. MRI showed medial compartment cartilage damage and a small amount of joint effusion. Treatment Plan: The recombinant cytokine bone repair injection used in this case was not selected. Instead, sodium hyaluronate injection (a routine clinical treatment drug) recommended as a first-line treatment in the "Guidelines for the Diagnosis and Treatment of Osteoarthritis" was selected and administered via intra-articular injection in the knee. The dosage was 2.5 mL / vial / time (sodium hyaluronate content 25 mg), once a week, with 5 vials constituting one complete course of treatment. The remaining basic nursing care and rehabilitation guidance were completely consistent with Case 1.

[0199] Treatment effect:

[0200] One month after the end of the treatment: the VAS pain score dropped to 32 points, the WOMAC score dropped to 45.7 points, the knee flexion and extension function improved slightly, and the patient could walk 100 meters continuously. However, pain and joint popping still occurred when going up and down stairs.

[0201] Six months after the end of the treatment: the pain VAS score rose to 46 points, the WOMAC score rose to 58.3 points, the functional improvement effect declined significantly, and MRI showed no clear repair of cartilage damage and no reversal of joint degeneration.

[0202] One year after the completion of the treatment course: The patient experienced recurring pain symptoms, requiring a second injection. MRI revealed that the knee cartilage damage had progressed to grade III, with mild narrowing of the joint space. The core difference between this and the treatment plan in this case: One year after the completion of the treatment course in this case, the patient maintained significant improvement in knee joint function and normal cartilage without degeneration; while conventional sodium hyaluronate treatment only provides short-term lubrication and symptom relief, without a clear cartilage repair effect, and the effect is short-lived, prone to symptom rebound, and unable to halt the degenerative process of the joint.

[0203] Case 2

[0204] Patient Information: Mr. Yang, male, 40 years old, suffered a shoulder injury in a car accident.

[0205] Treatment regimen: Targeted injection of 2 mL / vial of the single recombinant cytokine bone repair injection solution from Example 2 into the shoulder joint cavity. A course of treatment consisting of 2 mL x 5 vials yields significant results in shoulder function.

[0206] Comparative Example 2

[0207] This comparative example corresponds to Case 2 above: a shoulder injury caused by a car accident, and a control group treated with routine sports injuries.

[0208] Patient Information: Mr. Liu, 41 years old, male, suffered a rotator cuff tear with periarticular bone contusion due to a car accident. His baseline data matched that of Mr. Yang. His shoulder joint movement was severely limited, with abduction <60° and flexion <90°. His pain VAS score (0-100) was 65. MRI showed a grade II tear of the rotator cuff tendon, bone contusion of the greater tuberosity of the humerus, and joint effusion.

[0209] Treatment plan: The recombinant cytokine bone repair injection used in this case will not be used. Instead, the routine clinical treatment plan for sports injuries will be adopted: targeted injection of platelet-rich plasma (PRP) into the shoulder joint cavity, 3 mL / vial / time, once every 2 weeks, for a total of 3 injections for one complete course of treatment. At the same time, shoulder joint brace fixation and routine rehabilitation training will be used. All other nursing measures will be completely consistent with the original case 2.

[0210] Treatment effect:

[0211] One month after the end of the treatment: the pain VAS score dropped to 28 points, the range of motion of the shoulder joint improved slightly, abduction reached 90°, and flexion reached 120°. However, pain still existed when exerting force, and the patient was unable to lift heavy objects or perform overhead movements.

[0212] Three months after the end of treatment: the pain VAS score dropped to 22 points, the improvement of shoulder joint function entered a plateau period, MRI showed that the rotator cuff tear had no obvious healing, the bone contusion was only partially absorbed, and histological repair was not achieved.

[0213] Six months after the end of the treatment: Patients experienced recurrence of symptoms and required a second injection treatment. Some patients required arthroscopic minimally invasive surgery due to poor functional recovery.

[0214] The core difference between this treatment and the previous one is that the previous treatment only required 5 injections to achieve significant improvement in shoulder joint function and complete targeted repair of bone and soft tissue; while PRP treatment has problems such as large individual differences, limited repair effect, and easy to enter a plateau period after the course of treatment, which cannot achieve complete healing of damaged tissues, and severe patients still need surgical intervention.

[0215] Case 3

[0216] Patient Information: Ms. Fang, 56 years old, female patient.

[0217] Disease symptoms: Due to occupational reasons, the patient has KL grade II knee osteoarthritis (OA) with a pain VAS score of 60, limited function, a WOMAC score of 79.6, and can only walk 40 meters.

[0218] Treatment regimen: Inject 2 mL / vial / time of the single recombinant cytokine bone repair injection of Example 2, 2 mL × 4 vials for one treatment.

[0219] Comparative Example 3

[0220] Corresponding to Case 3 above: K-MRI showed grade II degeneration of knee cartilage, joint effusion, and no joint deformity.

[0221] Treatment plan: The recombinant cytokine bone repair injection used in this case will not be used. Instead, the routine symptomatic treatment plan for knee osteoarthritis will be selected: oral celecoxib capsules (nonsteroidal anti-inflammatory drug, 200mg / time, once daily, for 4 consecutive weeks) + intra-articular injection of triamcinolone acetonide injection (glucocorticoid, 1mL / time, 40mg, single injection). The remaining rehabilitation guidance and lifestyle interventions will be completely consistent with the original case.

[0222] Treatment effect:

[0223] Two weeks after treatment: pain was rapidly relieved, VAS score dropped to 18 points, WOMAC score dropped to 52.3 points, walking distance increased to 100 meters, but morning joint stiffness still existed;

[0224] One month after treatment: Pain rebounded, VAS score rose to 35 points, WOMAC score rose to 61.8 points. Glucocorticoids only have short-term anti-inflammatory effects and no cartilage protection effect. The patient's walking ability did not improve further.

[0225] Six months after treatment: The pain VAS score rose to 52 points, and the WOMAC score rose to 72.5 points, basically returning to the baseline level before treatment. MRI showed that the degeneration of knee cartilage had further worsened and the joint space had narrowed slightly.

[0226] One year after treatment: The patient's cartilage degeneration progressed to KL grade III, with persistent pain and limited joint movement, requiring long-term oral analgesics to control symptoms, and no cartilage repair effect was observed. The core difference between this treatment and the previous one: The previous treatment only required 4 injections to achieve complete pain relief (VAS=0) and complete functional recovery (WOMAC=2), allowing for unrestricted walking of 200 meters, and an MRI one year later showed normal cartilage without degeneration; while conventional anti-inflammatory symptomatic treatment only provides short-term symptom relief, has no cartilage repair effect, carries the risk of gastrointestinal and cardiovascular adverse reactions with long-term use, and cannot stop the cartilage degeneration process, leading to continued disease progression.

[0227] Therefore, this invention uses a high-purity single effective ingredient formulation with an effective ingredient purity ≥95.0%, clearly defined components, and no excess proteins or complex additives, reducing the probability of adverse reactions from the source. The product is prepared through a rigorous purification process, with impurity content controlled at extremely low levels, exhibiting excellent biocompatibility and low immunogenicity, significantly reducing the risk of immune response and inflammatory reactions. Furthermore, the product's biosafety has been fully verified, with a low risk of ectopic ossification, good stability and high safety during in vivo application, providing reliable assurance for clinical use and tissue repair.

[0228] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A single recombinant cytokine bone repair injection, characterized in that, It consists of the following components by mass percentage: 0.001%-0.05% single recombinant cytokine, 2%-5% mannitol, 0.5%-2% human serum albumin, 0.1%-0.3% phosphate buffer, and the balance being sterile water for injection.

2. The single recombinant cytokine bone repair injection according to claim 1, characterized in that, It consists of the following components by mass percentage: 0.03% single recombinant human cytokine, 2.2% mannitol, 1% human serum albumin, 0.2% phosphate buffer, and the balance being sterile water for injection.

3. The single recombinant cytokine bone repair injection according to claim 1, characterized in that, It consists of the following components by mass percentage: 0.001% single recombinant human cytokine, 5% mannitol, 0.5% human serum albumin, 0.3% phosphate buffer, and the balance being sterile water for injection.

4. The single recombinant cytokine bone repair injection according to claim 1, characterized in that, It consists of the following components by mass percentage: 0.05% single recombinant human cytokine, 2% mannitol, 2% human serum albumin, 0.1% phosphate buffer, and the balance being sterile water for injection.

5. The single recombinant cytokine bone repair injection according to any one of claims 1-4, characterized in that, The single cytokine is selected from any one of BMP-2, PDGF-BB, FGF-2, and VEGF.

6. The method for preparing the single recombinant cytokine bone repair injection according to any one of claims 1-5, characterized in that, The procedure includes the following steps: Take 0.001%-0.05% of a single recombinant cytokine, 2%-5% of mannitol, 0.5%-2% of human serum albumin, 0.1%-0.3% of phosphate buffer, and the remainder as sterile water for injection, according to the following mass percentages. Mix thoroughly, adjust pH and osmotic pressure; filter sterilely, and aseptically fill to obtain the single recombinant cytokine bone repair injection solution.

7. The method for preparing the single recombinant cytokine bone repair injection according to claim 6, characterized in that, The method for preparing the single cytokine includes the following steps: Step 1: Construction of engineered cells Step 1.1: Target gene codon optimization and fragment acquisition Based on the natural gene sequence of a single recombinant human cytokine, codon bias optimization was performed on the E. coli expression system, rare codons were removed, and the mRNA secondary structure was optimized. The optimized sequence was then synthesized into a whole genome, with restriction endonuclease sites NcoI and XhoI and protective bases introduced upstream and downstream, respectively, and PCR amplification was performed. The PCR products were detected by 1% agarose gel electrophoresis and purified using a gel extraction kit to obtain the target gene fragment. Step 1.2: Double digestion of the target gene and vector The purified target gene fragment from step 1.1 was combined with pET-28a(+) plasmid and verified by double digestion with NcoI / XhoI. The digestion products were subjected to 1% agarose gel electrophoresis, and the linearized vector and the target fragment were recovered separately. Step 1.3: Ligation and transformation of Escherichia coli DH5α The linearized vector was mixed with the target fragment, T4 DNA ligase was added, and ligation was carried out overnight; the ligation product was transformed into DH5α competent cells, spread on LB solid medium containing 50 μg / mL kanamycin, and cultured upside down. Step 1.4: Screening of positive clones, double enzyme digestion verification, and bidirectional sequencing Single colonies were extracted for colony PCR identification; plasmids were extracted after the positive strains were expanded and cultured, and verified by double digestion with NcoI / XhoI; bidirectional sequencing was performed on the correctly digested plasmids, and the sequencing results were 100% consistent with the optimized sequence, which is the positive recombinant plasmid. Step 1.5: Transformation and expression of Escherichia coli BL21 DE3 The positive recombinant plasmid was transformed into BL21 DE3 competent cells and cultured on LB-Kan plates. Single colonies were picked and verified to be correct by colony PCR and double enzyme digestion of plasmid, thus obtaining the original engineered bacteria. Step 1.6: Preservation of microbial strains The original engineered bacteria were inoculated into LB-Kan liquid medium, shaken overnight, and then frozen for later use. Step 2: Inducing Expression Step 2.1: Seed liquid preparation Step 1: Activate the frozen engineered bacteria by streaking onto LB-Kan plates; pick a single colony and inoculate it with 3-5 mL of LB-Kan liquid medium, incubate overnight to obtain the seed culture; Step 2.2: Expanded Culture and Induction Seed culture was inoculated into the expansion medium and cultured until OD2000. 600 =0.6–0.8, add IPTG to a final concentration of 0.5 mM, induce at 25°C for 12 h; collect bacterial cells by centrifugation, wash twice with PBS, and store at -20°C; Step 3: Protein sample processing, purification, and injection solution preparation Step 3.1: Cell disruption The bacterial cells were resuspended in lysis buffer, sonicated, centrifuged, and the supernatant and precipitate were collected separately. Step 3.2: SDS-PAGE and Western blot identification The supernatant and precipitate samples were subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining; Western blot was performed using the corresponding cytokine-specific primary antibody to verify the correct expression of the target protein; grayscale analysis was used to calculate the expression level and soluble percentage. Step 3.3: Western Blot protein purification Expression was confirmed by Western blotting using Anti-His antibody or target protein-specific antibody. The supernatant was purified by Ni affinity chromatography, concentrated by ultrafiltration, and filtered to obtain a single recombinant cytokine.

8. The method for preparing the single recombinant cytokine bone repair injection according to claim 6, characterized in that, The pH value is 7.2-7.4, and the osmotic pressure is 280mOsm / L-320mOsm / L.

9. The method for preparing the single recombinant cytokine bone repair injection according to claim 6, characterized in that, The pore size of the filter membrane used for sterile filtration is 0.22 μm.

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